**4. The effects of heavy metals on soil microorganisms**

Metals without biological function are generally tolerated only in minute concentrations, whereas essential metals with biological functions, are usually tolerated in higher concentra‐ tions [9]. They have either metabolic functions as constituents of enzymes or meet structural demands, e.g. by supporting the cell envelope. Frequently the concentration and the speciation of metal determine whether it is useful or harmful to microbial cells [9].

Microorganisms are the first biota that undergoes direct and indirect impacts of heavy metals. Some metals (e.g. Fe, Zn, Cu, Ni, Co) are of vital importance for many microbial activities when occur at low concentrations. These metals are often involved in the metabolism and redox processes. Metals facilitate secondary metabolism in bacteria, actinomycetes and fungi [9; 57]. E.g. chromium is known to have stimulatory effect on both actinorhodin production and growth yield of the model actinomycete *S. coelicolor* [58]. However, high concentrations of heavy metals may have inhibitory or even toxic effects on living organisms [59]. Adverse effects of metals on soil microbes result in decreased decomposition of organic matter, reduced soil respiration, decreased diversity and declined activity of several soil enzymes [60]. Some of the general changes in morphology, the disruption of the life cycle and the increase or decrease of pigmentation are easy to observe and evaluate [9]. Rajapaksha et al. [61] compared the reactions of bacteria and fungi to toxic metals in soils (Zn and Cu). They concluded, that bacterial community is more sensitive to increased concentrations of heavy metals in soils than the fungal community. The relative fungal/bacterial ratio increased with increasing metal levels. Those authors also noticed the varying effect of soil pH on the microbial reaction to soil pollution, i.e. that lower pH in contaminated soils enhanced the negative effect on bacteria, but not on fungi.

**Heavy metals Sources**

768 Environmental Risk Assessment of Soil Contamination

Cr Electroplating industry, sludge, solid waste, tanneries

soils and geological materials

wood burning

**Table 2.** Different sources of heavy metals in soils [56]

industrial wastes enriched in Pb, paints

synthesis (e.g., varnish, pigment formulation)

Zn Electroplating industry, smelting and refining, mining, biosolids

**4. The effects of heavy metals on soil microorganisms**

of metal determine whether it is useful or harmful to microbial cells [9].

plants, herbicides, volcanoes, mining and smelting

application of phosphate fertilizers, sewage sludge

Cu Electroplating industry, smelting and refining, mining, biosolids

As Semiconductors, petroleum refining, wood preservatives, animal feed additives, coal power

Cd Geogenic sources, anthropogenic activities, metal smelting and refining, fossil fuel burning,

Pb Mining and smelting of metalliferous ores, burning of leaded gasoline, municipal sewage,

Hg Volcano eruptions, forest fire, emissions from industries producing caustic soda, coal, peat and

Ni Volcanic eruptions, land fill, forest fire, bubble bursting and gas exchange in ocean, weathering of

Metals without biological function are generally tolerated only in minute concentrations, whereas essential metals with biological functions, are usually tolerated in higher concentra‐ tions [9]. They have either metabolic functions as constituents of enzymes or meet structural demands, e.g. by supporting the cell envelope. Frequently the concentration and the speciation

Microorganisms are the first biota that undergoes direct and indirect impacts of heavy metals. Some metals (e.g. Fe, Zn, Cu, Ni, Co) are of vital importance for many microbial activities when occur at low concentrations. These metals are often involved in the metabolism and redox processes. Metals facilitate secondary metabolism in bacteria, actinomycetes and fungi [9; 57]. E.g. chromium is known to have stimulatory effect on both actinorhodin production and growth yield of the model actinomycete *S. coelicolor* [58]. However, high concentrations of heavy metals may have inhibitory or even toxic effects on living organisms [59]. Adverse effects of metals on soil microbes result in decreased decomposition of organic matter, reduced soil respiration, decreased diversity and declined activity of several soil enzymes [60]. Some of the general changes in morphology, the disruption of the life cycle and the increase or decrease of pigmentation are easy to observe and evaluate [9]. Rajapaksha et al. [61] compared the reactions of bacteria and fungi to toxic metals in soils (Zn and Cu). They concluded, that bacterial community is more sensitive to increased concentrations of heavy metals in soils than

Se Coal mining, oil refining, combustion of fossil fuels, glass manufacturing industry, chemical

The toxic concentration of heavy metals may cause enzyme damage and consequently their inactivation, as the enzymes-associated metals can be displaced by toxic metals with similar structure [59]. Moreover, heavy metals alter the conformational structures of nucleic acids and proteins, and consequently form complexes with protein molecules which render them inactive. Those effects result in disruption of microbial cell membrane integrity or destruc‐ tion of entire cell [62]. Heavy metals also form precipitates or chelates with essential metabolites [63].

