**2. The complexity of microbial community in soils**

Except for occasional insects or earthworms, once visible traces of plant biomass are removed, soil appears as a lifeless mass, that is composed of mineral particles and organic residues. However, even desert soils are abundant source of living microorganisms. This seemingly lifeless matter contains complex microbial community, including bacteria, fungi, protozoa and viruses. The integrity of the aboveground and belowground ecosystems depends on the stability, resilience and function of the soil microbial community [5].

Soil is an interesting medium for growing microorganisms, as it contains various nutrients that the microbes need for their metabolism. Unfortunately, nutrients are not always readily available [6]. However, it is one of the richest reservoirs of microorganisms, i.e. 1 gram of agricultural soil may contain even several billion colony forming units (CFUs) of microorgan‐ isms belonging to thousands of different species [7], and even though microorganisms constitute less than 0.5% of the soil mass, they have a major impact on soil properties and processes [5]. Table 1 presents the average numbers of soil microorganisms in a "typical" temperate soil. Destruction of the soil microbiota through mismanagement or environmental pollution causes decline or even death of the aboveground plant and animal populations.


**Table 1.** Relative numbers and approximate biomass of the soil microbiota in a fertile soil [8].

The most characteristic feature of microbial habitats is the great micro-spatial variability of environmental parameters, like temperature or nutrient availability. Many basic requirements of heterogeneous microorganisms are satisfied by various soil microhabitats. This is the reason why, in ecological terms, a number of varying microbial niches can be described. Therefore, the microbial community is composed of diverse taxa with different nutritional demands

within small microenvironments [9]. Analysis of the spatial distribution of bacteria at micro‐ habitat levels showed that in soils subjected to different fertilization treatments, the majority of bacteria were located in micropores of stable soil micro-aggregates (2 – 20 µm), as they contained over 80% of cells [10]. Such microhabitats offer the most favorable conditions for microbial growth in terms of water and substrate availability, gas diffusion and protection against predation. The microhabitat-adapted groups of microorganisms form so-called consortia which are held together by mutually facilitating metabolic processes. The consortia are characterized by more or less sharp boundaries, and variable level of interaction with each other and with other parts of the soil biota. Numerous investigations emphasize the impact of soil structure and spatial isolation on microbial diversity and community structure [11]. Some studies indicate that the soil particle size affects the diversity of microorganisms and com‐ munity structure to a greater extent than other factors such as bulk pH and the type or amount of available organic compounds [12]. Other investigations show that the type and amount of available organic substrates strongly affect the abundance of microbial groups and their functional diversity in soils [13]. Fierer and Jackson [14] claim that the structure of soil bacterial communities is not random also at continental scale and that the diversity and composition of soil bacterial communities at large spatial scales can be predicted to a large extent by a single variable, that is soil pH. The diversity of soil microorganisms comprises different levels of biological organization. It includes genetic variability among taxa (species), number (richness), relative abundance (evenness) of taxa and functional groups within communities [11]. The overall biodiversity of soil microflora comprises bacteria, fungi, actinomycetes and photosyn‐ thetic microorganisms [6].

The next part concerns major sources of heavy metals in soils with particular emphasis on the most important source of soil pollution, i.e. human activity (and more precisely – industry and mining). The following part discusses the effects that toxic levels of heavy metals may have on the microbial population in soils. The last two parts of this chapter describe the ways of dealing with heavy metal pollution – one introduces the term of phytoremediation (soil remediation with the use of plants) and the other one focuses on the use of microorganisms

Except for occasional insects or earthworms, once visible traces of plant biomass are removed, soil appears as a lifeless mass, that is composed of mineral particles and organic residues. However, even desert soils are abundant source of living microorganisms. This seemingly lifeless matter contains complex microbial community, including bacteria, fungi, protozoa and viruses. The integrity of the aboveground and belowground ecosystems depends on the

Soil is an interesting medium for growing microorganisms, as it contains various nutrients that the microbes need for their metabolism. Unfortunately, nutrients are not always readily available [6]. However, it is one of the richest reservoirs of microorganisms, i.e. 1 gram of agricultural soil may contain even several billion colony forming units (CFUs) of microorgan‐ isms belonging to thousands of different species [7], and even though microorganisms constitute less than 0.5% of the soil mass, they have a major impact on soil properties and processes [5]. Table 1 presents the average numbers of soil microorganisms in a "typical" temperate soil. Destruction of the soil microbiota through mismanagement or environmental pollution causes decline or even death of the aboveground plant and animal populations.

