**3.4 Microbial СО2 production in soil**

Total rates of microbial mineralization of SOM and oil hydrocarbons in soil were determined by the rate of CO2 production (μg C-CO2 g-1 DS h-1).

In controls 1 and 2, the rates of SOM mineralization both by aboriginal soil microorganisms and the mixture of these microorganisms plus introduced strain *P. aureofaciens* BS1393(pBS216) were within the range of 0.2 0.02 μg C-CO2 g-1 DS h-1 and practically did not change during the 67-day observation (Fig. 1, control 1 and control 2). In soil with added oil hydrocarbons (experiments 1 and 2), the rate of mineralization of total organic carbon significantly increased and reached the maximum value of about 3.2 μg C-CO2 g-1 DS h-1 on days 7-8 after the beginning of the exposure . (Fig. 1, Exp. 1 and Exp. 2). In experiment 2, with the bacterium P*. aureofaciens* BS1393(pBS216) added to the indigenous microbiota, there are two maximums of CO2 production rate: the first in 3 days and the second one in 8 days after the beginning of exposure (Fig. 1, Exp. 2). In the experiment 1 with aboriginal microbiota (Fig. 1, Exp. 1) only one maximum of CO2 production rate was observed in 7 days after the beginning of exposure. It is supposed that this special feature was responsible for the availability of the introduced bacteria P*. aureofaciens* BS1393(pBS216) to consume the oil hydrocarbons.

corresponding dilutions was inoculated onto Petri dishes with LB medium. The colonyforming units (CFU) on the plates were counted and their mean values in the control and

As seen from Table 2, in one day after introduction of the strain *P. aureofaciens* BS1393(pBS216) experiments (soil with oil) and controls (soil without oil) showed a decrease of the quantity of cells of this strain from 106 cells g-1 soil to 104 cells g-1 soil measured as colony-forming units (CFU). However, in 7 days after the beginning of the experiment, the CFU number of the bacteria introduced in the experiment with oil was about 2.7 ×106 cells g-1 DS, i.e. more than 17-fold higher than the CFU of the same bacterium in the control soil without oil (Table 2). These results indicate the ability of the strain introduced for biodegradation of oil hydrocarbons to utilize them as a growth substrate. In 14-21 days, the CFU of the introduced strain noticeably decreased again and by day 28 reached the initial

> Colony-forming units (x104)/g of soil \*1 d 7 d 14 d 21 d 28 d 35 d

Control 8.0 (1.7) 15.7 (5.8) 4.0 (0.9) 1.6 (0.6) 3.1 (3.5) 2.2 (0.9)

Experiment 4.7 (3.4) 268.0 (149) 31.7 (18.7) 21.1 (14.4) 2.4 (1.4) 1.5 (0.5)

\*Times after bacteria culture was introduced into soil. (Standard deviations from 3 parallels are given in

Total rates of microbial mineralization of SOM and oil hydrocarbons in soil were

In controls 1 and 2, the rates of SOM mineralization both by aboriginal soil microorganisms and the mixture of these microorganisms plus introduced strain *P. aureofaciens* BS1393(pBS216) were within the range of 0.2 0.02 μg C-CO2 g-1 DS h-1 and practically did not change during the 67-day observation (Fig. 1, control 1 and control 2). In soil with added oil hydrocarbons (experiments 1 and 2), the rate of mineralization of total organic carbon significantly increased and reached the maximum value of about 3.2 μg C-CO2 g-1 DS h-1 on days 7-8 after the beginning of the exposure . (Fig. 1, Exp. 1 and Exp. 2). In experiment 2, with the bacterium P*. aureofaciens* BS1393(pBS216) added to the indigenous microbiota, there are two maximums of CO2 production rate: the first in 3 days and the second one in 8 days after the beginning of exposure (Fig. 1, Exp. 2). In the experiment 1 with aboriginal microbiota (Fig. 1, Exp. 1) only one maximum of CO2 production rate was observed in 7 days after the beginning of exposure. It is supposed that this special feature was responsible for the availability of the introduced bacteria P*. aureofaciens* BS1393(pBS216)

Table 2. Growth of *Pseudomonas aureofaciens* BS1393(pBS216) without (control) and with crude oil hydrocarbons (experiment) to a concentration of 106 colony-forming units g-1 of

experiments were calculated.

level of 104 cells g-1 DS.

soil introduced into arable soil.

