**4. Mechanisms associated with bioremediation by microorganisms**

into appropriate bioremediation agents. Recombinant DNA technology explores the use of different approaches including PCR, antisense RNA technique, and site-directed mutagenesis.

Engineered strains of *Deinococcus geothermalis* have been developed for the bioremediation of environments containing mixed radioactive waste at high temperatures. Recombinant strain of *Acenitobacter baumanii* was found to enhance degradation rates at sites contaminated with crude oil [45]. In the presence of metals, some higher organisms produce cysteine-rich peptides, such as glutathione (GSH), phytochelatins (PCs), and metallothioneins (MTs), which can bind and sequester metal ions in biologically inactive forms. The overexpression of MTs in re‐ combinant bacterial cells resulted in enhanced metal accumulation, thus offering a promising

Recent studies show that certain GEMs have increased ability to metabolize specific chemicals

Genetic engineering techniques and studies on the metabolic potential of microorganisms have allowed the design of genetically modified microorganisms capable of degrading specific contaminants. This approach offers an opportunity to create an artificial combination of genes that do not exist together in nature. The most commonly used techniques include engineering with single genes or operons, pathway construction, and alternation of the sequences of existing genes [22]. Genetic and biochemical techniques, such as PCR, *in situ* hybridization, and use of antibodies, can also contribute greatly to our knowledge regarding the potential activity of the microorganisms present at polluted sites. DNA tests can indicate the presence

1 2

5

19 those involving biosorption.

strategy for the development of microbial-based biosorbents [12].

**Figure 3.** Scientific publications on bioremediation using microorganisms.

8 Advances in Bioremediation of Wastewater and Polluted Soil

such as hydrocarbons and pesticides [12, 23].

Figure 4 shows the major groups of microorganisms commonly used for the bioremediation of metals, which include bacteria, microalgae, fungi, and yeast.

3 Figure 4. Microorganisms employed in the bioremediation and processes/mechanisms involved in the case of dead and living 4 biomass. **Figure 4.** Microorganisms employed in the bioremediation and processes/mechanisms involved in the case of dead and living biomass.

 Bioremediation can be separated into two categories, biosorption and bioaccumulation. Biosorption is a passive adsorption mechanism that is fast and reversible [6, 49]. The metals are retained by means of physicochemical interaction (e.g., ion exchange, adsorption, complexation, precipitation, and crystallization) between the metal and the functional groups present on the cell surface [6, 47–50]. Several factors can affect the biosorption of metals, such as pH, ionic strength, biomass concentration, temperature, particle size, and presence of other ions in the solution [48]. Both living and dead biomass can occur for biosorption because it is independent of cell metabolism. On the other hand, bioaccumulation includes both intra- and extracellular processes where passive uptake plays only a limited and not very well-defined role [6]. Therefore, living biomass can only occur for bioaccumulation. Table 2 shows a comparison of the main parameters associated with biosorption and bioaccumulation processes. In general, the biosorption process needs inexpensive cost because the biomass can be obtained from industrial waste, and it can be regenerated and reused in many cycles. Bioaccumulation, on the other hand, needs expensive cost because the process occurs in the presence of living cells in which reuse is limited. Also, important factors to be considered include selectivity of metals and the potential for regeneration. The selectivity in biosorption is generally low because the bind only occurs by physicochemical interaction. It can be increased through modification of the biomass. Nevertheless, processes involving bioaccumulation generally perform better than

 The structure of the cell wall of a microorganism contains various macromolecules, such as polysaccharides and proteins, with a high number of charged functional groups, including carboxyl, imidazole, sulfydryl, thioether, phenol, carbonyl, amide, ester sulfate, amino, and hydroxyl groups [51–53]. The positively charged metal present in the solution gravitates toward these functional groups and adsorption occurs. The form in which microorganisms are cultivated can influence the cell wall composition, and this can be exploited to improve the adsorption capacity of the microorganisms [6]. Bacteria can remove heavy metals from wastewater via functional groups, such as ketones, aldehydes, and carboxyl groups present in their cell walls and thereby produce less chemical sludge [54]. Both gram-positive and gram-negative bacteria are used for the uptake of metals. Green, red, and brown algae are also used as biosorbents. Some functional presents in bacteria such as uronic acid of carboxyl groups and sulfate groups, xylans, galactans, and alginic acid are capable of performing ion exchange. The advantage of using algae as biosorbents is that they generally