**Numbers**

**Per m2 Per g**

The most characteristic feature of microbial habitats is the great micro-spatial variability of environmental parameters, like temperature or nutrient availability. Many basic requirements of heterogeneous microorganisms are satisfied by various soil microhabitats. This is the reason why, in ecological terms, a number of varying microbial niches can be described. Therefore, the microbial community is composed of diverse taxa with different nutritional demands

Bacteria 1013-1014 108-109 300-3000 Actinomycetes 1012-1013 107-108 300-3000 Fungi 1010-1011 105-106 500-5000 Microalgae 109-1010 103-106 10-1500

**Table 1.** Relative numbers and approximate biomass of the soil microbiota in a fertile soil [8].

**Biomass [wet kg×ha-1]**

resistant to heavy metals in the process of soil remediation.

760 Environmental Risk Assessment of Soil Contamination

**2. The complexity of microbial community in soils**

stability, resilience and function of the soil microbial community [5].

**Organisms**

Bacteria constitute the most numerous group of soil microbes – a teaspoon of productive soil contains between 100 million and 1 billion bacterial cells. As soil environment changes rather drastically, spore-forming bacteria tend to be the most common. When environmental conditions become too difficult for normal growth, the bacteria form spores and remain dormant until the environment returns to proper conditions [6]. They facilitate various processes in soils, e.g. those related to water dynamics, nutrient cycling or disease suppression [15]. Soil-dwelling bacteria may be divided into different groups based on:


**•** Classification based on phyla: based on morphology, barcode DNA sequences, physiolog‐ ical requirements and biochemical characteristics, bacteria have been classified into 12 phyla. Each phylum corresponds to a number of bacterial species and genera [15].

Tate [5] lists the most commonly encountered soil bacterial genera as: *Acinetobacter, Agrobac‐ terium, Alcaligenes, Arthrobacter, Bacillus, Brevibacterium, Caulobacter, Cellulomonas, Clostridium, Corynebacterium, Flavobacterium, Hyphomicrobium, Metallogenium, Micrococcus, Mycobacterium, Pseudomonas, Sarcina, Streptococcus* and *Xanthomonas*. These are the heterotrophic bacteria that are augmented in soil by autotrophic and mixotrophic representatives, including nitrifiers, *Thiobacillus* species and iron bacteria.

Bacteria facilitate a number of physical and biochemical alterations or reactions in soils and thereby directly or indirectly support the development of higher plants. Their performance is vital for a variety of processes that include: decomposition of cellulose or other carbohydrates (e.g. *Bacillus*, *Achromobacter*, *Cellulomonas*, *Clostridium*, *Methanococcus*), ammonification (*Bacillus*, *Pseudomonas*), nitrification (*Nitrosomonas*, *Nitrobacter*), denitrification (*Achromobacter*, *Pseudomonas*, *Bacillus*, *Micrococcus*) and nitrogen fixation (symbiotic *Rhizobium*, *Bradyrhizobi‐ um* etc., non-symbiotic *Azotobacter*, *Beijerinckia*) [16].

On the other hand, soil fungi form three functional groups: decomposers, mutualists and pathogens. Fungi, along with bacteria, are important decomposers of hard to digest organic matter and they increase nutrient uptake of phosphorus. Mycorrhizal fungi support plants by promoting root branching and increasing nitrogen, phosphorus and water uptake. They improve plant resilience to pests, diseases or drought and improve soil structure, as fungal hyphae binds soil particles together to create water-stable aggregates. They in turn create the pore spaces in the soil that enhance water retention and drainage [17]. The most common fungi found in soil belong to the *Penicillium* and *Aspergillus* genera together with the representatives of the Zygomycetes and the mycorrhizae-associated Ascomycetes and Basidiomycetes [5].

Actinomycetes are a large group of microorganisms, systematically identified as bacteria, that grow as hyphae. They decompose a wide range of substances, but they are particularly important in degrading recalcitrant (difficult to degrade) compounds such as chitin, lignin, keratin and cellulose. Moreover, they produce a number of secondary metabolites such as antibiotics i.e. streptomycin [18] or geosmine which is responsible for "earthy" smell after soil plowing [15]. Actinomycetes are important in forming stable humus, which enhances soil structure, improves soil nutrient storage and increases water retention in soils. According to Tate [5], the most commonly encountered soil actinomycetes belong to *Nocardia* and *Strepto‐ myces* genera.