**3.4 Microbial СО2 production in soil** 

to consume the oil hydrocarbons.

determined by the rate of CO2 production (μg C-CO2 g-1 DS h-1).

Variants

parenthesis)

Fig. 1. Rates of СО2 production by microbial mineralization of substrates in experiments simulating microbial utilization of oil hydrocarbons. Control 1 (aboriginal microflora); Control 2 (aboriginal microflora + introduced bacteria); Experiment 1 (aboriginal microflora + oil); Experiment 2 (aboriginal microflora + introduced bacteria + oil)

Two to 3 days (Exp. 2) and 5 to 6 days (Exp. 1) days after the start of exposure,, the crude oil introduced into agricultural soil caused an exponential increase in the CO2 emission rate indicating microbial growth after lag-phase (Fig. 2).

Fig. 2 Substrate-induced respiratory response of the microbial community during incubation of soil treated with crude oil hydrocarbons: 1 - the initial CO2 emission by growth of native soil microbiota and 2- the initial CO2 emission by growth of mixture of native soil microbiota with strain *P. aureofaciens* BS1393(pBS216)

The Waste Oil Resulting from Crude Oil Microbial Biodegradation in Soil 79

The absence of any significant differences in СО2 production in controls 1 and 2 was considered as an evidence of insignificant additional mineralization of SOM attributable to the introduced strain of *P. aureofaciens* BS1393(pBS216 ). In the case of oil-containing soils, the amounts of metabolic СО2 in experiments 1 and 2 exceeded 6.8-fold that of controls 1 and 2, being 167.0 and 238 mg C-CO2 (Exp. 1) and 174.0 and 251 mg C-CO2 (Exp. 2) during 47- and 67-day exposure, respectively (Table 4). The data also showed that the additional introduction of the hydrocarbon-oxidizing strain *P. aureofaciens* BS1393(pBS216) into oilcontaining soil (Exp. 2) promoted the increase of metabolic СО2 amount (4 - 13 %) compared

Total СО2 production in experiments 1 and 2 included microbial mineralization of SOM and oil hydrocarbons, therefore the share of СО2 formed by mineralization of each of the above substrates was determined by measuring values 13C, both in the carbon isotope characteristics of SOM and oil products and in the metabolic carbon dioxide formed during

In experiments 1 and 2, the 13C values of the metabolic CO2 released from soil in the 3 days before oil hydrocarbons introduction into soil were –23.53 0.21 %o and –23.56 0.25 %o, respectively, and actually identical to the isotopic characteristics of СО2 in the controls (Fig. 3).

Time, d

Fig. 3. Carbon isotope characteristics (13C, ‰) of CO2 produced in experiments of microbial mineralization of SOM and oil hydrocarbons introduced into soil: Control 1 (aboriginal microflora); Control 2 (aboriginal microflora + introduced bacteria); Experiment 1 (aboriginal

microflora + oil); Experiment 2 (aboriginal microflora + introduced bacteria + oil)

0 10 20 30 40 50 60 70

time, d vs Control 1 time, d vs control 2 time, d vs exp 1 time, d vs exp 2

to the aboriginal soil microorganisms.

**3.5 Analysis of the origin of soil СО2 using 13C/12C ratios** 

this process.