 Fungi and yeasts also used for the adsorption. The most advantage of fungi is highly variable, ranging in size from mushrooms to microscopic molds. They are easy to grow and produce a substantial biomass. The cell walls of fungi are rich in polysaccharides and glycoproteins, which contain, for instance, amine, imidazole, phosphate, sulfate, sulfhydryl, and hydroxyl groups [56, 57]. However, the cell walls of yeasts contain a microfibrillar structure composed of more than 90% polysaccharides.

29 do not produce toxic substances, unlike other microorganisms such as bacteria or fungi [55].

34 The main groups present in these walls are amine, hydroxide, carboxyl, sulfate, and phosphate groups [58].

Bioremediation can be separated into two categories, biosorption and bioaccumulation. Biosorption is a passive adsorption mechanism that is fast and reversible [6, 49]. The metals are retained by means of physicochemical interaction (e.g., ion exchange, adsorption, com‐ plexation, precipitation, and crystallization) between the metal and the functional groups present on the cell surface [6, 47–50]. Several factors can affect the biosorption of metals, such as pH, ionic strength, biomass concentration, temperature, particle size, and presence of other ions in the solution [48]. Both living and dead biomass can occur for biosorption because it is independent of cell metabolism. On the other hand, bioaccumulation includes both intra- and extracellular processes where passive uptake plays only a limited and not very well-defined role [6]. Therefore, living biomass can only occur for bioaccumulation.

Table 2 shows a comparison of the main parameters associated with biosorption and bioac‐ cumulation processes. In general, the biosorption process needs inexpensive cost because the biomass can be obtained from industrial waste, and it can be regenerated and reused in many cycles. Bioaccumulation, on the other hand, needs expensive cost because the process occurs in the presence of living cells in which reuse is limited. Also, important factors to be considered include selectivity of metals and the potential for regeneration. The selectivity in biosorption is generally low because the bind only occurs by physicochemical interaction. It can be increased through modification of the biomass. Nevertheless, processes involving bioaccu‐ mulation generally perform better than those involving biosorption.

The structure of the cell wall of a microorganism contains various macromolecules, such as polysaccharides and proteins, with a high number of charged functional groups, including carboxyl, imidazole, sulfydryl, thioether, phenol, carbonyl, amide, ester sulfate, amino, and hydroxyl groups [51–53]. The positively charged metal present in the solution gravitates toward these functional groups and adsorption occurs. The form in which microorganisms are cultivated can influence the cell wall composition, and this can be exploited to improve the adsorption capacity of the microorganisms [6]. Bacteria can remove heavy metals from wastewater via functional groups, such as ketones, aldehydes, and carboxyl groups present in their cell walls and thereby produce less chemical sludge [54]. Both gram-positive and gramnegative bacteria are used for the uptake of metals. Green, red, and brown algae are also used as biosorbents. Some functional presents in bacteria such as uronic acid of carboxyl groups and sulfate groups, xylans, galactans, and alginic acid are capable of performing ion exchange. The advantage of using algae as biosorbents is that they generally do not produce toxic substances, unlike other microorganisms such as bacteria or fungi [55].

Fungi and yeasts also used for the adsorption. The most advantage of fungi is highly variable, ranging in size from mushrooms to microscopic molds. They are easy to grow and produce a substantial biomass. The cell walls of fungi are rich in polysaccharides and glycoproteins, which contain, for instance, amine, imidazole, phosphate, sulfate, sulfhydryl, and hydroxyl groups [56, 57]. However, the cell walls of yeasts contain a microfibrillar structure composed of more than 90% polysaccharides. The main groups present in these walls are amine, hydroxide, carboxyl, sulfate, and phosphate groups [58].


