Influence of heavy metals pollution on the formation of microbial community in gray forest soil

The state of microbial communities of gray forest soil contaminated with heavy metals at a dose of 5, 10, 100 MPC, in the presence and absence of vegetative corn biomass has been investigated in model experiments. The protective function of the phytocenosis concerning several groups of microorganisms, in particular, azotobacter and polysaccharide-synthesizing bacteria, has been shown. The number of azotobacter in the rhizosphere of plants exceeds the indices of soil without plants: in the control by 3.33 %, at 5 MPC – 36.6, at 10 MPC by 95.6 %. The indicative function of azotobacter has been confirmed concerning soil pollution with heavy metals. The number of azotobacter is decreased with increasing the pollutant dose in the soil without plants: at 5 MPC by 2.64 times, at 10 MPC by 6.67 times, the corresponding indicators for plants rhizosphere are 2.05 and 3.52 times. Azotobacter is not detected by the method of overgrowing soil lumps at the maximum level of soil contamination with heavy metals (100 MPC). The mycelial forms of microorganisms (micromycetes) are highly resistant to contamination with heavy metals that have been confirmed. The activity of mineralization of organic substances, including hummus, is decreased with the increasing of pollutant dose in the soil has been shown. The pedotrophy index decreases at 5 MPC by 3.16%, at 10 MPC – 30.9, and 100 MPC by 46.8 % in soil without plants. Similar numbers for variants with plants fluctuate between 14.2 and 105.4 %. The conclusion about a lesser susceptibility to microbial mineralization of complexes of humic acids and heavy metals has been confirmed. With an increase in the dose of the pollutant, the activity of humus mineralization decreases in soil without plants: at 5 MPC by 16.7 %, at 10 MPC – 12.6, at 100 MPC by 74.7 %; with plants: at 5 MPC by 14.3 %, at 10 MPC – 8.33, at 100 MPC by 113.7 %. It is also drawn attention to the fact that the activity of humus mineralization in the soil rhizosphere is lower than in the soil without plants. In our opinion, the reason for this is the presence of easily utilized substrates in the composition of plant root exudates. It makes the mineralization of hard-to-reach humus molecules inappropriate.


Introduction
The problem of environment, soil and food pollution with heavy metals is one of the most urgent today. The number of publications in the scientific literature on this topic is only inferior to the most studied problem -fixation of atmospheric nitrogen by prokaryotic organisms. The studies of complex systems, which are included not only the soil and microorganisms living in it, but also pla nts that are directly effected on the formation of microbial communities under conditions of soil pollution with heavy metals have been actualized recently (Epelede et al., 2010;Xiong et al., 2010). Modern methods such as measuring of the basal respiration level, substrateinduced respiration, studying of the communities' structure by the PCR analysis and the distribution of functional genes are used to assess the response of microorganisms to contamination with heavy metals. The aim of our research was to study the changes of microbial community functional structure of gray forest soil under the influence of increasing concentrations of heavy metals.

Materials and Methods
The model experiment was established with the using of gray forest large-silty light-loamy soil of the stationary experiment of the National Scientific Center "Institute of Agriculture of NAAS" (experimental farm "Chabany", Kiev-Svyatoshinsky district, Kiev region). The soil of the "intensive agriculture soil" option, which includes a field crop rotation with a saturation of mineral fertilizers of N96P108K112.5 based on the plowing background of crop by-products (2010 crop -oats) was used. The 0-20 cm soil layer contains: humus -1.75 %, alkaline hydrolyzed nitrogen (according to Cornfield) -6.86 mg, nitrate nitrogen -6.46, ammonium nitrogen -0.20, mobile phosphorus -60.0 mg and exchangeable potassium -25.4 mg per 100 g of air dry soil, pH (KCl) -4.9. The soil was sampled in the fall and its biological activity was restored by moistening in thermostat at 25 °C for 21 days before carrying out a model experiment. Variants with artificially created backgrounds of zinc and lead were investigated: No 3 and 4 -5 times excess of MPC; No 5 and 6exceeding the MPC by 10 times; No 7 and 8 -exceeding the MPC by 100 times. The control sample was the soil with a natural concentration of heavy metals. The concentration of metals was calculated using the acid-soluble fraction at the creating the background pollution, since it is this fraction is considered the main technogenic component of the stock of heavy metals in the soil. The control vessels were filled with a KNO3 solution at an appropriate concentration is to equalize the nitrogen content. Maize seeds were sown in half of the vessels 8 days before the introduction of heavy metals. The corn plants were in the 3-4-leaf phase by the time the heavy metals were added. The state of microbial communities was studied 32 days after the addition of heavy metals. The number of microorganisms of individual ecological, trophic and functional groups was assessed by inoculation a soil suspension on appropriate nutrient media (Tepper et al., 2004). The intensity indices of mineralization processes of nitrogen-and carbon-containing compounds, phytotoxic properties of the soil were determined in accordance with the previously described (Malynovska et al., 2011).

