INTRODUCTION

In recent decades, special attention has been paid to the problems of anthropogenic pollution of soils and remediation of contaminated soils. Among the large number of pollutants, heavy metals (HMs) should be singled out: soil contamination with HMs is a global problem because of their long half-life and persistence in the environment. This leads to the accumulation of HMs in soils to the levels, when they begin to exert a toxic effect on living organisms and soil biological activity [9, 13, 79, 80]. The appearance of HMs in soils may be related to weathering of the parent rock, but the main factors of the soil contamination with HMs are the aerial emissions of metallurgical enterprises and motor vehicles, hydrogenic pollution with untreated wastewater and sewage sludge, the use of pesticides and fertilizers, the improper disposal of industrial waste, etc. [43, 56].

Another priority pollutant is oil. Emergency spills may occur during its extraction, transportation, storage, and processing. The input of hydrocarbons to a soil exerts a significant negative impact on the soil physicochemical and biological properties, as well as on the soil micro- and macrobiota [8, 9, 12, 15, 77]. In addition, there are cases of combined pollution, when HMs and oil are simultaneously present in the soil, which makes difficult its remediation [34, 50].

Bioremediation with the use of the metabolic potential of biological objects is a promising method of ecosystem rehabilitation, because this efficient and inexpensive method enables the removal of pollutants with minimal damage to the environment [57]. Numerous experiments have already been performed to determine the remediation impact on the biological activity of different types of soils contaminated with oil and HMs [16, 18, 24, 49], in the Republic of Bashkortostan, in particular [4, 14, 15]. Bioaugmentation is one of the variants of bioremediation; it implies the treatment of disturbed areas by particular microorganisms or their consortia with the required enzymatic properties and adaptive potential. Hydrocarbon-oxidizing bacterial strains resistant to increased concentrations of HMs in the environment are of particular interest for purification of soils contaminated with both oil and HMs [26, 42, 50, 59]. However, there are few studies of the biological activity of soils purified from oil and HMs by applied microorganisms [7].

The aim of this work is to study the effect of oil and various doses of lead on the enzymatic activity and microbiological parameters of leached chernozem, as well as to evaluate the efficiency of the application of oil-destructing bacteria for its recovery.

OBJECTS AND METHODS

Research objects. We studied the top 20-cm-thick layer of leached chernozem (Luvic Chernozem) from Ufa district of the Republic of Bashkortostan in a model experiment. The soil was characterized by the following parameters: pHKCl 6.3, Ntotal 0.61%, the humus content 6.8%, and available (0.2 N KCl extract) P2O5 and K2O 94.5 and 101.7 mg/kg soil, respectively. Coarse roots and other plant residues were preliminarily removed from the soil samples, which were then air-dried, sifted through a sieve (mesh size was 1 cm), and placed in pots (3 kg each). The water-air regime was optimized by drainage; the soil water content was maintained at the level of 60% of the total water capacity during the experiment, and regular soil loosening was performed. We applied oil (density, 852 kg/m3; viscosity, 28 mPa s; the contents of paraffins 3.3 wt %, resins 8.5 wt %, and asphaltenes 5–9 wt %) at the rate of 50 g/kg soil. This variant of the experiment corresponds to the range of oil concentrations, which is considered a stress zone for the soil microbial system characterized by an increase in catabolic activity and in the number of hydrocarbon-oxidizing microorganisms (HCOMs) [11]. Bioremediation with the use of active hydrocarbon-oxidizing strains of microorganisms is usually very efficient at this level of soil contamination [3, 6]. Higher concentrations of petroleum hydrocarbons may inhibit bacterial growth, which results in a low intensity of biodegradation and even in the death of hydrocarbon-oxidizing bacteria [54]. We also applied lead (Pb2+) in the form of chemically pure salt dissolved in water (Pb(CH3COO)2·3H2O) (Reakhim, Russia) at the rates of 450, 900, and 1800 mg Pb2+/kg, which corresponded to 15, 30, and 60 MPC (at present, the maximum permissible concentration (MPC) of lead in soil in the Russian Federation is 30 mg/kg), and a liquid culture of bacteria at the rate of 2 × 106 CFU/g. A corresponding amount of distilled water was added to the variants without application of solutions of the lead salt and/or inoculum. The experiment was performed in triplicate. The variant without lead salt, oil, and bacteria (background variant) was used as a control. Soils were sampled on the third, 45th, and 95th days of the experiment. The samples were air dried, sieved through a 2-mm sieve for homogeneous mixture, and stored in plastic bags at 4°C for the analyses.