Algae are the most common among photosynthetic microorganisms found in soil. They are found only near soil surface, where light is readily available [6]. The most common genera of green algae found in soil are: *Chlorella, Chlamydomonas, Chlorococcum, Protosiphon* etc. and that of diatoms are *Navicula, Pinnularia. Synedra, Frangilaria.* Their functions include the mainte‐ nance of soil fertility, increasing water retention capacity of soil, prevention of soil erosion due to the fact that they act as cementing agents in binding soil particles. They add organic matter to soil after the cell death and thus increase the amount of organic carbon, while their photo‐ synthetic activity release large quantity of oxygen that facilitate the aeration in submerged soils or oxygenate the soil environment. They also take part in weathering rocks, thus building up the soil structure [19]

**•** Classification based on phyla: based on morphology, barcode DNA sequences, physiolog‐ ical requirements and biochemical characteristics, bacteria have been classified into 12

Tate [5] lists the most commonly encountered soil bacterial genera as: *Acinetobacter, Agrobac‐ terium, Alcaligenes, Arthrobacter, Bacillus, Brevibacterium, Caulobacter, Cellulomonas, Clostridium, Corynebacterium, Flavobacterium, Hyphomicrobium, Metallogenium, Micrococcus, Mycobacterium, Pseudomonas, Sarcina, Streptococcus* and *Xanthomonas*. These are the heterotrophic bacteria that are augmented in soil by autotrophic and mixotrophic representatives, including nitrifiers,

Bacteria facilitate a number of physical and biochemical alterations or reactions in soils and thereby directly or indirectly support the development of higher plants. Their performance is vital for a variety of processes that include: decomposition of cellulose or other carbohydrates (e.g. *Bacillus*, *Achromobacter*, *Cellulomonas*, *Clostridium*, *Methanococcus*), ammonification (*Bacillus*, *Pseudomonas*), nitrification (*Nitrosomonas*, *Nitrobacter*), denitrification (*Achromobacter*, *Pseudomonas*, *Bacillus*, *Micrococcus*) and nitrogen fixation (symbiotic *Rhizobium*, *Bradyrhizobi‐*

On the other hand, soil fungi form three functional groups: decomposers, mutualists and pathogens. Fungi, along with bacteria, are important decomposers of hard to digest organic matter and they increase nutrient uptake of phosphorus. Mycorrhizal fungi support plants by promoting root branching and increasing nitrogen, phosphorus and water uptake. They improve plant resilience to pests, diseases or drought and improve soil structure, as fungal hyphae binds soil particles together to create water-stable aggregates. They in turn create the pore spaces in the soil that enhance water retention and drainage [17]. The most common fungi found in soil belong to the *Penicillium* and *Aspergillus* genera together with the representatives of the Zygomycetes and the mycorrhizae-associated Ascomycetes and Basidiomycetes [5].

Actinomycetes are a large group of microorganisms, systematically identified as bacteria, that grow as hyphae. They decompose a wide range of substances, but they are particularly important in degrading recalcitrant (difficult to degrade) compounds such as chitin, lignin, keratin and cellulose. Moreover, they produce a number of secondary metabolites such as antibiotics i.e. streptomycin [18] or geosmine which is responsible for "earthy" smell after soil plowing [15]. Actinomycetes are important in forming stable humus, which enhances soil structure, improves soil nutrient storage and increases water retention in soils. According to Tate [5], the most commonly encountered soil actinomycetes belong to *Nocardia* and *Strepto‐*

Algae are the most common among photosynthetic microorganisms found in soil. They are found only near soil surface, where light is readily available [6]. The most common genera of green algae found in soil are: *Chlorella, Chlamydomonas, Chlorococcum, Protosiphon* etc. and that of diatoms are *Navicula, Pinnularia. Synedra, Frangilaria.* Their functions include the mainte‐ nance of soil fertility, increasing water retention capacity of soil, prevention of soil erosion due to the fact that they act as cementing agents in binding soil particles. They add organic matter to soil after the cell death and thus increase the amount of organic carbon, while their photo‐

phyla. Each phylum corresponds to a number of bacterial species and genera [15].

*Thiobacillus* species and iron bacteria.

762 Environmental Risk Assessment of Soil Contamination

*myces* genera.

*um* etc., non-symbiotic *Azotobacter*, *Beijerinckia*) [16].

