**7. Application of microorganisms to remediate heavy metal-polluted soils**

Another approach for biological remediation of heavy metal-polluted soils includes the use of microorganisms to detoxify metals by valence transformation, extracellular chemical precipi‐ tation or volatilization etc. [56]. Bioleaching is the method that uses microorganisms to solubilize heavy metal pollutants either by direct bacterial processes, or as a result of interac‐ tions with metabolic products, or both [76]. It can be used *in situ* or *ex situ* to help to remove the pollutants from soils. This process is based on mobilization of metal cations from insoluble ores by biological oxidation and complexation. This process was adapted from mining industry for the use in soil remediation and a general term covering both bioleaching and biooxidation techniques could be "biomining". This technique is mainly employed for copper, cobalt, nickel, zinc and uranium, which are extracted either from insoluble sulfides or (in the case of uranium) from oxides [86]. The classical bioleaching bacteria belong to the genus *Acidithiobacillus* (*A. thiooxidans* and *A. ferrooxidans*), *Acidiphilium*, *Acidimicrobium*, *Ferromicrobi‐ um* or *Sulfobacillus* [86].

Another solution for soil bioremediation using microorganisms is to apply microbiallymediated biochemical processes, such as oxidation/reduction or methylation reactions [87]. Often, biostimulation and bioaugmentation are the components of bioremediation strategies. Biostimulation is a form of *in situ* bioremediation which uses growth rate stimulation nutrients, electron donors or acceptors to encourage the growth of site-specific indigenous microorgan‐ isms capable of degrading environmental pollutants. Common electron donors and acceptors used in biostimulation include: acetate, sulfate, nitrate and ethanol [88]. Bioaugmentation is the introduction of specific competent microorganisms to the local microbial population in order to increase the metabolic capacities needed for remediation [89]. Biosorption is a physicochemical process that occurs naturally and allows to passively concentrate and bind contaminants onto the microbial cell structure [90]. Metal biosorption by living organisms is a complicated process that consists of two steps. In the first step, metal ions are adsorbed on the cell surface by interactions between metals and cell surface functional groups. Biosorption of metal ions occurs primarily on the outer surface of microbial cells and is the first step in the interactions between metals and microbial cell walls [4]. The cell wall consists of a variety of polysaccharides and proteins, and hence offers a number of active sites capable of binding metal ions [91]. Differences in the cell wall composition among various microbial groups, i.e. algae, bacteria, cyanobacteria and fungi, cause significant differences in the type and amount of metal ions binding to them [91]. Physical adsorption via electrostatic or van der Waals forces allow to retain metal ions on the outer surfaces of bacterial cells. In addition to physical adsorption, ion exchange and complexation are believed to be the dominant mechanisms involved in metal biosorption [4]. The first step, passive biosorption, is metabolism-independ‐ ent and proceeds rapidly by any one or a combination of metal binding mechanisms. In the second step, due to active biosorption, metal ions penetrate the cell membrane and enter into the cells. This is, however, a slowly occurring process. Active mode is metabolism-dependent and related to metal transport and deposition [91]. There are several microbial genera and species capable of metal biosorption. Fungi were found to be efficient biosorbent organisms, as their cells are characterized by a high percentage of cell wall material, which shows excellent metal binding properties [92]. *Aureobasidium pullulans*, *Cladosporium resinae*, *Aspergillus niger*, *Aspergillus versicolor* or *Rhizopus nigricans* are the fungal species proved to be effective in heavy metal biosorption [91]. Numerous studies also identified several species of bacteria as efficient metal accumulating microorganisms. For instance, *Bacillus* spp. has been reported to have a high potential of metal sequestration and has been used in commercial biosorbent preparation [91]. Other bacterial species capable of metal transformation include, among others: *Escherichia coli*, *Pseudomonas maltophilia*, *Shewanella putrefaciens*, *Pseudomonas aeruginosa*, *Enterobacter cloacae* [4].

**7. Application of microorganisms to remediate heavy metal-polluted soils**

Another approach for biological remediation of heavy metal-polluted soils includes the use of microorganisms to detoxify metals by valence transformation, extracellular chemical precipi‐ tation or volatilization etc. [56]. Bioleaching is the method that uses microorganisms to solubilize heavy metal pollutants either by direct bacterial processes, or as a result of interac‐ tions with metabolic products, or both [76]. It can be used *in situ* or *ex situ* to help to remove the pollutants from soils. This process is based on mobilization of metal cations from insoluble ores by biological oxidation and complexation. This process was adapted from mining industry for the use in soil remediation and a general term covering both bioleaching and biooxidation techniques could be "biomining". This technique is mainly employed for copper, cobalt, nickel, zinc and uranium, which are extracted either from insoluble sulfides or (in the case of uranium) from oxides [86]. The classical bioleaching bacteria belong to the genus *Acidithiobacillus* (*A. thiooxidans* and *A. ferrooxidans*), *Acidiphilium*, *Acidimicrobium*, *Ferromicrobi‐*