Strains of microorganisms. Strains of bacteria from the collection of microorganisms of the Ufa Institute of Biology, Ufa Federal Research Center, Russian Academy of Sciences—Thalassospira xiamenensis UOM 2 (UOM 2), Enterobacter sp. UOM 3 (UOM 3), Pseudomonas songnenensis UOM 4 (UOM 4), and a microbial composition (MC) of all of these strains—were applied to the contaminated soil.

Strain Enterobacter sp. UOM 3 is close to typical strains of the species E. asburiae and E. ludwigii [23]. Representatives of these species have been described as typical endophytic and rhizospheric bacteria and recommended for their application in agriculture [68, 83]. Numerous studies of recent years, ours in particular, have shown the efficiency of Enterobacter strains for oil decomposition in soils because of their high viability hydrocarbon-oxidizing capacity [23, 27, 35, 38, 47, 73].

These microorganisms proved to be not antagonists towards one another; they were tolerant to both oil and lead and could synthesize the phytohormone indolyl-3-acetic acid [23, 31]. Bacteria were cultivated in a thermostatically controlled shaker on meat-peptone broth at 180 rpm and a temperature of 28°C during 72 h [19]. Liquid cultures of each strain were mixed at the ratio of 1 : 1 : 1 to create a microbial composition. It was determined that the number of cells of each bacterial strain cultured on the Raymond medium with oil (5%) [67] and lead acetic acid (Pb2+ content was 2.5 mg/mL) reached at least 1 × 106 CFU/mL within five days of incubation. This indicates that the strains are resistant to increased concentrations of Pb in the medium and can use petroleum hydrocarbons as a source of carbon.

We isolated microorganisms by a conventional method of inoculation on nutrient agarized media: nutrient agar (peptone, 10 g/L; yeast extract, 5 g/L; NaCl, 5 g/L; glucose, 1 g/L; agar. 15 g/L; and 1000 mL of distilled water) for heterotrophic microorganisms; Ashby medium for oligonitrophils and nitrogen fixers; Hutchinson medium for cellulolytic microorganisms; starch–ammonia agar for actinomycetes; Tsukamura medium for HCOMs (100 µL of sterile diesel fuel was applied to the medium surface as a carbon source in each Petri dish); and acidified Czapek’s medium (pH 4.5) for micromycetes [19, 21].

Phytotoxicity of the samples of leached chernozem was assessed by biotests on seed germination and growth of radish (Raphanus sativus L.) of Pink-red variety with white tip sterilized in a 1% solution of potassium permanganate. Water extracts from the soil were prepared by shaking weighed soil samples with distilled water (with t the weight to volume ratio of 1 : 10) for 1 h followed by filtering red [66]. Filter paper was placed in Petri dishes, 5 mL of soil water extract was applied to it, and 20 radish seeds were put. Seeds treated with distilled water were used as a control. Seed germination was evaluated according to [75]. Petri dishes were incubated for 72 hours at 22°C. When root appeared, the seeds were considered sprouted. The percentage of seed germination, root elongation, and germination index were calculated according to [66]:

Seed germination (%) = (number of germinated seeds in the sample extract/number of germinated seeds in the control extract) × 100.

Root elongation (%) = (mean root elongation in the sample/mean root elongation in the control extract) × 100.

Germination index (%) = (seed germination (%) × root elongation (%))/100.

The soil water-retention capacity was determined according to GOST 26713-85.

We used the following methods to analyze the activity of soil enzymes: by Galstyan for catalase and invertase activity and by Shcherbakov and Raikhinshtein for urease activity [22].

The soil reaction (pH) was evaluated by the potentiometric method according to GOST 27979-88.

The content of petroleum products in the soil was determined gravimetrically as described in [58].

The data were statistically processed using standard MS Excel programs. Mean dada ± standard error were presented in figures and tables. The reliability of differences was assessed by the Student t-criterion.

RESULTS AND DISCUSSION

Phytotoxicity. The germination index of tested seeds on the 95th day of the experiment was 91.4% for the background soil and 48.4% for the soil with oil (Table 1). The low germination index was related to the high concentration of toxic components in the pollutant, which exerted an inhibitory effect on seed germination and root growth [39]. In addition, oil pollution reduced water availability due to the formation of an oily film on the surface of seeds, which prevented their germination and restricted further plant growth [30, 61].