**Table 2.** Comparison of biosorption and bioaccumulation processes [51].

Bioremediation can be separated into two categories, biosorption and bioaccumulation. Biosorption is a passive adsorption mechanism that is fast and reversible [6, 49]. The metals are retained by means of physicochemical interaction (e.g., ion exchange, adsorption, com‐ plexation, precipitation, and crystallization) between the metal and the functional groups present on the cell surface [6, 47–50]. Several factors can affect the biosorption of metals, such as pH, ionic strength, biomass concentration, temperature, particle size, and presence of other ions in the solution [48]. Both living and dead biomass can occur for biosorption because it is independent of cell metabolism. On the other hand, bioaccumulation includes both intra- and extracellular processes where passive uptake plays only a limited and not very well-defined

Table 2 shows a comparison of the main parameters associated with biosorption and bioac‐ cumulation processes. In general, the biosorption process needs inexpensive cost because the biomass can be obtained from industrial waste, and it can be regenerated and reused in many cycles. Bioaccumulation, on the other hand, needs expensive cost because the process occurs in the presence of living cells in which reuse is limited. Also, important factors to be considered include selectivity of metals and the potential for regeneration. The selectivity in biosorption is generally low because the bind only occurs by physicochemical interaction. It can be increased through modification of the biomass. Nevertheless, processes involving bioaccu‐

The structure of the cell wall of a microorganism contains various macromolecules, such as polysaccharides and proteins, with a high number of charged functional groups, including carboxyl, imidazole, sulfydryl, thioether, phenol, carbonyl, amide, ester sulfate, amino, and hydroxyl groups [51–53]. The positively charged metal present in the solution gravitates toward these functional groups and adsorption occurs. The form in which microorganisms are cultivated can influence the cell wall composition, and this can be exploited to improve the adsorption capacity of the microorganisms [6]. Bacteria can remove heavy metals from wastewater via functional groups, such as ketones, aldehydes, and carboxyl groups present in their cell walls and thereby produce less chemical sludge [54]. Both gram-positive and gramnegative bacteria are used for the uptake of metals. Green, red, and brown algae are also used as biosorbents. Some functional presents in bacteria such as uronic acid of carboxyl groups and sulfate groups, xylans, galactans, and alginic acid are capable of performing ion exchange. The advantage of using algae as biosorbents is that they generally do not produce toxic

Fungi and yeasts also used for the adsorption. The most advantage of fungi is highly variable, ranging in size from mushrooms to microscopic molds. They are easy to grow and produce a substantial biomass. The cell walls of fungi are rich in polysaccharides and glycoproteins, which contain, for instance, amine, imidazole, phosphate, sulfate, sulfhydryl, and hydroxyl groups [56, 57]. However, the cell walls of yeasts contain a microfibrillar structure composed of more than 90% polysaccharides. The main groups present in these walls are amine,

role [6]. Therefore, living biomass can only occur for bioaccumulation.

10 Advances in Bioremediation of Wastewater and Polluted Soil

mulation generally perform better than those involving biosorption.

substances, unlike other microorganisms such as bacteria or fungi [55].

hydroxide, carboxyl, sulfate, and phosphate groups [58].

Most heavy metals cannot be biodegraded and they tend to accumulate in the microorganism [59]. Several factors influence metal accumulation, such as the degree of exposure, metal concentration, temperature, and salinity, and therefore it is difficult to obtain detailed infor‐ mation on how the accumulation occurs in the bioremediation [60]. The process of accumula‐ tion is complex and varied according the pathway of metabolism is regulated by the metal concentration [61]. Mechanisms of metal ion uptake based on surface binding and metals ions entering the cell membrane have been proposed [62–65].
