*6.1.1. Bacteria*

Among microorganisms, bacteria constitute of being the most abundant, versatile, most diverse creature on this planet earth [48, 62]. They are basically classified on the basis of their morphology as rod, cocci or spirillum [48, 63]. A bacterium has relatively simple morphology consisting of cell wall, cell membrane, capsule, slime layer and internal structures mitochondria, Golgi apparatus, ribosomes, endoplasmic reticulum. Slime layer contains functional groups like carboxyl, amino, phosphate or sulfate for metals chelation [48, 62]. Cell wall in general, is responsible for surface binding sites and binding strength for different metal ions depending on different binding mechanisms. Various bacterial species e.g. *Bacillus*, *Pseudomonas*, *Escherichia* [48] exhibit biosorption property because of their small size and ability to grow in different environmental conditions [64–66].

Gram classification divides bacteria in two broad categories; Gram positive and Gram negative. Gram negative mostly constitute pathogens although pathogens are also reported in Gram positive. Gram positive bacteria are comprised of thick peptidoglycan layer connected by amino acid bridges, also known to contain polyalcohols and teichoic acids. Overall, Gram positive bacterial cell wall comprised of 90% peptidoglycan. Some teichoic acids are linked to lipids of lipid bilayer forming lipoteichoic acid. These lipoteichoic acids are linked to lipids of cytoplasmic membrane. They constitute linkage of peptidoglycan to cytoplasmic membrane. This results in cross linking of peptidoglycan forming a grid like structure. These teichoic acids are responsible for negative charge on cell wall due to presence of phosphodiester bonds between teichoic acid monomers [48]. On the other hand, Gram negative bacterial cell wall contains an additional outer membrane composed of phospholipids and lipopolysaccharides. Gram negative cell wall contains 10–20% peptidoglycan. The negative charge on the Gram negative bacteria is due to lipopolysaccharides, teichoic acids, teichuronic acids. Extracellular polysaccharides also exhibit the property of metal binding. They are not present in all Gram negative bacteria. Moreover, those species that contain them, they can be easily removed by chemical washing or mechanical disruption [49, 67].

## *6.1.1.1. Bacterial biosorption*

As cell surface encounter metal ion, formation of a complex takes place, which is a pre-requisite for uptake of metals by the organism [59, 60]. Once surface sorption takes place, the metal is transported into the periplasmic space of Gram-negative cells and transported further into the cytoplasm [60]. When cell encounters high concentration of any heavy metal, the heavy metal ion is transported into the cytoplasm, accumulated inside the cell due to one type of metal uptake which is fast, unspecific, constitutively expressed and does not require ATP [61]. The cations of heavy metals interact with physiological ions Cd2+ with Zn2+ or Ca2+, Ni2+ and Co2+ with Fe2+, Zn2+ with Mg2+ thus inhibit the function of respective physiological cations.

Biosorbents can be classified as living or non- living organic materials. They are discussed

Among microorganisms, bacteria constitute of being the most abundant, versatile, most diverse creature on this planet earth [48, 62]. They are basically classified on the basis of their morphology as rod, cocci or spirillum [48, 63]. A bacterium has relatively simple morphology consisting of cell wall, cell membrane, capsule, slime layer and internal structures mitochondria, Golgi apparatus, ribosomes, endoplasmic reticulum. Slime layer contains functional groups like carboxyl, amino, phosphate or sulfate for metals chelation [48, 62]. Cell wall in general, is responsible for surface binding sites and binding strength for different metal ions depending on different binding mechanisms. Various bacterial species e.g. *Bacillus*, *Pseudomonas*, *Escherichia* [48] exhibit biosorption property because of their small size and abil-