Table 1.   The effect of bioremediation on phytotoxicity of leached chernozem contaminated with oil and lead (on the 95th day of the experiment)
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The use of HCOMs exerted a favorable effect on the germination of radish seeds in the oil-contaminated soil. This could be related to an enhanced decomposition of hydrocarbons in the soil with a corresponding decrease in the amount of toxic oil components. The germination index of seeds reached its maximum (67.6%) after the application of the strain Thalassospira xiamenensis UOM 2.

Soil contamination with lead caused a decrease in the germination index of radish seeds as compared to the control. The germination index decreased with an increase in the lead content. High lead content (1800 mg/kg) significantly inhibited seed germination. This is one of the proven symptoms of the toxicity of this HM related to its effect on the enzymatic activity of plant cells, damage of membranes, etc. [52]. Lead caused intensive development of microscopic fungi and infection of radish seedlings, which negatively affected the growth and development of plants.

In the variants with combined oil and lead contamination, seed germination was also lower than in the control: the germination index varied within 39.2–54.4%. However, it was higher under the combined influence of pollutants than in the case of contamination with lead only. This is in agreement with an earlier study [53], in which a decrease in phytotoxicity under the impact of combined contamination with HM and organic substances was explained by the binding of lead ions with oil components with a decrease in lead bioavailability.

Application of bacteria to the soil with combined contamination improved radish germination. In the case of the minimum lead content (450 mg/kg), the germination index was maximal, when strains UOM 3 and UOM 4 were applied: 61.6 and 62.1%, respectively. With an increase in the lead contents to 900 and 1800 mg/kg, the germination index was the highest, when the soil was treated with the strain UOM 2 and MC. This could be related to the biological decomposition of toxic oil components and possible deactivation of lead cations due to their binding by microorganisms. The ability to bind metal ions by extracellular biopolymers was previously described for representatives of the families Pseudomonadaceae and Enterobacteriaceae [46]. In addition, the capacity of pseudomonades to absorb HMs on the cell walls and inside the cells was revealed [74].

The germination index is considered to be an indicator of the environment phytotoxicity (<50%), moderate phytotoxicity (50–80%), or its absence (>80%) [66]. It may be concluded that soils contaminated with oil at the concentration of 5% and with lead at the concentration >450 mg/kg are phytotoxic. The application of bacteria to the soil with oil and combined (oil and HM) pollution decreases its phytotoxicity and transforms it to a moderately toxic state.

Degradation of petroleum hydrocarbons. The survival of introduced bacteria is an important parameter of the efficiency of the biological preparation.

To assess the survival of the studied strains in the soil, we preliminarily studied samples of sterile soil for excluding the development of an indigenous hydrocarbon-oxidizing microbiota. On the 14th day after the application of the studied strains of microorganisms, the number of HCOM was 106–107 CFU/g, whereas they were not detected in the oil-contaminated sterile soil.

The amount of HCOMs after 45 days was greater in the treated samples than in the untreated samples, which indirectly characterized the adaptation and survival of the applied HCOMs.

According to our data, the number of HCOMs remained sufficient for their destructive activity (Table 2). Biodegradation is usually considered the main mechanism for removing pollutants from soil [36], although other mechanisms (for example, volatilization) may be involved in this process.

Table 2.   Dynamics of the number of hydrocarbon-oxidizing microorganisms, the degree of biodegradation of hydrocarbons, and changes in pH in leached chernozem during bioremediation
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In the oil-contaminated soil, the degradation of hydrocarbons was more intensive than in the soil with combined contamination. The application of bacterial preparations increased the decomposition of hydrocarbons by 6.2–30.8% as compared to the untreated variant. By the end of the experiment, biodegradation in the oil-contaminated soil reached maximum in the variant with the introduction of microorganisms UOM 2 and UOM 4 (83.8 and 79.8%, respectively).

In the presence of lead at all the studied concentrations, the decomposition of hydrocarbons decreased by 4.4–11.2%. The application of HCOMs exerted a favorable effect on the processes of oil degradation in the soil with combined contamination.. The most efficient cultures were UOM 3 and UOM 4 for the soil with oil and lead acetate concentration of 450 mg/kg and UOM 2 and MC for the soil with oil and lead acetate concentration of 900 and 1800 mg/kg. A direct dependence of the degree of biodegradation of hydrocarbons on the lead concentration in the soil with combined contamination was absent.