Results and Discussion
Plants with their root secretions act as protectors against microorganisms of several ecological and trophic groups, in particular, azotobacter. The number of azotobacter in the rhizosphere of plants exceeds the parameters of soil without plants: in the control by 3.33 %, at 5 MPC-36.6, with 10 MPC -by 95.6 % ( Table 1). The higher the level of soil contamination with heavy metals, the more pronounced the protective function of plants relative to azotobacter. Azotobacter belongs to microorganisms -indicators of the ecological purity of the soil, from our point of view. Its number is decreased when the soil is contaminated with both oil products and heavy metals (Malynovska et al., 2011;Malynovska et al., 2012). The number of azotobacter decreases with the increasing of pollutant dose in soil without plants: at 5 MPC -by 2.64 times, at 10 MPC -by 6.67 times in the presented data. The corresponding indicators for the plants rhizosphere are 2.05 and 3.2 times. Azotobacter is not detected by the fouling of soil lumps at the maximum level of soil contamination with heavy metals (100 MPC). We showed earlier, when studying gray forest soil in a state of fallow that not only the abundance of azotobacter changes under the influence of a dose of heavy metals, but also the physiological and biochemical ac tivity of its cells significantly decreases under the influence of increasing doses of heavy metals (Malynovska et al., 2012). Thus, the number and physiological and biochemical activity of azotobacter cells are diagnostic indicators of the intensity of contamination of gray forest soil with heavy metals has been shown in a series of model experiments (Malynovska et al., 2012;2013;2013a). Table 1. Influence of heavy metals on the number of microorganisms in gray forest soil contaminated for 32 days (intensive agriculture soil, million CFU*/g of dry soil.
The number of acid-forming microorganisms is much higher in the plants rhizosphere in comparison with the soil without phytocenosis: without contamination -2.96 times, with 5 MPC -8.23, with 10 MPC -9.40, with 100 MPC -12.7 times (Table 1). Organic and mineral acids take an active part in the dissolution of mineral elements from their poorly soluble forms. This fact can be considered the evidence of the regulation of the number of acid-forming microorganisms by plants and, consequently, the intensification of the process of transferring mineral elements into a state accessible to plants. Soil contamination with heavy metals, for an unknown reason, enhances this process, which is also confirmed by the studies carried out by us using gray forest soil in a state of fallow (Malynovska et al., 2012). The number and physiological-biochemical activity of polysaccharide-synthesizing bacteria depend on the level of soil contamination with heavy metals has been shown earlier, using the example of a gray forest soil deposit. In particular, the number of polysaccharide-synthesizing bacteria increases in the rhizosphere of plants at 5 MPC -2.24 times, at 10 MPC -6.89, at 100 MPC -1.67 times (Malynovska et al., 2012). We explained this by the protective function of bacterial polysaccharides relative to producer cells. We obtained somewhat different patterns when studying the contamination of gray forest soil with heavy metals for the same period (32 days), however, using soil that is in intensive agricultural crop rotation (see research methods). Therefore, the number of polysaccharide-synthesizing bacteria, in soil without plants, are decreased proportionally the dose of heavy metals: at 5 MPC -2.98 times, at 10 MPC -3.76, at 100 MPC -7.41 times (Table 1). However, the number of polysaccharide-synthesizing bacteria increases in the rhizosphere of plants, with an increase of the pollutant dose, as in the case of fallow land. At the same time, the regul arity of the influence of plant cultivation is clearly traced: the number of polysaccharide-synthesizing bacteria is always higher in the rhizosphere than in soil without plants, and the higher the level of contamination with heavy metals, the greater the degree of influence of plants on the number of bacteria. Previously, it was also found that the number of polysaccharide-synthesizing bacteria decreases with an increase of heavy metals dose in short-term soil contamination (one day), since the synthesis of exopolysaccharides has not yet become a selective advantage of bacteria of this group (Malynovska et al., 2013). Thus, the number of polysaccharide-synthesizing bacteria depends on the period of soil contamination, the dose of heavy metals, the presence or absence of plants, as well as the method of use (fallow, extensive or intensive agricultural soil) and agrochemical characteristics of the contaminated soil.