Gram classification divides bacteria in two broad categories; Gram positive and Gram negative. Gram negative mostly constitute pathogens although pathogens are also reported in Gram positive. Gram positive bacteria are comprised of thick peptidoglycan layer connected by amino acid bridges, also known to contain polyalcohols and teichoic acids. Overall, Gram positive bacterial cell wall comprised of 90% peptidoglycan. Some teichoic acids are linked to lipids of lipid bilayer forming lipoteichoic acid. These lipoteichoic acids are linked to lipids of cytoplasmic membrane. They constitute linkage of peptidoglycan to cytoplasmic membrane. This results in cross linking of peptidoglycan forming a grid like structure. These teichoic acids are responsible for negative charge on cell wall due to presence of phosphodiester bonds between teichoic acid monomers [48]. On the other hand, Gram negative bacterial cell wall contains an additional outer membrane composed of phospholipids and lipopolysaccharides. Gram negative cell wall contains 10–20% peptidoglycan. The negative charge on the Gram negative bacteria is due to lipopolysaccharides, teichoic acids, teichuronic acids. Extracellular polysaccharides also exhibit the property of metal binding. They are not present in all Gram negative bacteria. Moreover, those species that contain them, they can be easily removed by chemical washing or mechanical disruption [49, 67].

This result in oxidative stress in the cell [1].

ity to grow in different environmental conditions [64–66].

**6. Types of biosorbents**

**6.1. Living organic materials**

below in detail.

26 Biosorption

*6.1.1. Bacteria*

Bacterial cell wall encountering the metal ion is the first component of biosorption. The metal ions get attached to the functional groups (amine, carboxyl, hydroxyl, phosphate, sulfate, amine) present on the cell wall [49, 67]. The general metal uptake process involves binding of metal ions to reactive groups present on bacterial cell wall followed by internalization of metal ions inside cell [48]. More metal is uptaken by Gram positive bacteria due to presence of glycoproteins. Less metal uptake by Gram negative bacteria is observed due to phospholipids and LPS [68, 69]. Biosorption of various metals by different bacteria is given in **Table 2**.



*6.1.2.1. Biosorption by algae*

2. Cadmium *Bifurcaria* 

3. Chromium *Pithophora spp.*

5. Copper *Calotropis procera*

7. Lead *Calotropis procera*

Oocystis *Pithophora spp.* (filamentous) *Fucus vesiculosus* (brown algae)

Oocystis *Sargassum filipendula* Microalgae *Sargassum* sp. (brown algae) *Fucus vesiculosus* (brown algae) *Ascophyllum nodosum*

(filamentous) *Sargassum* sp. *Spirogyra* sp. (green algae) *Sargassum* sp. (brown algae)

**Metals Algae Temperature** 

*bifurcate* Oocystis *Pithophora spp.* (filamentous) *Sargassum* sp. (brown algae) *Sargassum tenerrimum Fucus vesiculosus* (brown algae) *Ascophyllum nodosum*

**(°C)**

**Sr. No.**

According to Abbas et al., [48], algal cell wall is made up of polysaccharides (alginic acid, chitin, xylan, mannan) which provides functional groups (sulfate, hydroxyl, phosphate, imidazole, amino, amine) known to act as metal binding sites [74]. As far as metal binding mechanism is concerned, ionic charge and covalent bonding are hypothesized. Carboxyl and sulfate groups are involved in ionic bonding whereas amino and carboxyl groups are involved in covalent bonding between metal ion and functional group. In response to metal ions, phytochelatins are produced inside the algal body [48]. Biosorption of various metals by different bacteria is given in **Table 3**.

1. Arsenic *Spirogyra hyalina* 25 — 180 2 1 9.8 [128]

4.5 7.5 - 5 6 6 6

4. Cobalt *Spirogyra hyalina* 25 — 180 2 2.5 7.856 [128]

4 5.5 4.5 - 4 5 4

4 5.5 - 5

150 60–80 - -

150 60–80 175 150 150 - -


175 72 - 150 150 - -

**pH Agitation Time Wt (g/L) q(mg/g) or %** 

2.5 28–51 0.17–14 - 4 0.25 0.5–1

9 days 6 3 -


2 4.4–6.0 5 5 - 0.25 0.5–1

2 16–80 0.12–0.13 0.25

**removal**

95 - - 22.2 0.4 mmol/g 1.12 mmol/g 114.9


14.5 - - 0.66 18.6 0.97 70.9

22.8 - - 1.04 **References**

[129] [130] [131] [132] [133] [134] [135]

Biosorption of Heavy Metals

29

http://dx.doi.org/10.5772/intechopen.72099

[131] [136] [132] [132]

[137] [130] [138] [139] [132] [134] [135]

[137] [130] [131] [134]

**Where,** Wt = weight of used adsorbent; Q = uptake removal of pollutant (mg/g); Agitation = speed of shaker (rpm); T = Temperature of the experiment (°C).

