**5. Considerations regarding metal uptake capacity of microorganisms**

The pathway via which the metal binds to a specific site of the biomass is of great importance in relation to the efficiency of a bioremediation process. For example, the ingestion of sediments by microorganisms is considered a principal route of exposure to metals, although free metal ions in sediment pore waters are generally considered to be the most bioavailable form of metals. Thus, metal accumulation is affected by the feeding behavior of microorganisms [61]. After the ingestion of heavy metals, a process of metal excretion and/or detoxify begins to avoid potential toxic effects. However, microorganisms will not suffer the toxic effects of the presence of metals when they are stored in detoxified forms [61]. Moreover, the metal–biomass interaction is dependent on the type of metal that can bind to oxygen-containing or S- and Ncontaining ligands. Although this may be a simple overview of the mechanisms involved, it can act as a starting point for proposing new approaches related to the efficiency of metal uptake by microorganisms [50].

Otherwise, microorganisms can synthesize metal binding proteins, such as MTs or PCs, and the proteins are strongly related to the capacity of metal adsorption, accumulation, and resistance [50]. In particular, metalloproteins are a large group of these proteins, which play an important role mainly in regulating the amount of metals within the cells.

Metal binding proteins present outside of cell membrane attract metal ions exist in solution and assist the transport to cytosol, where metallochaperones (specialized protein chelators) transfer metals to the appropriate receptor protein. The binding sites of the metal binding proteins have been improved to other protein, such as heterologous metalloproteins by using genetic technique. Some researchers developed heterologous metalloproteins with higher affinity and metal-binding capacity and/or specificity and selectivity, which was expressed in bacteria to improve their capacity to adsorb metals [50]. The technique changing the proteins on the cell surface, into heterogeneous one by using recombinant DNA has emerged as a novel approach to enhance the capacity of adsorption. Both bacteria and yeasts have been investi‐ gated for this purpose. A wide diversity of metal-binding proteins, such as glutathione (GSH), GSH-related phytochelatins (PCs), cysteine-rich metallothioneins (MTs), and synthetic phytochelatins (ECn), have been used to enhance the bioaccumulation of heavy metals [66]. For example, the recombinant bacterial strain cloned mercury operon, which coded the regulatory gene (MerR) and other genes involved in the transport, was constructed. The strain showed high resistant to mercury by the detoxification of mercury ions within the cell [66].

The expression of metal-binding proteins or peptides in microorganisms to enhance heavy metal accumulation and/or tolerance has great potential. Several different peptides and proteins have been explored [20, 50]. Different resistance mechanisms can be activated, for example, the production of peptides of the family of metal binding proteins, such as MTs or phytochelatins (PCs); the regulation of the intracellular concentration of metals, with the expression of transporters of proteins of ligand–metal complexes from the cytoplasm to the inside of vacuoles; and the efflux of metal ions by ion channels present in the cell wall. The genes to show the tolerance toward toxicity of metals are often encoded on the transposons or plasmids, which facilitate their dispersion from cell to cell [12]. The tolerance is caused by either the activity bacterial metal resistance result from either the active efflux pumping of the toxic metal out of the cell or enzymatic detoxification (generally via redox chemistry) where a toxic ion is converted into a less toxic or less available metal ion.

Several metal-binding peptides have been studied with the aim of increasing Cd resistance or accumulation by *E. coli* cells. Naturally occurring Cd-binding proteins and peptides, such as MTs and PCs, are very rich in cysteine residues. In addition, histidines are known to have high affinity for transition metal ions such as Zn2+, Co2+, Ni2+ and Cu2+. Therefore, various peptides comprising different sequences of cysteines or histidines was used to bind Cd [20], and consequently Cd tolerance and accumulation could be enhanced in *E. coli* cells. It would be of interest to evaluate Cd-binding peptides and proteins engineered into more environmentally robust bacteria, such as *Pseudomonas*, for their potential use in bioremediation [20].