13C, %o







At the initial stages of microbial oil mineralization in experiments 1 and 2, the specific rates of metabolic CO2 emission (µ) were determined using the approximating equation [1] and lag periods (tlag) were calculated by the equation [2] (Table 3). The values of parameters K as an index of catabolism of microbial cells in soil were calculated from the analysis of CO2 production at the initial stages of microbial oil mineralization. The values of parameters K (Table 3) show the close rates of initial production of metabolic CO2 in these experiments. At the same time, parameter *r* indicating the presence of growing microorganisms in soil is higher by three orders of magnitude in experiment 2 with introduced bacteria compared to experiment 1 with native microbiota in soil. Parameter µ showing specific rates of CO2 production in experiments 1 and 2 has close values within the measurement error. As one would expect, the lag period of test substrate consumption and CO2 production in experiment 2 with the introduced bacterium P*. aureofaciens* BS1393(pBS216) was about 2,5±03 days, i.e., significantly less than in experiment 1 with native microbiota only (the lag period of 6,2±0,5 days).


Table 3. Parameters of the equations [1] and [2] characterized the respiration rates of native soil microbiota (Experiment 1) and mixture microbiota after bioagmentation with strain *P. aureofaciens* BS1393(pBS216) (Experiment 2) after crude oil addition to the agricultural soil. Standard deviation intervals are in brackets

Beginning from day 25 to day 67 from exposure, the rate of СО2 production in experiments 1 and 2 decreased slightly and stabilized at a level of 1.25 0.25 μg C-CO2 g-1 DS h-1 (Fig. 1). Total СО2 production in controls (control 1 and 2) for the 47-day and for 67-day periods of observation was 24.8 ±1.2 mg C-CO2 and 35.5 1.2 mg C-CO2 (Table 4).


\*Total production Qtotal=(24·vaverage (μg С-СО2 g-1 DS h-1)· t (days))x100 g DS

\*\*Time after the crude oil addition to soil. Standard deviations of three parallel determinations are given in brackets.

Table 4. Mean rates of СО2 emission (μg С-СО2 g-1 DS per h) and total production of С-СО<sup>2</sup> during the time experiment (mg С-СО2 per 100 g DS)

At the initial stages of microbial oil mineralization in experiments 1 and 2, the specific rates of metabolic CO2 emission (µ) were determined using the approximating equation [1] and lag periods (tlag) were calculated by the equation [2] (Table 3). The values of parameters K as an index of catabolism of microbial cells in soil were calculated from the analysis of CO2 production at the initial stages of microbial oil mineralization. The values of parameters K (Table 3) show the close rates of initial production of metabolic CO2 in these experiments. At the same time, parameter *r* indicating the presence of growing microorganisms in soil is higher by three orders of magnitude in experiment 2 with introduced bacteria compared to experiment 1 with native microbiota in soil. Parameter µ showing specific rates of CO2 production in experiments 1 and 2 has close values within the measurement error. As one would expect, the lag period of test substrate consumption and CO2 production in experiment 2 with the introduced bacterium P*. aureofaciens* BS1393(pBS216) was about 2,5±03 days, i.e., significantly less than in experiment 1 with native microbiota only (the lag

Native soil microbiota (Experiment. 1) Agricultural soil 0.6085 8.991·10-6 1.7814 6.2 (0.5) Native soil microbiota + *P. aureofaciens* BS1393(pBS216) (Experement. 2) Agricultural soil 0.4906 7.445·10-3 1. 6913 2.5 (0.3) Table 3. Parameters of the equations [1] and [2] characterized the respiration rates of native soil microbiota (Experiment 1) and mixture microbiota after bioagmentation with strain *P. aureofaciens* BS1393(pBS216) (Experiment 2) after crude oil addition to the agricultural soil.

Beginning from day 25 to day 67 from exposure, the rate of СО2 production in experiments 1 and 2 decreased slightly and stabilized at a level of 1.25 0.25 μg C-CO2 g-1 DS h-1 (Fig. 1). Total СО2 production in controls (control 1 and 2) for the 47-day and for 67-day periods of

\*\*Time after the crude oil addition to soil. Standard deviations of three parallel determinations are given

Table 4. Mean rates of СО2 emission (μg С-СО2 g-1 DS per h) and total production of С-СО<sup>2</sup>

*tlag*, d K r µ

\*Total production,

25.7 (0.6) 36.7 (0.6) 24.03 (0.6) 34.25 (0.6) 167 (6) 238 (6) 174 (5) 251 (5)

mg С-СО2 \*\*Time, days

period of 6,2±0,5 days).