Soil pH. Oil, lead, and their combined effect caused some acidification of the soil by the middle of the experiment (to pH 5.65–6.0), which probably enhanced the adverse effect of the pollutants (Table 2). The use of lead in the form of acetic acid salt could cause an acid soil reaction. In oil-contaminated variants, this could be related to the decomposition of hydrocarbons and the formation of acid compounds and free cations, which corresponded to data of other researchers [28]. Soil pH is an important parameter, because it often exerts a strong effect on the solubility of metals in soil and soil solution. A decrease in pH by a unit causes about twofold increase in the concentration of metals [63].

However, by the end of the experiment, pH shifted to the alkaline range and became close to that in the background soil. This tendency to alkalization was obviously explained by the high acid-base buffer capacity of chernozems [10]. In general, the soil reaction was favorable for the development of microorganisms and the decomposition of organic substances, including oil, since the pH from 6 to 8 is considered optimal for their growth [60].

The number of ecological-trophic groups of microorganisms. The number of microorganisms is an important parameter of soil biological activity and may be an indicator of soil status. Oil and combined contamination of soil caused an increase in the number of heterotrophic microorganisms, which was probably related to the role of oil as an additional carbon source (Table 3). It is obvious that the contribution of HCOMs to the rise in the number of heterotrophs was the greatest. Bioaugmentation of oil-contaminated soil also caused an increase in the number of heterotrophic microorganisms, probably due to the intensive development of the used hydrocarbon-oxidizing microbiota, which was confirmed by published data [32]. Contamination with lead at various rates did not affect the number of heterotrophic microorganisms in the soil.

Table 3.   Dynamics of the number of various ecological-trophic groups of microorganisms in leached chernozem during bioremediation
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Oil and lead in the soil (individually and in combination) inhibited the development of actinomycetes: their number decreased by 1–2 orders of magnitude as compared to the control. On one hand, this decrease in the polluted and remediated soil could indicate a strong sensitivity actinomycetes to pollutants. It was found [1] that an increased content of mobile forms of HMs in soils caused structural and functional changes in the communities of actinomycetes: a decrease in their number and species diversity. On the other hand, actinomycetes are rather sensitive to changes in the medium acidity [2], so their smaller number could be related to soil acidification. It is possible that the actinomycete complex was initially dominated by species adapted to a narrower pH range. Thus, the negative effect of soil pollution on the development of actinomycetes could be caused by a direct toxic effect of pollutants and by an indirect effect of changes in the medium conditions after oil and lead were added to the soil.

The number of microscopic fungi slightly increased in the first day after oil and lead application, which could be related to soil acidification. In addition, micromycetes are capable of using a wide range of organic compounds, including oil [20, 76], so its low concentrations do not exert a pronounced toxic effect on these microorganisms. The application of bacteria caused a decrease in the number of fungi, which could be related to their competition with HCOMs for food.

The number of oligonitrophilic, nitrogen-fixing, and cellulose-decomposing microorganisms reflected their negative response to pollutants in soil. The application of HCOMs sometimes exerted a positive effect on the development of these groups of microorganisms. For example, their number became close to the control by the end of the experiment as a result of the application of strain UOM 2 to the soil contaminated with oil and with oil and lead at the rate of 900 mg/kg. Thus, actinomycetes were highly sensitive to the oil and lead pollution. An increase in the number of heterotrophic microorganisms in variants with oil, combined pollution, and bioremediation was obviously related to active development of hydrocarbon-oxidizing microbiota.

Enzymatic activity. Soil enzymes are catalysts of important metabolic processes, including the decomposition of organic pollutants [25, 70]. Enzymes are considered to be sensitive to pollution and are easily determined without expensive and complex tools, so they are often used as indicators to assess various kinds of soil pollution [29]. Recent studies have shown that microorganisms can improve the enzymatic activity of soil [44, 62] and that the resistance of bacteria to HMs is related to increased activity of antioxidant enzymes and urease [33, 40, 69]. It is known that HMs may be precipitated by bacterial urease and thus removed from soils [51].

Catalase activity. Catalase is very sensitive to contamination with HMs and oil [37, 48], so its activity may be used to analyze the substrate toxicity. It is known that this bacterial enzyme causes degradation of reactive oxygen species, the formation of which is induced by HMs, oil, and some other xenobiotics [82].

In the oil-contaminated soil, the catalase activity (CA) initially increased, but then began to decrease and became lower than in the control (Fig. 1a). This generally corresponded to data on the inhibition of catalase activity by hydrocarbons [64]. When strain UOM 3 and MC were applied to the oil-contaminated soil, the CA remained constant (3 mL O2/g min), starting from the second half of the experiment, which could indicate the beginning of stabilization of soil conditions.