**Table 2.** Bacteria and their biosorption features regarding different metals [48, 126, 127].

#### *6.1.2. Algae*

Algae are aquatic plants that lack true roots and stems. It can range from micro algae to macroalgae. They are autotrophic. They can grow in big biomass even when less nutrition is provided. They are considered good biosorbent material [48, 70–73] because of their big size, high sorption capacity and no production of toxic substances. Mostly they are classified as microalgae (fresh water or green algae), macroalgae (marine or brown algae) and red algae. Among these three classes, brown alga is reported to have higher metal uptake capacity. The following features are responsible for binding of heavy metal ions to algae surface; algae species, ionic charge of metal and chemical composition of metal ion solution. Metal ion binding sites on algal surface includes sulfhydryl, hydroxyl, phosphate, sulfate, imidazole, amine, carboxyl groups [74]. The metal uptake mechanism of algae is similar to that of bacteria that is bonding of metal ions with the surface followed by internalization. According to Abbas et al., [48], either of two mechanisms in algal biosorption is involved: (1) ion exchange method where ions present on algal surface Ca, Mg, Na, K they are displaced by metal ions, (2) complexation between functional groups and metal ions.

## *6.1.2.1. Biosorption by algae*

*6.1.2. Algae*

**Sr. No.**

28 Biosorption

**Metals Bacteria Temperature** 

*Geobacillus thermodenitrificans*

10. Selenium *Cupriavidus metallidurans* CH34

11. Silver *Cupriavidus metallidurans* CH34

T = Temperature of the experiment (°C).

*Geobacillus thermodenitrificans* **(°C)**

7. Lead *Enterobacter cloacae* 25 5 240 2 0.1 67.9 [115]

8. Mercury *Enterobacter cloacae* 25 4 240 2 0.1 43.23 [115] 9. Nickel *Actinomycetes* sp. 30 5 150 24 5 36.55 [116]

12. Zinc *Pseudomonas aeruginosa* 25 — — — — 1.33 [119]

**Table 2.** Bacteria and their biosorption features regarding different metals [48, 126, 127].

*Bacillus* sp. 30 5–9 100 24 — 69.34 [124] *Pseudomonas* sp. 30 5–9 100 24 — 90.41 [124] *Micrococcus* sp. 30 5–9 100 24 — 84.27% [124] *Bacillus cereus* 25 5.5 — 24 1.0 36.71 [119]

*Stenotrophomonas maltophilia* 25 5.0 140 2 20 0.41 [116]

*Micrococcus* sp. 35 5 120 24 — 90% [117]

**pH Agitation Time Wt** 

25 4 100 12 — 53 [122]

— — — — — — [111–114]

— — — — — — [111–114]

25 5 100 12 — 18 [122]

**(g/L)**

**q(mg/g) or % removal**

**References**

groups and metal ions.

Algae are aquatic plants that lack true roots and stems. It can range from micro algae to macroalgae. They are autotrophic. They can grow in big biomass even when less nutrition is provided. They are considered good biosorbent material [48, 70–73] because of their big size, high sorption capacity and no production of toxic substances. Mostly they are classified as microalgae (fresh water or green algae), macroalgae (marine or brown algae) and red algae. Among these three classes, brown alga is reported to have higher metal uptake capacity. The following features are responsible for binding of heavy metal ions to algae surface; algae species, ionic charge of metal and chemical composition of metal ion solution. Metal ion binding sites on algal surface includes sulfhydryl, hydroxyl, phosphate, sulfate, imidazole, amine, carboxyl groups [74]. The metal uptake mechanism of algae is similar to that of bacteria that is bonding of metal ions with the surface followed by internalization. According to Abbas et al., [48], either of two mechanisms in algal biosorption is involved: (1) ion exchange method where ions present on algal surface Ca, Mg, Na, K they are displaced by metal ions, (2) complexation between functional