Control 1 Control 1 Control 2 Control 2 Experiment 1 Experiment 1 Experiment 2 Experiment 2

in brackets.

Type of soil µg CO2-C g-1 soil h-1

observation was 24.8 ±1.2 mg C-CO2 and 35.5 1.2 mg C-CO2 (Table 4).

μg С-СО2 g-1 DS h-1

0.228(0.013) 0.228(0.013) 0.213(0.013) 0.213(0.013) 1.480(0.122) 1.480(0.122) 1.546(0.100) 1.546(0.100)

\*Total production Qtotal=(24·vaverage (μg С-СО2 g-1 DS h-1)· t (days))x100 g DS

during the time experiment (mg С-СО2 per 100 g DS)

Standard deviation intervals are in brackets

Experiment Mean Production rate,

The absence of any significant differences in СО2 production in controls 1 and 2 was considered as an evidence of insignificant additional mineralization of SOM attributable to the introduced strain of *P. aureofaciens* BS1393(pBS216 ). In the case of oil-containing soils, the amounts of metabolic СО2 in experiments 1 and 2 exceeded 6.8-fold that of controls 1 and 2, being 167.0 and 238 mg C-CO2 (Exp. 1) and 174.0 and 251 mg C-CO2 (Exp. 2) during 47- and 67-day exposure, respectively (Table 4). The data also showed that the additional introduction of the hydrocarbon-oxidizing strain *P. aureofaciens* BS1393(pBS216) into oilcontaining soil (Exp. 2) promoted the increase of metabolic СО2 amount (4 - 13 %) compared to the aboriginal soil microorganisms.

Total СО2 production in experiments 1 and 2 included microbial mineralization of SOM and oil hydrocarbons, therefore the share of СО2 formed by mineralization of each of the above substrates was determined by measuring values 13C, both in the carbon isotope characteristics of SOM and oil products and in the metabolic carbon dioxide formed during this process.

## **3.5 Analysis of the origin of soil СО2 using 13C/12C ratios**

In experiments 1 and 2, the 13C values of the metabolic CO2 released from soil in the 3 days before oil hydrocarbons introduction into soil were –23.53 0.21 %o and –23.56 0.25 %o, respectively, and actually identical to the isotopic characteristics of СО2 in the controls (Fig. 3).

Fig. 3. Carbon isotope characteristics (13C, ‰) of CO2 produced in experiments of microbial mineralization of SOM and oil hydrocarbons introduced into soil: Control 1 (aboriginal microflora); Control 2 (aboriginal microflora + introduced bacteria); Experiment 1 (aboriginal microflora + oil); Experiment 2 (aboriginal microflora + introduced bacteria + oil)

g C-CO2 g-1 dw soil h-1

13C, %o

PE, %

0 ,0 0 ,5 1 ,0 1 ,5 2 ,0 2 ,5 3 ,0 3 ,5


introduced bacteria + oil).

The Waste Oil Resulting from Crude Oil Microbial Biodegradation in Soil 81

T im e , d v s C o n tro l1 T im e , d v s C o n tro l2 T im e , d v s E x p 1 T im e , d v s E x p 2

3

T im e , d v s C o n tro l\_ d e l1 T im e , d v s C o n tro l\_ d e l2 T im e , d v s E x p \_ d e l1 T im e , d v s E x p \_ d e l2

> T im e , d v s P E \_ 1 T im e , d v s P E \_ 2

0 2 4 6 8 1 0 1 2 1 4 1 6

0 2 4 6 81 0 1 2 1 4 1 6

T im e , d a y s 0 2 4 6 8 1 0 1 2 1 4 1 6

B

**A**

**B**

**C**

2

Fig. 4. Rates (A) and isotopic characteristics of СО2 resulting from SOM and oil

hydrocarbons mineralization (B), and priming effect (C) for 15 days after oil introduction into soil: control 1 (aboriginal microflora); control 2 (aboriginal microflora + introduced bacteria); experiment 1 (aboriginal microflora + oil); experiment 2 (aboriginal microflora +