Various metals may affect different microbial populations and the resulting impact may vary depending on the metal whose limit concentrations in soils were exceeded. For instance, the pollution of soils with copper affects microorganisms that take part in nitrification and mineralization of protein compounds [50]. Silver is one of the most toxic metals to heterotro‐ phic bacteria. This effect is used for the production of antiseptic preparations. However, there are some silver-resistant bacteria, both in clinical and natural conditions. Some strains of *Thiobacillus ferrooxidans* are able to accumulate particularly large amounts of silver [50]. About 100 ppm of zinc in soils may inhibit nitrification processes and about 1000 ppm inhibits the majority of microbiological processes in soils [64]. Microorganisms play vital role in circulation and transformation of mercury compounds in the environment. Numerous bacteria and fungi show high tolerance (also acquired) to increased concentrations of mercury in soils. However, some microorganisms are sensitive to excess mercury, e.g. the concentration of <10 ppm Hg may have toxic effects on nitrifiers in soils [50]. Increased concentrations of lead in surface soil layers negatively affect soil microflora. Processes of organic matter decomposition, particularly cellulose, are inhibited as a result of decreased enzymatic activity of microorganisms. This results in soil degradation. Biosorption of lead by soil microorganisms reaches on average 0.2% of this metal, but in some cases it may reach even 40% of biomass and may be used for biological remediation [50]. Some studies indicate that long-term contamination of soils with heavy metals has adverse effects on soil microbial activity. For instance, Juwarkar et al. [65] while researching the remediation strategies for cadmium and lead contaminated soils, compared the numbers of the selected groups of microorganisms in natural and heavy metal spiked soils. The results that they obtained indicated that the examined microbial groups were much less abundant in contaminated soils than in natural ones [Table 3]. On the other hand, Lenart and Wolny-Koładka [66] recorded significantly variable numbers of the selected microbial groups while analyzing the uncontaminated and heavy metal contaminated soils of ArcelorMittal steelworks in Cracow. Similarly, their results indicated that except for fungi, the soil-dwelling microorganisms were much less abundant in heavy metal polluted soils than in uncontami‐ nated soils (Table 4). Heavy metal contamination results in reduction of microbial biomass and even if they do not cause the reduction in their number – they reduce biodiversity or disturb the community structure [64].


**Table 3.** Microbiological characteristics of natural and heavy metal spiked spoil samples in Nagpur (India) [65]


**Table 4.** Ranges of the selected microbial groups in heavy metal contaminated and uncontaminated soils of ArcelorMittal steelworks in Cracow, Poland [66].

However, one of the reasons of decreasing biodiversity of microorganisms in heavy metal polluted soils is the selection for tolerant species or strains. Metal exposure may lead to the establishment of tolerant microbial populations, that are often represented by several Grampositive genera such as *Bacillus*, *Arthrobacter* and *Corynebacterium* or Gram-negatives, e.g. *Pseudomonas*, *Alcaligenes*, *Ralstonia* or *Burkholderia* [68]. It was shown that the impact of heavy metals on the bacterial metabolism depends on the growth form. The resistance towards metals seems higher in consortia than in pure cultures [69]. A great number of heavy metal-resistant bacteria, such as e.g. *Cupriavidus metallidurans* possess efflux transporters that excrete toxic or overconcentrated metals outside the cell [70]. Efflux transporters have high substrate affinity and can therefore maintain low cytosolic concentration of metals [9]. Alternatively, microbial cells may prevent the intoxication by the release of metal-binding compounds into the extracellular surroundings. In that case, metals are chelated outside the cell and thus blocked from entering the cell through the membrane transporters that otherwise facilitate the influx [9]. Some fungal and bacterial species are able to keep metals outside their cells by the extracellularly active melanin [71]. It is a secondary metabolite that has strong cation chelating properties through the anionic function such as carboxyl and deprotonated hydroxyl groups [9]. A substantial number of soil microorganisms, such as widespread fungus *Aspergillus niger*, solubilize metals by the release of organic acids or by the immobilization of metals through excretion of different compounds, such as oxalates [72]. Some microorganisms possess the abilities to protect their cells by a cytosolic sequestration mechanisms. These mechanisms are activated once the metal enters the cell and cannot be excreted. In this case internal inclusion bodies, e.g. polyphosphate granules (volutin) bind large amounts of metal cations [73]. Investigation and understanding of microbial resistance mechanisms towards heavy metals are crucial for the potential application of microorganisms for remediation of polluted soils.