**Where,** Wt = weight of used adsorbent; Q = uptake removal of pollutant (mg/g); Agitation = speed of shaker (rpm);

According to Abbas et al., [48], algal cell wall is made up of polysaccharides (alginic acid, chitin, xylan, mannan) which provides functional groups (sulfate, hydroxyl, phosphate, imidazole, amino, amine) known to act as metal binding sites [74]. As far as metal binding mechanism is concerned, ionic charge and covalent bonding are hypothesized. Carboxyl and sulfate groups are involved in ionic bonding whereas amino and carboxyl groups are involved in covalent bonding between metal ion and functional group. In response to metal ions, phytochelatins are produced inside the algal body [48]. Biosorption of various metals by different bacteria is given in **Table 3**.



ranging from forests to polluted soils and water bodies. They uptake the metals in their fruiting bodies, mycelia and sporocarps [48]. Biosorption of various metals by different fungi and

Yeasts are famous organisms while studying biosorption. *Saccharomyces cerevisiae* is well known yeast which is considered a model system to study biosorption. They are easy to grow, non-pathogenic and give high biomass yield using simple growth medium [80]. The availability of complete genome information makes its genetic engineering an easy job [75, 81]. They are also considered ideal experimental organism in molecular biology experimentation [75, 82–84]. The property of biosorption by yeast cells is affected by various factors including properties of metal ions (valency, radius), cell age of *S. cerevisiae* cells, conditions of culture (composition of growth medium, carbon source), biosorption conditions (initial concentration of metals and biomass, availability of metal ions, temperature, pH, other ions in growth medium) [75]. Moreover, the large size of yeast makes them promising candidates for metal bioremediation. *Saccharomyces cerevisiae* is a widely studied yeast strain. Its different forms are already studied for its biosorption properties including immobilized versus fess cell, living versus dead cells, engineered versus non

**pH Agitation Time Wt** 

2 6 0.33

1 3 0.75 - 4

3 1 0.41


8 8 **(g/L)**

0.4 0.7 3–9

10 2 3.75 - 2

2 1 4

25 - 2

10 10 **q(mg/g) or % removal**

23.2 13 15

16.39 1.97 4.66 - 34.8

4.0 32.2 -

80.8 71 4.84

95.3% 95.3% **References**

[142] [143] [141]

Biosorption of Heavy Metals

31

http://dx.doi.org/10.5772/intechopen.72099

[150] [151] [152]

[153] [154] [155]

[156] [140]

[144, 145] [146] [147] [148] [149]

mushrooms is given in **Tables 4** and **5** respectively.

engineered cells, cultural versus waste cells, etc. [69, 85–89].

**(°C)**

25 25 25

25 25 35

25 25 25

30 30

**Table 4.** Fungi and their biosorption features regarding different metals [48].

1. Arsenic *Penicillium chrysogenum* 25 3–4 190 — 1 24.5 [140]

6 4.75 5

4.5 4.5 6 5.5 6

4.5 5.5 6

5.5 7 5.5

5.5 5.5

7. Nickel *Aspergillus niger* 25 4.5 150 3 1 7.69 [157]

120 125 150

150 200 180

225 - -

100 100

**Metals Bacteria Temperature** 

*Aspergillus niger Hydrilla verticillata*

*Pleurotus ostreatus Trichoderma viride*

*Penicillium canescens*

*Aspergillus fumigatus*

*Fomes fasciatus Aspergillus lentulus*

*Trichoderma longibrachiatum Pleurotus ostreatus*

2. Cadmium *Aspergillus cristatus*

3. Chromium *Aspergillus niger*

4. Copper *Pleurotus ostreatus*

5. Lead *Rhizopus nigricans*

6. Mercury *Aspergillus flavus*

*Mucor*

*6.1.4. Yeasts*

**Sr. No.**

**Table 3.** Algae and their biosorption features regarding different metals [48].

#### **6.1.3. Fungi**

Fungi are eukaryotic living organism which includes yeasts, mushrooms, molds, etc. The cell wall structure of fungi offers good metal binding properties. Fungi in living and dead both forms can be used as biosorbent material [48, 75]. Metal uptake by fungi involves two processes (i) active uptake or bioaccumulation or intracellular uptake, it is dependent on cell metabolism and (ii) biosorption or passive uptake which involves binding of metal ions to surface of cell wall and it is independent of cell metabolism. The energy independent metal uptake mechanism can be affected by temperature, metabolic inhibitors, etc. Metal uptake by fungi was reported both active and passive. Active uptake occurred only with living cells. In this case, the interaction of metal ions with cell surface functional groups may involves ionexchange, complexation or just physical adsorption.