After oil hydrocarbons addition to soil, the share of 13С isotope in metabolic carbon dioxide abruptly dropped, which was an evidence of СО2 production partly from oil hydrocarbons containing less 13С isotope compared to SOM. The maximum depletion of 13С isotope in metabolic СО2 was registered during the days 11-15 from the beginning of exposure in experiments 1 and 2. This was considered a result of the mineralization of mainly alkane oil fractions. Our assumption that the major part of aliphatic hydrocarbons from the introduced crude oil had already been utilized by that period is evidenced by the carbon isotope characteristics of the metabolic carbon dioxide with the value of 13C = -28.5 0.5 %o (Fig. 3). After 15 days and until the end of the experiment (67 days), the isotopic characteristic of СО2 was at around the value of 13C = -26.8 0.5 %o. Using equation [4], the average weighted isotope composition of СО2 produced by microbial mineralization of total organic products (oil hydrocarbons and SOM) in experiments 1 and 2 during 67-days was characterized by 13C values about of -26.6 0.1 %o, which significantly differed from the carbon isotope characteristics of oil (13C = -28.4 0.2%o) and SOM (13C = -23.01 0.2 ‰,). It can be said with confidence that metabolic CO2 was produced during microbial mineralization of a part of SOM and a part of oil hydrocarbons.

#### **3.6 Priming effect of oil hydrocarbons**

The *kinetic* PE was calculated by comparing СО2 amounts generated by microbial mineralization of SOM and oil products (Exp. 1 and 2) to СО2 amounts generated in the controls in the corresponding periods of observation [Eq. 11].

In order to quantify both the extent and direction of PE of oil hydrocarbons, we have compared the rates of СО2 production by microbial mineralization of SOM before and after introduction of oil hydrocarbons into soil at the initial period of maximum microbial activity, i.e., during 15 days after addition of crude oil to soil (Fig. 4). As shown in Figure 4 (A), the activation of the metabolism of aboriginal hydrocarbon-oxidizing soil microorganisms in experiments 1 took about 6 days from the introduction of the hydrocarbon substrate, when microbial rate of СО2 production increased to a rate closer to that of experiment 2 with the *P. aureofaciens* BS1393(pBS216) addition. The mass isotope balance data showed that during these days in experiment 1 the mineralization of oil hydrocarbons was insignificant and the rate of SOM mineralization was less the rate in control (negative PE) (Fig. 4, C PE\_1). Experiment 2, in contrast to experiment 1, showed the utilization of oil hydrocarbons in the initial period of exposure was accompanied by a noticeable increase of SOM mineralization rate compared to the initial value (positive PE) (Fig. 4, C PE\_2). However, PE became negligible in both experiments during 6-8 day exposure; it is possibly the mineralization time of aliphatic hydrocarbons or their partially oxidized products. The negative PE has been demonstrated previously in the processes of the microbial mineralization of n-hexadecanoic acid introduced into soil (Zyakun et al. 2011). At the next period of the exposure, the PE values demonstrate the positive values of 300 % in experiment 1 and about 400 % in experiment 2. On completion experiments, the total PE has been calculated using Eq. {12}. Taking into account the CO2 quantity registered in experiments 1 and 2 during the whole period of exposure (Table 4, Qtotal) and the share of CO2 under microbial utilization of SOM (Table 5, FSOM), we find the quantity of CO2 form as a result of SOM mineralization in the experiments (Table 5, [CO2]SOM)

