**4. Bioaccumulation and biosorption**

In view of the disadvantages associated with conventional methods for metal removal, there is a need for alternative, cost-effective technologies. In recent years, biosorption/bioaccumulation processes have been considered as novel, economic, efficient, and eco-friendly alternative treatment technologies for the removal of heavy metals from contaminated wastewaters generated from various industries.

#### **4.1. Bioaccumulation**

Bioaccumulation is a metabolism-mediated active process in which the metal ions accumulate the biosorbent intracellularly in the living cells. The process occurs in two steps: the first step is the adsorption of metal ions onto cells, which is quick and identical to biosorption, and the later step is slower which includes the transport of metal species inside the cells by active transport [25]. Unlike biosorption, it is an irreversible, complex process which depends on the metabolism of the cells. The process of bioaccumulation occurs by cultivating the biomass of a microorganism in the vicinity of the metal to be accumulated. Since the solution contains the growth medium, the organism begins its metabolic processes and activates the intracellular transport systems for the accumulation of the sorbate. However, the major limitation of the process is that the nutritive medium for growth of the microorganism contains organic carbon sources [26, 27]. Bioaccumulation is an active process which requires a living biosorbent and is mediated by the metabolism of the microorganism used. The process operates by cultivating the microbe in the presence of a metal ion which has to be removed. Part of the biosorbate accumulates inside the cell which enables the biomass to increase and bind greater amounts of metal ions. The organisms which are capable of resisting high loads of metal ions are best suited for accumulating metal species. They do not possess any mechanisms for hindering the accumulation of metal ions in large quantities [28]. They may possess special mechanisms for synthesizing special intracellular binding regions rich in thiol groups as a response to metal ions in their surviving environment. It was found that morphology and physiology of the cell changes upon increase in concentration of the metal ion to be accumulated [29]. Efficient bioaccumulation can be achieved by selecting the microbes that are screened from polluted environments [30]. *Pichia stipitis* yeast was capable of bio-accumulating Cu (II) and Cr (III) with the maximum uptake capacity of 15.85 and 9.10 mg/g, respectively, from aqueous solutions with an initial concentration of 100 ppm at pH 4.5 [31]. *Aspergillus niger* was capable of removing Cu (II) and Pb (II) with the maximum uptake capacity of 15.6 and 34.4 mg/g, respectively [32]. **Table 1** summarizes some more examples of biosorbents used for metal bioaccumulation.


a Since the process of bioaccumulation is achieved with the living organisms, the uptake capacity was determined with the wet weight of the biosorbent.

**Table 1.** Use of microorganisms for bioaccumulation of metal ions.

**Nanofiltration (NF)** is used for the separation of large molecules possible by small pores when they are within the molecular weight range from 300 to 500 Da with a pore diameter of 0.5–2 nm. A commercially available nanofiltration membrane NF270 was used for the removal

**Reverse osmosis (RO)** is a pressure-driven membrane separation process that forces the solution to pass through a semi-permeable membrane for the removal of heavy metals from various industries. Reverse osmosis was used for the removal of Cu (II), Ni (II), and Zn (II) by

**Electrodialysis (ED)** is a novel liquid hybrid membrane separation process used for the separation of ionized species in the solution that passes through an ion exchange membrane when electric potential is applied or due to concentration gradient. The removal of heavy metal ions in groundwater in Korea was achieved by an ED system for the removal of arsenic, lead,

**Photocatalysis** is used for the rapid and efficient destruction of environmental pollutants by using semiconductors which are non-toxic. This method is achieved by a five-step process: transfer, adsorption to the surface of the semiconductor, photocatalytic reactions at the surface, and finally decomposition and removal of the pollutants at the interface region. The heavy metals present in the pharmaceutical waste were photocatalytically degraded and removed by using selenium-doped ZnO nanocomposite semiconductor and the removal capacity was found to be 0.421 (Cu), 0.211 (Cr), 0.147 (Pb), and 0.097 (Cd) per 0.5 g of ZnO/Se