#### *6.1.3.1. Biosorption by fungi*

According to Das et al., [69] fungal cell wall exhibit excellent metal biding properties due to its components. The cell wall of fungus is composed mainly of chitins, mannans, glucans, in addition to lipids, polysaccharides, pigments e.g. melanin [48, 76–78]. Fungal cell wall is reported to be made up of 90% polysaccharides. The functional groups which are involved in metal binding includes carboxyl, phosphate, uranic acids, proteins, nitrogen containing ligands, chitin or chitosan [48, 79]. Biosorption ability of fungal cells can be manipulated by physical of chemical treatments including autoclaving, heat processes or dimethyl sulfoxide, laundry detergent, orthophosphoric acid, formaldehyde, gluteraldehyde, NaOH, respectively [69]. Macrofungi also called as mushrooms, grow wild in all types of environments ranging from forests to polluted soils and water bodies. They uptake the metals in their fruiting bodies, mycelia and sporocarps [48]. Biosorption of various metals by different fungi and mushrooms is given in **Tables 4** and **5** respectively.

#### *6.1.4. Yeasts*

**6.1.3. Fungi**

**Sr. No.**

30 Biosorption

**Metals Algae Temperature** 

(brown algae) *Cladophora fascicularis Spirogyra hyaline*

(brown algae) *Fucus vesiculosus* (brown algae) *Ascophyllum nodosum*

*Sargassum* sp. (brown algae) *Ascophyllum nodosum*

(brown algae)

8. Mercury *Sargassum* sp.

9. Nickel *Sargassum* sp.

12. Zinc Microalgae

13. Iron *Sargassum* sp.

**(°C)**

30 25 25

30 25 25

30 30 25

**Table 3.** Algae and their biosorption features regarding different metals [48].

4 - -

5 5 6


150 - -

150 150 -

Fungi are eukaryotic living organism which includes yeasts, mushrooms, molds, etc. The cell wall structure of fungi offers good metal binding properties. Fungi in living and dead both forms can be used as biosorbent material [48, 75]. Metal uptake by fungi involves two processes (i) active uptake or bioaccumulation or intracellular uptake, it is dependent on cell metabolism and (ii) biosorption or passive uptake which involves binding of metal ions to surface of cell wall and it is independent of cell metabolism. The energy independent metal uptake mechanism can be affected by temperature, metabolic inhibitors, etc. Metal uptake by fungi was reported both active and passive. Active uptake occurred only with living cells. In this case, the interaction of metal ions with cell surface functional groups may involves ion-

**pH Agitation Time Wt (g/L) q(mg/g) or %** 



5 - 0.5–1




30 3 150 — — 14.6 [132]

**removal**

14.8 20 39.2

26.1 0.80 50

0.72 mmol/g 15.4 53.2

**References**

[132] [128] [128]

[132] [134] [135]

[139] [132] [135]

According to Das et al., [69] fungal cell wall exhibit excellent metal biding properties due to its components. The cell wall of fungus is composed mainly of chitins, mannans, glucans, in addition to lipids, polysaccharides, pigments e.g. melanin [48, 76–78]. Fungal cell wall is reported to be made up of 90% polysaccharides. The functional groups which are involved in metal binding includes carboxyl, phosphate, uranic acids, proteins, nitrogen containing ligands, chitin or chitosan [48, 79]. Biosorption ability of fungal cells can be manipulated by physical of chemical treatments including autoclaving, heat processes or dimethyl sulfoxide, laundry detergent, orthophosphoric acid, formaldehyde, gluteraldehyde, NaOH, respectively [69]. Macrofungi also called as mushrooms, grow wild in all types of environments

exchange, complexation or just physical adsorption.