Influence of the level of heavy metals
Ukrainian Journal of Ecology, 10(6), 2020 have protective functions against the action of heavy metals on bacterial cells (Dudman, 1977); however, the degree of manifestation of this function depends on the characteristics of the soil that has been contaminated. The data published in the scientific literature on the effect of heavy metals on soil microorganisms are often contradictory. The discrepancy between the patterns, identified by the researchers, may be due to different methods, different doses of heavy metals, different compositions of media for the cultivation of one or another group of microorganisms, and even different terminology . Therefore, Zvyagintsev et al. (1987) concluded that pollution with heavy metals significantly reduces the number of oligonitrophils in sod-podzolic soil. At the same time, MPA medium diluted 100 times was used to determine the amount of oligonitrophils, while Mishustin's medium with leached agar (to remove residual nitrogen compounds) is the generally accepted medium for the cultivation of oligonitrophils. Our data indicate that oligonitrophils do not belong to microorganisms sensitive to heavy metals in both short and medium term contamination (Malynovska et al., 2012;2013;2013a). According to Chugunova et al. (1990), nitrifies and cellulolytic are the most sensitive to the inhibitory action of heavy metals. According to our data, the number of nitrifies decreases significantly only at the highest dose of heavy metals studied -100 MPC (Table 1). The number of cellulolytic also depends largely on the presence of plants or the remains of their roots than on the dose of heavy metals. Some researchers consider mycelial forms of microorganisms is more resistant to the action of heavy metals (Zvyagintsev et al ., 1997;Selivanovskaya et al., 2002). This conclusion is confirmed both for options without plants, and for growing corn (Table 1) according to the results of our studies. The number of CFU of micromycetes increases especially strongly at 100 MPC ; it exceeds the number of fungi in the control without plants by 2.3 times, with plants -by 1.29 times. The number of representatives of another mycelial formactinomycetes -decreases with an increasing of heavy metals dose, maximum -at 2-3 times. At the same time, the number of actinomycetes decreases more sharply than the total number of prokaryotes, which decreases at 5 MPC by 12 .5 %, at 10 MPC -17.8, at 100 MPC -by 23.4 % (without plants). Heavy metals slow down (inhibit) the mineralization of organic matter in the soil according to well-established ideas and the results of our own research (Malynovska et al., 2012;El-Shinnawi et al., 1976;Landa et al., 1978). The obtained data confirm this pattern: the pedotrophy index decreases in soil without plants at 5 MPC by 3.16 %, at 10 MPC -30.9, and at 100 MPC -by 46.8 % (  Stepanova (1976) showed that the content of heavy metals in soils correlates with the humus content. It is possibly due to the formation of complexes between heavy metal ions and molecules of humic acids, which are less susceptible to mineralization th an molecules of humic acids outside the complexes. An increase of the humus content in soils contaminated with heavy metals was noted in the works of Bezuglova et al. (1999), which the authors associate with the negative influence of pollutants on the state o f microbial cenosis and a decrease in the overall biological activity of the soil. Gamaley et al. (2008) also confirmed this conclusion on the example of clear gray and chernozem soils of roadside strips of highways. They showed qualitative differences between the humus of polluted and relatively clean soils: in the soils of roadside strips, humus is formed, enriched with more soluble humic compounds, which have greater mobility and more pronounced acidic properties. Our data confirm the conclusion about a lesser susceptibility to microbial mineralization of complexes of humic acids and heavy metals (