Fig. 1.
figure 1

Catalase activity of the soil contaminated with (a) oil and lead at the concentrations of (b) lead 450 mg/kg soil, (c) 900 mg/kg soil, and (d) 1800 mg/kg soil (d) and with both pollutants.Here and in the following figures: BS is background soil, OS is oil-polluted soil, and Pb is lead acetate.

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In the variants with lead, the CA was higher than that in the control. It became maximum (6.1 mL O2/g min) on the 45th day of the experiment in the variant with the lead rate of 1800 mg/kg soil (Fig. 1d). This confirmed that the CA could increase parallel to the concentration of HMs [81]. The activation of this enzyme in the presence of lead acetate was probably caused by a reaction between Pb2+ and the functional groups of catalase.

Similarly to oil, the combined contamination caused an increase in the CA at the initial stage; later, the activity of the enzyme decreased (Figs. 1b, 1c).

The CA of the soil contaminated with oil and lead (450 mg/kg) was higher after bioaugmentation in the middle and at the end of the experiment as compared to the untreated variant, the highest values being achieved by HCOM 4 and MC. The application of bacteria to the soil contaminated with oil and lead in high concentration (1800 mg/kg) exerted a positive effect on the enzyme activity. There was even an increase in the CA in variants with the strain UOM 4 and MC added to the soil contaminated with oil and lead in high concentration by the end of the experiment, which indirectly attested to the normalization of oxidation-reduction processes.

Thus, the soil contaminated with oil and with oil and lead was characterized by a general tendency to a decrease in the CA. The individual application of lead, on the contrary, stimulated the CA. The use of microorganisms was favorable for the CA: it was higher than in the variant without bacterization.

Invertase activity. Invertase is one of the enzymes resistant to the excessive content of HMs and oil in soil [8, 17]. Despite this, its activity is considered informative for the integral assessment of the soil status [5, 78].

In our experiment, the invertase activity (IA) decreased in the oil-contaminated soil, and this was also observed after the application of bacteria (Fig. 2a).

Fig. 2.
figure 2

Invertase activity of the soil contaminated with (a) oil and lead at the concentrations of (b) lead 450 mg/kg soil, (c) 900 mg/kg soil, and (d) 1800 mg/kg soil (d) and with both pollutants, during bioremediation in particular.

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This could be related to the destruction of bacterial cells by toxic substances in the pollutant [14]. Since the invertase origin is extracellular, the death of some bacteria could cause a decrease in its synthesis and in the IA of the soil [65]. The only exception was observed in the variant with strain UOM 2: the IA exceeded that in the control by 10% on the 95th day. A decrease in the IA with time was also seen for the soil with combined contamination (Figs. 2b–2d). In most cases, the IA was higher in the variants with lead than in the control (mainly during the first half of the experiment). The application of the bacterial strain UOM 4 to the oil-contaminated soil with lead (450 mg/kg) caused an increase in the IA to 5 mg of glucose/g of soil by the end of the experiment (Figs. 2c and 2d). The restoration of the IA level after initial inhibition could be related to the growth of the introduced microbial population as a result of adaptation [41] and to the ability of this strain to utilize a wider range of oil hydrocarbons.

It was revealed that the IA directly depends on the amount of soil microbes [45], because it slightly correlates with the number of heterotrophic microorganisms (r = 0.35, p > 95%) by the end of this experiment.

Thus, oil suppressed the IA. Soil contamination with lead, on the contrary, increased the activity of this enzyme at the initial stage of contamination. The combined pollution caused various reactions of soil invertase, however, bioaugmentation sometimes exerted a positive effect on the IA in the soil with both oil and combined contamination.

Urease activity. The urease activity (UA) in soil characterizes the potential of urea mineralization. The enzyme forms stable complexes (urease–humus) and contributes much to soil fertility [55]. Its activity may be an adequate indicator of combined contamination (HMs, polycyclic aromatic hydrocarbons, etc.), at the early stages, in particular [72, 81].

Oil exerted a negative impact on the UA of the soil (Fig. 3a).

Fig. 3.
figure 3

Urease activity of soil contaminated with (a) oil and lead at the concentrations of (b) lead 450 mg/kg soil, (c) 900 mg/kg soil, and (d) 1800 mg/kg soil (d) and with both pollutants.