*6.1.3.1. Biosorption by fungi*

Yeasts are famous organisms while studying biosorption. *Saccharomyces cerevisiae* is well known yeast which is considered a model system to study biosorption. They are easy to grow, non-pathogenic and give high biomass yield using simple growth medium [80]. The availability of complete genome information makes its genetic engineering an easy job [75, 81]. They are also considered ideal experimental organism in molecular biology experimentation [75, 82–84]. The property of biosorption by yeast cells is affected by various factors including properties of metal ions (valency, radius), cell age of *S. cerevisiae* cells, conditions of culture (composition of growth medium, carbon source), biosorption conditions (initial concentration of metals and biomass, availability of metal ions, temperature, pH, other ions in growth medium) [75]. Moreover, the large size of yeast makes them promising candidates for metal bioremediation. *Saccharomyces cerevisiae* is a widely studied yeast strain. Its different forms are already studied for its biosorption properties including immobilized versus fess cell, living versus dead cells, engineered versus non engineered cells, cultural versus waste cells, etc. [69, 85–89].


**Table 4.** Fungi and their biosorption features regarding different metals [48].


**6.2. Non-living organic materials**

*6.2.1. Wastes of agricultural or food industry*

**7. Factors affecting biosorption**

affects metal uptake process [48, 93–95].

promised [48].

size and EPS formation.

sites [48, 101].

isms prefer it over wall adsorption.

Biosorption process is affected by following factors.

The wastes of agriculture or food industry includes agricultural byproducts as corn cobs, soya bean hulls, cotton seeds hulls [92] or fruit peels. They contain cellulosic material in their cell wall which is known to contain functional groups like phenolics or carboxylic. On the basis of cation exchange between functional groups and metal ions, the binding of metal ion with functional group results in biosorption and thus removal of metal ion from medium [49].

Biosorption of Heavy Metals

33

http://dx.doi.org/10.5772/intechopen.72099

**Temperature**: For efficient removal of metal ions from environment, the optimum temperature needed to be investigated. It is generally assumed that biosorption is carried out between 20 and 35°C. High temperatures above 45°C may results in damage to proteins which in turn

**pH**: It is a very important parameter. It affects solubility of metal ions and binding sites of biomass. At lower pH, the biosorption of metals is affected [96, 97]. General range of pH for metal uptake is between 2.5–6. Above this limit, metal uptake ability of biosorbent gets com-

**Nature of biosorbents**: Metal uptake is reported in different forms like biofilms, freely suspended microbial cells or immobilization of microbial cells. It can be altered by physical or chemical treatments. Physical treatments include autoclaving, drying, boiling, sonication, etc. Chemical treatment as the name indicates involves chemicals like acid or alkali to improve biosorption capacity. According to Wang and Chen, [75], the fungal cells are deacetylated which affects the structure of chitin resulting in the formation of chitosan-glycan complexes which have results high metal affinities. Abbas et al., [48] also report about effect of age, growth medium components on biosorption as they might result in cell wall composition, cell

**Surface area to volume ratio**: This property plays an important role in efficient removal of heavy metal from medium. The surface area property plays a significant role in case of biofilms [48]. The binding of metal ions with microbial cell wall is previously reported [98]. Although intracellular metal adsorption is energy-consuming process but still microorgan-

**Concentration of biomass**: The concentration of biomass is directly proportional to the metal uptake [48, 98, 99]. It is reported that electrostatic interaction between the cells plays an important role in metal uptake. At a given equilibrium, the biomass adsorbs more metal ions at low cell densities than at high densities [100]. Metal uptake depends on biding sites. More biomass concentration or more metal ions restricts the access of metal ions to binding

**Table 5.** Mushrooms and biosorption of different metals [48].

#### *6.1.4.1. Biosorption by yeast*

The free form of yeast cells is not considered good candidates for biosorption [86]. Free cells face the problem of separation of solid liquid phase. This problem seems to be less effective in flocculating cell [90]. Pretreatment of yeast cells can result in increased surface to volume ration for binding of metal with the metal binding sites. It is reported that pH above 5 optimizes the metal biosorption in yeast cells [91]. According to Abbas et al., [48] in yeasts, higher concentration of heavy metals can be accumulated by bioaccumulation process than biosorption. However, general biosorption is responsible for the major uptake of heavy metals for many filamentous fungi. Biosorption of various metals by different yeasts is given in **Table 6**.


**Table 6.** Yeasts and their biosorption features regarding different metals [48].