After oil hydrocarbons addition to soil, the share of 13С isotope in metabolic carbon dioxide abruptly dropped, which was an evidence of СО2 production partly from oil hydrocarbons containing less 13С isotope compared to SOM. The maximum depletion of 13С isotope in metabolic СО2 was registered during the days 11-15 from the beginning of exposure in experiments 1 and 2. This was considered a result of the mineralization of mainly alkane oil fractions. Our assumption that the major part of aliphatic hydrocarbons from the introduced crude oil had already been utilized by that period is evidenced by the carbon isotope characteristics of the metabolic carbon dioxide with the value of 13C = -28.5 0.5 %o (Fig. 3). After 15 days and until the end of the experiment (67 days), the isotopic characteristic of СО2 was at around the value of 13C = -26.8 0.5 %o. Using equation [4], the average weighted isotope composition of СО2 produced by microbial mineralization of total organic products (oil hydrocarbons and SOM) in experiments 1 and 2 during 67-days was characterized by 13C values about of -26.6 0.1 %o, which significantly differed from the carbon isotope characteristics of oil (13C = -28.4 0.2%o) and SOM (13C = -23.01 0.2 ‰,). It can be said with confidence that metabolic CO2 was produced during microbial

The *kinetic* PE was calculated by comparing СО2 amounts generated by microbial mineralization of SOM and oil products (Exp. 1 and 2) to СО2 amounts generated in the

In order to quantify both the extent and direction of PE of oil hydrocarbons, we have compared the rates of СО2 production by microbial mineralization of SOM before and after introduction of oil hydrocarbons into soil at the initial period of maximum microbial activity, i.e., during 15 days after addition of crude oil to soil (Fig. 4). As shown in Figure 4 (A), the activation of the metabolism of aboriginal hydrocarbon-oxidizing soil microorganisms in experiments 1 took about 6 days from the introduction of the hydrocarbon substrate, when microbial rate of СО2 production increased to a rate closer to that of experiment 2 with the *P. aureofaciens* BS1393(pBS216) addition. The mass isotope balance data showed that during these days in experiment 1 the mineralization of oil hydrocarbons was insignificant and the rate of SOM mineralization was less the rate in control (negative PE) (Fig. 4, C PE\_1). Experiment 2, in contrast to experiment 1, showed the utilization of oil hydrocarbons in the initial period of exposure was accompanied by a noticeable increase of SOM mineralization rate compared to the initial value (positive PE) (Fig. 4, C PE\_2). However, PE became negligible in both experiments during 6-8 day exposure; it is possibly the mineralization time of aliphatic hydrocarbons or their partially oxidized products. The negative PE has been demonstrated previously in the processes of the microbial mineralization of n-hexadecanoic acid introduced into soil (Zyakun et al. 2011). At the next period of the exposure, the PE values demonstrate the positive values of 300 % in experiment 1 and about 400 % in experiment 2. On completion experiments, the total PE has been calculated using Eq. {12}. Taking into account the CO2 quantity registered in experiments 1 and 2 during the whole period of exposure (Table 4, Qtotal) and the share of CO2 under microbial utilization of SOM (Table 5, FSOM), we find the quantity of CO2 form as

mineralization of a part of SOM and a part of oil hydrocarbons.

controls in the corresponding periods of observation [Eq. 11].

a result of SOM mineralization in the experiments (Table 5, [CO2]SOM)

**3.6 Priming effect of oil hydrocarbons** 

Fig. 4. Rates (A) and isotopic characteristics of СО2 resulting from SOM and oil hydrocarbons mineralization (B), and priming effect (C) for 15 days after oil introduction into soil: control 1 (aboriginal microflora); control 2 (aboriginal microflora + introduced bacteria); experiment 1 (aboriginal microflora + oil); experiment 2 (aboriginal microflora + introduced bacteria + oil).