Besides these conventional methods, techniques like coagulation/flocculation [21], electrocoagulation [22], electro-floatation [23], and electro-deposition [24] have been used for the removal of heavy metals from contaminated water resources. However, all the above-mentioned technologies are associated with various disadvantages like incomplete metal removal, generation of sludge, high reagent and energy requirements, and aggregation of metal pre-

In view of the disadvantages associated with conventional methods for metal removal, there is a need for alternative, cost-effective technologies. In recent years, biosorption/bioaccumulation processes have been considered as novel, economic, efficient, and eco-friendly alternative treatment technologies for the removal of heavy metals from contaminated wastewaters

Bioaccumulation is a metabolism-mediated active process in which the metal ions accumulate the biosorbent intracellularly in the living cells. The process occurs in two steps: the first

of Cd (II), Mn (II), and Pb (II) with an efficiency of 99, 89, and 74%, respectively [17].

manganese, and nitrate nitrogen with 73.9, 89.9, 98.9, and 95.1%, respectively [19].

using a polyamide thin-film composite membrane TW30-1812-50 [18].

nanocomposite [20].

72 Biosorption

cipitates and fouling of the membranes.

generated from various industries.

**4.1. Bioaccumulation**

**4. Bioaccumulation and biosorption**

#### **4.2. Biosorption**

Biosorption can be defined as a simple metabolically passive physicochemical process involved in the binding of metals ions (biosorbate) to the surface of the biosorbent which is of biological origin [25]. Biological removal includes the use of microorganisms, plantderived materials, agriculture or industrial wastes, biopolymers, and so on. It is a reversible rapid process involved in binding of ions onto the functional groups present on the surface of the biosorbent in aqueous solutions by means of various interactions rather than oxidation through aerobic or anaerobic metabolism [37]. The advantages of this process include are simple operation, no additional nutrient requirement, low quantity of sludge generation, low operational cost, high efficiency, regeneration of biosorbent, and no increase in the chemical oxygen demand (COD) of water, which are otherwise the major limitations for most of the conventional techniques [27]. Biosorption can remove contaminants even in dilute concentrations and has special relevance with respect to heavy metal removal owing to toxicity at ppb levels. Microorganisms (live and dead) and other industrial and agriculture byproducts can be used as biosorbents for the process of biosorption.

The first stage in biosorption is that biosorbent should be suspended in the solution containing the biosorbate (metal ions). After incubation for a particular time interval, equilibrium is attained. At this stage, the metal-enriched biosorbent would be separated [27]. The process of biosorption is advantageous because it is reversible, does not require nutrients, a single-stage process, of quick range, has no danger of toxic effects and cellular growth, allows intermediate equilibrium concentration of metal ions, and is not controlled by metabolism [26].

Biosorption capacity (mg/g) of the biosorbent can be defined as the amount of biosorbate (metal ions) biosorbed per unit weight of the biosorbent and can be expressed by using the following mass balance equation:

$$qe = \frac{\left(\text{Ci} - \text{Ce}\right)V}{m} \tag{1}$$

binding of metal ions by physical (electrostatic interaction or van der Waals forces) or chemical (displacement of either bound metal cations (ion exchange) or protons) binding, chelation, reduction, precipitation, and complexation (refer **Figure 1**). Biosorbents contain chemical/functional groups like amine, amide, imidazole, thioether, sulfonate, carbonyl, sulfhydryl, carboxyl, phosphodiester, phenolic, imine, and phosphate groups that can attract and sequester metal ions. The key factors controlling and characterizing these mechanisms

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• the chemical, stereochemical, and coordination characteristics of metal ions like molecular

• properties of the biosorbent, that is, the structure and nature (in case of microorganism—living/

• the process parameters like pH, temperature, concentration of sorbate and sorbent, and

The combined effects of the above parameters influence the metal speciation (the formation of

It is defined as the formation of a complex by the association of two or more species. Mononuclear (monodentate) complexes are formed between the metal ion and the ligands in which the metal atom occupies the central position. Polynuclear (multidenate) complex is formed by more than one metal ion in the center and the metal atom may carry a positive,

**Figure 1.** Hypothesis of different mechanisms of biosorption. M+: heavy metal ions, C: chelating agents, BE: molecules

with exchangeable ions, BM: molecules with metal ions, Tp: transport protein.

weight, ionic radius, and oxidation state of the targeted metal species;

are [38, 39]:

non-living);

**5.1. Complexation**

• type of the binding site (biological ligand)

new forms of metal as a result of biosorption).

other competing metal ions; and • availability of the binding sites.