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The addition of microorganisms stimulated the UA at the initial stage, but it decreased with time though remained higher than that in the soil without bioremediation and generally close to that in the control. The strain UOM 2 caused an increase in the UA by 64% on the third day and by 25% by the end of the experiment relative to the control.

Soil contamination with lead acetate inhibited soil urease. In the variant with combined contamination, the UA was lower that in the control and in the variants with only one pollutant. It is known that the UA decreases due to changes in the enzyme molecular structure caused by inhibitors. It is assumed that HMs bind sulfhydryl groups of the active center forming equivalents of metal sulfides, while organic ecotoxicants (including oil) can denature the entire protein structure [71]. The combined effect of xenobiotics probably resulted in a synergistic effect, which caused faster inhibition of the UA.

Bioaugmentation exerted a favorable effect on the UA, which became maximum after the application of the strain UOM 2 and MC: 0.54 N-NH4/g by the end of the experiment in both variants contrary to 0.43 N-NH4/g in the control (Fig. 3b). In the soil with oil and lead in the amount of 900 and 1800 mg/kg, the UA on the 95th day was two times lower as compared to the control (Figs. 3c and 3d). The use of microorganisms in these variants initially stimulated urease, but its activity decreased with time, which was probably explained by the depletion of the substrate for bacteria. The UA in samples with bacteria application was higher than in the untreated variants and sometimes reached control values.

Thus, the combined soil contamination with oil (5%) and lead at concentrations of 450–1800 mg/kg inhibited the UA stronger than in the soil contaminated with lead only. In the variant with combined contamination with oil and lead in the amount of 450 mg/kg, the UA initially sharply dropped and then gradually increased. A rise in the lead content caused a slow decrease in the UA. The use of strains of microorganisms exerted a favorable effect on the restoration of the UA both in the oil-contaminated soil and in the soils with combined contamination.

CONCLUSIONS

In a model experiment, soil pollution with oil, lead, and their combination, as well as bioaugmentation by cultures of hydrocarbon-oxidizing microorganisms (HCOMs) Thalassospira xiamenensis UOM 2, Enterobacter sp. UOM 3, and Pseudomonas songnenensis UOM 4 and by their microbial composition (MC) affected the biological activity of leached chernozem.

A pronounced phytotoxic effect of the pollutants was manifested in all the studied variants of pollution as judged from a poorer radish germination and inhibition of the growth of its roots. The application of HCOMs reduced phytotoxicity. The most efficient microorganisms were represented by the bacterial strain T. xiamenensis UOM 2 for the oil-contaminated soil; by the strains Enterobacter sp. UOM 3 and P. songnenensis UOM 4 for the soil with combined contamination at the lead concentration of 450 mg/kg; and by T. xiamenensis UOM 2 and the MC at higher lead concentrations. The use of bacteria enabled the transformation of the contaminated leached chernozem into the category of moderately phytotoxic soil.

The introduced HCOM survived well in the soil contaminated with both pollutants. In the oil-contaminated soil, the degradation of hydrocarbons was more intensive than in the soil with combined contamination without the introduction of bacteria. Bioremediation of leached chernozem enhanced the destruction of these xenobiotics. Their biodegradation in the oil-contaminated soil was maximal, when T. xiamenensis UOM 2 and P. songnenensis UOM 4 were applied. Under combined pollution, the decomposition of hydrocarbons was the greatest under the effect of Enterobacter sp. UOM 3 and P. songnenensis UOM 4 in the soil with the lead content of 450 mg/kg and of T. xiamenensis UOM 2 and MC in the soil with the lead content of 900 and 1800 mg/kg.

Oil and combined soil pollution caused an increase in the number of heterotrophic microorganisms, obviously due to the intensive development of hydrocarbon-oxidizing microbiota. Actinomycetes were most sensitive microorganisms to oil and lead pollution.

Soil with oil and combined contamination was characterized by a tendency to a decrease in the catalase activity. The individual introduction of lead acetate in the amount of 450, 900, and 1800 mg/kg into the soil stimulated the catalase activity. The use of microbial strains for bioremediation exerted a positive effect on this parameter.

At the initial stage of pollution, oil caused suppression of the invertase activity, while lead, on the contrary, increased it. The combination of pollutants resulted in different reactions of soil invertase. Bioaugmentation of the soil contaminated with oil and with oil and lead exerted a positive effect on the invertase activity in some cases.

The combined effect of pollutants inhibited the urease activity stronger than in the case of lead contamination. Application of microbial strains was generally favorable for the urease activity restoration both in the oil-contaminated soil and in the soil with combined pollution.