Although biomass of all microorganisms living in soil constitutes only several percent of organic matter content, they play an important role in the functioning of entire ecosystems [20]. They take part in soil formation, mineralize organic substances, provide plants with bioavail‐ able compounds, cooperate with plants or may be used as a source of insecticidal substances [21]. One of the most important and most widely studied microbial groups in terms of beneficial effects to soil and plants is the group of Plant Growth Promoting Rhizobacteria (PGPR) [22]. This group includes bacterial species from genera such as *Azotobacter*, *Azospiril‐ lum*, *Bacillus*, *Burkholderia*, *Enterobacter*, *Erwinia*, *Flavobacterium*, *Pseudomonas* and *Rhizobium* [23]. Activity of these bacteria significantly increases plant growth and yield due to a variety of mechanisms, such as phytohormone production, symbiotic and asymbiotic N2 fixation, production of siderophores, activity against phytopathogenic microorganisms, synthesis of antibiotics, enzymes and/or fungicidal compounds, as well as solubilization of mineral phosphates and other nutrients [24]. PGPR may improve plant growth, salinity and metal toxicity stress tolerance, as well as they are able to produce phytohormones such as indole-3 acetic acid (IAA) [25]. Some PGPR produce the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which hydrolyses ACC, the immediate precursor of ethylene in plants. By decreasing its concentration in seedlings and thus its inhibitory effect, these PGPR stimulate seedlings' root length [26]. Figure 1. shows the ways how Plant Growth Promoting Rhizobac‐ teria can stimulate plants. Bacteria from the genus *Rhizobium* form symbiotic associations with roots of leguminous plants like clovers, peas or alfalfa. These Gram-negative, rod-shaped bacteria infect growing root hairs, forming visible nodules. In this form of symbiosis, plants supply simple carbohydrates to bacteria while bacteria convert nitrogen (N2) from air into the forms (NO3 or NH4 + ) that plant can use. When leaves or roots from the plant decompose, nitrogen content increases in soil [15]. Some microbial species are capable of detergent decomposition, taking part in self-purification process of soils. Decomposers are particularly important in immobilizing or retaining nutrients in their cells, thus preventing the loss of nutrients, such as nitrogen, from the rooting zone.

Despite beneficial effects of numerous soil microbes on plant growth or development, soil structure and functioning, some soil-dwelling microorganisms may cause plant, animal and human diseases. Similarly to the beneficial soil microflora, soil pathogens include bacteria, fungi and viruses. One of the example of the most important or best known plant pathogens include *Agrobacterium tumefaciens* (whose updated scientific name is now *Rhizobium radiobact‐ er*) [27] which is the causal agent of crown gall disease of walnuts, grape vines, stone fruits and many others. These bacteria infect plant roots and induce cells to divide (due to overproduction of auxin and cytokinin), causing a tumor-like swellings that contain infected cells [28]. *Erwinia carotovora* (or now called *Pectobacterium carotovorum*) and *Erwinia amylovora*, the Gram-negative plant pathogens with a diverse host range cause infections of numerous agriculturally and scientifically important plant species, such as potato, apple, pear and some members of the family *Rosaceae*[29]. Soil is also an abundant source of fungal pathogens. Among them we may

**Figure 1.** Summary of mechanisms employed by Plant Growth Promoting Rhizobacteria to stimulate plant develop‐ ment.

distinguish *Rhizoctonia solani*, a plant pathogenic fungus with a wide host range and worldwide distribution. It causes collar rot, crown rot, root rot, damping off and wire stem [30]. It mainly attacks plant seeds below the soil surface, but may also infect leaves and stems. Due to a variety of hosts that this pathogen attacks, it is of great importance and is detrimental to a variety of crops. The *Armillaria* root rot, caused by several species of basidoimycete genus *Armillaria* – the honey fungus is, on the other hand, one of the greatest threat for woody plants [31]. Another example of soil-borne plant pathogens is an important genus of fungi – *Fusarium*, which contains a number of, worldwide distributed, phytopathogenic species [32]. Moreover, *Fusarium* has also been more recently reported as an emerging human pathogen for immuno‐ compromised patients [33]. *Clostridium tetani* is an example of one of the most dangerous soilborne human pathogens. It is a tetanus-causing Gram-positive bacterium, whose transmission occurs through the contamination of wounds with soil carrying its spores [34]. Generally, soil is a typical carrier of human bacterial and fungal pathogens. Another example of them is *Bacillus anthracis*, the causative agent of anthrax, which is found worldwide in a variety of soil environments. Inhalation of *B. anthracis* spores can be fatal. Nevertheless, the incidence of both of these fatal diseases has been largely controlled in developed countries due to the develop‐ ment of vaccines [35].

Undoubtedly, soil is an inexhaustible reservoir of microorganisms, both beneficial and pathogenic ones. Causing the imbalance between groups of soil macro- and microorganisms may be irreversible and result in a variety of effects, sometimes unpredictable. Such imbalance may be caused by soil pollution resulting from developing industry, therefore understanding the sources and effects of industrial soil pollution is an important element in preventing the environmental degradation.