The percent biosorption (R%) known as biosorption efficiency for the metal was evaluated from the following equation:

$$R\,\%=\frac{Ci-Ce}{Ci}\times 100\,\tag{2}$$

where qe is the amount of adsorbed metal ions of the adsorbent (mg g−1), C<sup>i</sup> is the initial concentration of metal ion in the solution (mg L−1), C<sup>e</sup> is the equilibrium concentration of metal ion in the solution (mg L−1), V is the volume of the medium (L), and m is the amount of the biomass used in the adsorption process (g).

#### **5. Mechanism of biosorption**

The mechanism of biosorption is a complex process which involves the binding of sorbate onto the biosorbent. Many natural materials can be used as biosorbents which involve the

binding of metal ions by physical (electrostatic interaction or van der Waals forces) or chemical (displacement of either bound metal cations (ion exchange) or protons) binding, chelation, reduction, precipitation, and complexation (refer **Figure 1**). Biosorbents contain chemical/functional groups like amine, amide, imidazole, thioether, sulfonate, carbonyl, sulfhydryl, carboxyl, phosphodiester, phenolic, imine, and phosphate groups that can attract and sequester metal ions. The key factors controlling and characterizing these mechanisms are [38, 39]:


The combined effects of the above parameters influence the metal speciation (the formation of new forms of metal as a result of biosorption).

### **5.1. Complexation**

**4.2. Biosorption**

74 Biosorption

metabolism [26].

where qe

following mass balance equation:

from the following equation:

be used as biosorbents for the process of biosorption.

*qe* = \_\_\_\_\_\_\_\_\_\_\_\_\_

*<sup>R</sup>* % <sup>=</sup> \_\_\_\_\_

centration of metal ion in the solution (mg L−1), C<sup>e</sup>

biomass used in the adsorption process (g).

**5. Mechanism of biosorption**

Biosorption can be defined as a simple metabolically passive physicochemical process involved in the binding of metals ions (biosorbate) to the surface of the biosorbent which is of biological origin [25]. Biological removal includes the use of microorganisms, plantderived materials, agriculture or industrial wastes, biopolymers, and so on. It is a reversible rapid process involved in binding of ions onto the functional groups present on the surface of the biosorbent in aqueous solutions by means of various interactions rather than oxidation through aerobic or anaerobic metabolism [37]. The advantages of this process include are simple operation, no additional nutrient requirement, low quantity of sludge generation, low operational cost, high efficiency, regeneration of biosorbent, and no increase in the chemical oxygen demand (COD) of water, which are otherwise the major limitations for most of the conventional techniques [27]. Biosorption can remove contaminants even in dilute concentrations and has special relevance with respect to heavy metal removal owing to toxicity at ppb levels. Microorganisms (live and dead) and other industrial and agriculture byproducts can

The first stage in biosorption is that biosorbent should be suspended in the solution containing the biosorbate (metal ions). After incubation for a particular time interval, equilibrium is attained. At this stage, the metal-enriched biosorbent would be separated [27]. The process of biosorption is advantageous because it is reversible, does not require nutrients, a single-stage process, of quick range, has no danger of toxic effects and cellular growth, allows intermediate equilibrium concentration of metal ions, and is not controlled by

Biosorption capacity (mg/g) of the biosorbent can be defined as the amount of biosorbate (metal ions) biosorbed per unit weight of the biosorbent and can be expressed by using the

The percent biosorption (R%) known as biosorption efficiency for the metal was evaluated

*Ci* − *Ce*

ion in the solution (mg L−1), V is the volume of the medium (L), and m is the amount of the

The mechanism of biosorption is a complex process which involves the binding of sorbate onto the biosorbent. Many natural materials can be used as biosorbents which involve the

is the amount of adsorbed metal ions of the adsorbent (mg g−1), C<sup>i</sup>

(*Ci* <sup>−</sup> *Ce*) *<sup>V</sup> <sup>m</sup>* (1)