Hexavalent chromium is mobile, highly toxic, and considered as a priority environmental pollutant. Chromate reductases found in chromium-resistant bacteria have the potential for use in bioremediation process because they are known to catalyze the reduction of Cr(VI) to Cr(III) [67]. The enzymatic reduction of Cr(VI) to Cr(III) involves the transfer of electrons from electron donors, like NAD(P)H, to Cr(VI) with the simultaneous generation of reactive oxygen species (ROS) [67]. Microorganisms that have the ability to reduce Cr(VI) are referred as chromium-reducing bacteria (CRB). Gram-positive CRB shows to have significant tolerance to the toxicity of Cr(VI) even at high concentrations, whereas gram-negative bacteria are much more sensitive to Cr(VI) [67]. Some genes responsible for resistance to Cr(VI) have been determined in bacteria. For example, the chrR gene located on the chromosome of *P. aerugi‐ nosa* confers resistance to chromate. *Ochrobactrum tritici* contains several genes associated with chromate resistance, namely, chrB, chrA, chrC, chrF, and ruvB. The presence of enzymes that play a role in reducing Cr(VI) have been reported for different microorganisms. The enzymes such as quinone reductases, nitroreductases, and NADPH-dependent enzymes vary in their ability to transform chromate and involve different pathways. Several bacteria reduce Cr(VI) through membrane-bound reductases, such as flavin reductase, cytochromes, and hydroge‐ nases. These enzymes can form part of the electron transport system and use chromate as the terminal electron acceptor [67]. Table 3 shows ability of typical microorganisms (algae, bacteria, fungi, and yeasts) to remove heavy metals from certain environments [68–80]. As can be seen, a wide range of microorganisms have been considered for the development of efficient technology for the removal of heavy metal ions from polluted effluents.

can act as a starting point for proposing new approaches related to the efficiency of metal

Otherwise, microorganisms can synthesize metal binding proteins, such as MTs or PCs, and the proteins are strongly related to the capacity of metal adsorption, accumulation, and resistance [50]. In particular, metalloproteins are a large group of these proteins, which play

Metal binding proteins present outside of cell membrane attract metal ions exist in solution and assist the transport to cytosol, where metallochaperones (specialized protein chelators) transfer metals to the appropriate receptor protein. The binding sites of the metal binding proteins have been improved to other protein, such as heterologous metalloproteins by using genetic technique. Some researchers developed heterologous metalloproteins with higher affinity and metal-binding capacity and/or specificity and selectivity, which was expressed in bacteria to improve their capacity to adsorb metals [50]. The technique changing the proteins on the cell surface, into heterogeneous one by using recombinant DNA has emerged as a novel approach to enhance the capacity of adsorption. Both bacteria and yeasts have been investi‐ gated for this purpose. A wide diversity of metal-binding proteins, such as glutathione (GSH), GSH-related phytochelatins (PCs), cysteine-rich metallothioneins (MTs), and synthetic phytochelatins (ECn), have been used to enhance the bioaccumulation of heavy metals [66]. For example, the recombinant bacterial strain cloned mercury operon, which coded the regulatory gene (MerR) and other genes involved in the transport, was constructed. The strain showed high resistant to mercury by the detoxification of mercury ions within the cell [66].

The expression of metal-binding proteins or peptides in microorganisms to enhance heavy metal accumulation and/or tolerance has great potential. Several different peptides and proteins have been explored [20, 50]. Different resistance mechanisms can be activated, for example, the production of peptides of the family of metal binding proteins, such as MTs or phytochelatins (PCs); the regulation of the intracellular concentration of metals, with the expression of transporters of proteins of ligand–metal complexes from the cytoplasm to the inside of vacuoles; and the efflux of metal ions by ion channels present in the cell wall. The genes to show the tolerance toward toxicity of metals are often encoded on the transposons or plasmids, which facilitate their dispersion from cell to cell [12]. The tolerance is caused by either the activity bacterial metal resistance result from either the active efflux pumping of the toxic metal out of the cell or enzymatic detoxification (generally via redox chemistry) where a

Several metal-binding peptides have been studied with the aim of increasing Cd resistance or accumulation by *E. coli* cells. Naturally occurring Cd-binding proteins and peptides, such as MTs and PCs, are very rich in cysteine residues. In addition, histidines are known to have high affinity for transition metal ions such as Zn2+, Co2+, Ni2+ and Cu2+. Therefore, various peptides comprising different sequences of cysteines or histidines was used to bind Cd [20], and consequently Cd tolerance and accumulation could be enhanced in *E. coli* cells. It would be of interest to evaluate Cd-binding peptides and proteins engineered into more environmentally

robust bacteria, such as *Pseudomonas*, for their potential use in bioremediation [20].

toxic ion is converted into a less toxic or less available metal ion.

an important role mainly in regulating the amount of metals within the cells.

uptake by microorganisms [50].

12 Advances in Bioremediation of Wastewater and Polluted Soil



**Table 3.** Sorption potential of certain microorganisms to remove heavy metals.