Another solution for soil bioremediation using microorganisms is to apply microbiallymediated biochemical processes, such as oxidation/reduction or methylation reactions [87]. Often, biostimulation and bioaugmentation are the components of bioremediation strategies. Biostimulation is a form of *in situ* bioremediation which uses growth rate stimulation nutrients, electron donors or acceptors to encourage the growth of site-specific indigenous microorgan‐ isms capable of degrading environmental pollutants. Common electron donors and acceptors used in biostimulation include: acetate, sulfate, nitrate and ethanol [88]. Bioaugmentation is the introduction of specific competent microorganisms to the local microbial population in order to increase the metabolic capacities needed for remediation [89]. Biosorption is a physicochemical process that occurs naturally and allows to passively concentrate and bind contaminants onto the microbial cell structure [90]. Metal biosorption by living organisms is a complicated process that consists of two steps. In the first step, metal ions are adsorbed on the cell surface by interactions between metals and cell surface functional groups. Biosorption of metal ions occurs primarily on the outer surface of microbial cells and is the first step in the interactions between metals and microbial cell walls [4]. The cell wall consists of a variety of polysaccharides and proteins, and hence offers a number of active sites capable of binding metal ions [91]. Differences in the cell wall composition among various microbial groups, i.e. algae, bacteria, cyanobacteria and fungi, cause significant differences in the type and amount of metal ions binding to them [91]. Physical adsorption via electrostatic or van der Waals forces allow to retain metal ions on the outer surfaces of bacterial cells. In addition to physical adsorption, ion exchange and complexation are believed to be the dominant mechanisms involved in metal biosorption [4]. The first step, passive biosorption, is metabolism-independ‐ ent and proceeds rapidly by any one or a combination of metal binding mechanisms. In the second step, due to active biosorption, metal ions penetrate the cell membrane and enter into the cells. This is, however, a slowly occurring process. Active mode is metabolism-dependent and related to metal transport and deposition [91]. There are several microbial genera and species capable of metal biosorption. Fungi were found to be efficient biosorbent organisms, as their cells are characterized by a high percentage of cell wall material, which shows excellent

*um* or *Sulfobacillus* [86].

774 Environmental Risk Assessment of Soil Contamination

Mechanisms involved in biochemical interactions between bacteria and metal ions involve specific enzymes that catalyze the oxidation, reduction, methylation, dealkylation and precipitation reactions. Microorganisms transform a substantial number of metals and metalloids by reducing or oxidizing them directly to a lower or higher redox state. Addition‐ ally, indirect oxidation or reduction is an alternative for immobilization of toxic metals in the environment. Methylation is an important process involved in geochemical cycling of metals and the removal of metal pollutants from soils. Methylation processes derive the methyl group from methylocarbolamine (CH3B12) which is implicated in the methylation of multiple metals and metalloids, such as Pb, Sn, Pd, Pt, Au, Ti, As, Se and Te [93]. Methylation of Hg, Sn and Pb can be mediated by a range of microbes, including *Clostridium* spp., methanogens and sulfate-reducing bacteria under anaerobic conditions and principally by fungi (e.g. *Penicilli‐ um* spp. and *Alternaria* spp.) under aerobic conditions. Methyl groups are enzymatically transferred to metals and a given species may transform a number of different metals [94]. Methyl-metal compounds are generally highly volatile and available to plants [50]. Another mechanism that has the potential for the application in heavy metal-polluted sites is the production of siderophores by different microbial genera. Siderophores are the largest class of compounds that can bind and transport Fe. They are highly specific Fe(III) ligands and are excreted by a wide variety of fungi and bacteria to aid Fe assimilation [94].

Microorganisms play an important role in the environmental biogeochemical cycle of metals and their properties are of significant interest in the remediation of contaminated sites. The microbial ability to absorb and transform metals is a promising aspect in respect of solving the pollution problems [4]. The potential of numerous microbial metal transformations in treat‐ ment of environmental pollution may be employed and some processes are already in commercial operation. However, many processes are still at the laboratory scale and yet to be tested in a rigorous applied and/or commercial context [94]. Another interesting aspect of the microbial community is their ability to multiply even under undesirable environmental conditions. These microorganisms sometimes affect soil environment more quickly than abiotic processes can. Therefore, the structure of soil microbial populations may be useful as a highly sensitive bioindicator of soil disturbance and progress of remediation [95].

Facing the increasing heavy metal pollution severity accompanied by rising land prices the communities around the world need to struggle for available investment grounds. This is mostly the problem of big cities, especially those with limited opportunities for development due to geographical barriers such as seashores, mountain ranges or desert areas. In such situations the polluted industrial areas cannot be left unused for long time to recover naturally. This creates a need for the development of various remedial procedures adjusted to changing contamination level, environmental conditions, available time and funding. Thus, remedial measures need to be almost always modified in order to meet those criteria. This makes that the continuous effort should be made to increase the effectiveness, flexibility and decrease the cost and side effects of the procedures available today. Although a number of measures was developed to remove the even toxic level of contamination, there are many degenerated areas that still cannot be successfully treated now. Those cases involve sites where remediation would be too expensive, time consuming or even technically disputable with currently available treatment procedures.