*Ci* × 100 (2)

is the equilibrium concentration of metal

is the initial con-

It is defined as the formation of a complex by the association of two or more species. Mononuclear (monodentate) complexes are formed between the metal ion and the ligands in which the metal atom occupies the central position. Polynuclear (multidenate) complex is formed by more than one metal ion in the center and the metal atom may carry a positive,

**Figure 1.** Hypothesis of different mechanisms of biosorption. M+: heavy metal ions, C: chelating agents, BE: molecules with exchangeable ions, BM: molecules with metal ions, Tp: transport protein.

negative, or neutral charge depending on the number of binding ligands involved. The complex formation to the monodentate ligand is more preferable than multidentate because the latter contains multiple ligands which may lead to multiple species binding. The metal ion interacts with the ligands by covalent bonds. The attenuated total reflection infrared spectral (ATR-IR) analysis of *Cyanobacterium microcystis* after the biosorption of antimony (III) suggested the involvement of carboxyl, hydroxyl, and amine groups through surface complexation [40]. A similar mechanism of biosorption was reported by other studies by using *Acidiphilium*, *Termitomyces clypeatus,* and alkali-modified sewage sludge for the removal of Cd (II), Cr (VI), and Cd (II), respectively [41–43].

**5.5. Precipitation**

**5.6. Reduction**

**6. Types of biosorbents**

ideal biosorbent are [56]:

The metal ions form precipitates with the functional groups present on the surface of the microbial cells and remain intact or penetrate into the microbial cell. Most cases involve the formation of insoluble inorganic metal precipitates. Organic metal precipitates may be formed when microbial cells are used. Most of the extracellular polymeric substances excreted by the microbes are involved in the formation of organic precipitates. Precipitation of Cu (II) onto *Mesorhizobium amorphae* causes deformation, aggregation, and damage to the cell surface as shown by scanning electron microscope-energy dispersive X-ray (SEM-EDX) analysis [48]. This mechanism of precipitation for biosorption of metal ions was reported by other studies using soybean meal, watermelon rind, and green tomato husk (*Physalis Philadelphia lam*) for the removal of Cr (III)

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In this process, the metal interacts with the functional groups like carboxyl, gets reduced, and leads to the growth of crystals. Elements like gold and palladium have been obtained by the process of reduction. The metal gets reduced once it binds to the biosorbent at discrete places. Removal of toxic hexavalent chromium can be done by the process of reduction. Many organisms remove Cr (VI) by reduction to Cr (III) by biosorption from the aqueous solution [50–52]. The mechanism of biosorption can be studied using different techniques. The acidic and basic properties of the functional groups that are present on the material surface and ion exchange properties can be determined by Boehm method or potentiometric titration [53]. Fourier transform infrared spectrometry (FTIR) offers important information about the functional groups that are present on the surface of biosorbents like carboxyl, amino, amide, hydroxyl, sulfate, carbonyl, ether, ester, and the nature of the bond that are involved in biosorption [54]. Scanning electron microscope (SEM) is a powerful technique for qualitative evaluation of the structure and morphological changes of the biosorbent before and after metal biosorption. Energy dispersive X-ray (EDX) technique provides valuable information about the availability of various elements on the surface of the biosorbent. X-ray photoelectron spectroscopy (XPS) is a quantitative spectroscopic technique for analyzing the surface chemistry of the biosorbent, that is, electronic state and empirical formula of the elements present and oxidative state of the biosorbed metal ion [55].

Identification of biosorbents for the process of biosorption is a major challenge. It is desirable to develop/obtain biosorbents with the capacity to bind/uptake metal ions with greater affinities [56]. A wide variety of materials available in nature can be used as biosorbents for the removal of metals from contaminated water resources. Any kind of plant, animal, and microbial biomass and their derivatives; plant, industrial and agriculture wastes; and byproducts discharged from various industries can be employed as biosorbents. It is important to select a biosorbent from the large spectrum of available materials. The desired characteristics of an

and Cu (II); Cu (II), Zn (II), and Pb (II); and Fe and Mn, respectively [45, 47, 49].