The Waste Oil Resulting from Crude Oil Microbial Biodegradation in Soil 83

Previously (Zyakun et al. 2003), it was shown that during the growth of microbial cells on hydrocarbons the ratio of biomass and CO2 carbon quantities was corresponding 1:1. In view of the above, we believe that the quantity of oil hydrocarbons taken up for the biosynthesis of cell biomass and organic exometabolites in soil during the 67-day exposure will be close to the carbon quantity of CO2 production and make no less then 16.7 and 17.5 percents of the oil introduced in experiments 1 and 2, respectively. By this is meant that the oil hydrocarbon consumption by microbial pool in soil amounts up 33.4 and 35 percent of

Extrapolation of the obtained data (Table 6) to a 6-month season, when the temperature conditions in the Krasnodar region provide for the metabolic activity of soil microbiota, shows that the uptake of crude oil hydrocarbons by native soil microbiota may reach no more than 92±2 % of the total oil hydrocarbon quantity in the oil. At a positive PE of oil hydrocarbons in soil, there is more intensive microbial degradation of SOM compared to the processes in native soil. On the other hand, oil hydrocarbons consumed by microorganisms are spent both for CO2 production and for the biosynthesis of biomass and organic exometabolites, which then are included in SOM and transform the structure of soil. The newly synthesized metabolites and microbial biomass components can be used by other biological systems (plants, macro- and microorganisms) that are incapable of direct utilization of oil hydrocarbons. The quantitative and isotopic data obtained in the experiments were used as a basis for estimation of the degree of replacement of part of SOM mineralized to CO2 by the newly synthesized products under microbial utilization of oil hydrocarbons. Table 6 shows the rates of microbial degradation and production of cell biomass and organic exometabolites in model experiments with microbial utilization of crude oil as a substrate. As a result of oil consumption both by native soil microbiota (Exp. 1) and introduced the bacterium strain *P. aureofaciens* BS1393(pBS216) (Exp. 2), the quantity of the newly synthesized organic products (carbon of cell biomass and exometabolites) nearly 1.6-fold exceeds the carbon quantity of SOM taken up for the CO2 mineralization (Table 6, R). It means that microbial transformation of oil hydrocarbons into products available as substrates for other living systems may be a peculiar source of organic fertilizers. In addition, there is more and more evidence that the bioremediation of oil-

With the proviso that crude oil carbon content no more than 1.4-fold higher than the SOM carbon amount, the soil microbiota is able to mineralize up to 17 % of crude oil hydrocarbons and 15 % of SOM during the 67-day experiments. Using mass isotope balance and differences between the 13C values of SOM and oil hydrocarbons, the quantities of CO2 produced during microbial mineralization of SOM and oil hydrocarbons have been determined. According to the highest depletion of 13C in CO2 evolved from soil during the initial time of the exposure with crude oil, it is suggested that at this time the aliphatic oil fraction predominantly participates in mineralization. Microbial consumption of oil hydrocarbons activates the process of SOM mineralization and demonstrates the presence of PE of oil hydrocarbons. During a 67-day period of the crude oil exposure in soil, the average values of PE reached over 150 % in soil with native soil microbiota and over 180 % in soil with the mixture of native microbiota and introduced bacteria *P. aureofaciens*

polluted soils is companied by plant growth stimulation.

total oil, respectively.

**4. Conclusion** 


\*13Cave is an average weighted of isotope characteristic of СО2 was calculated [Eq. 4] \*\*FSOM is a share of metabolic СО2 formed by microbial mineralization of SOM; # PE is a priming effect was calculated according to [ Eq. 11]; ##Time after the crude oil addition to soil. Standard errors of three parallel calculations are given in brackets.

Table 5. Average weighted characteristics (13Cave) of carbon isotope composition and fraction of СО2 formed by SOM mineralization and priming effect (PE) in experiments 1 and 2 relative to controls

Using the equation [12], we calculate the value of PE(total) by comparing CO2 production during microbial SOM utilization in the experiments and controls. As follows from Table 5, during 67-day exposure of oil hydrocarbons in soil the PE value reached 150 % in experiment 1 with native soil microbiota and 180 % in experiment 2 with mixed microbiota (soil microorganisms and the bacterium strain *P. aureofaciens* BS1393(pBS216)). Thus, addition of crude oil to the soil activates to a large extent the microbial mineralization of native soil organic matter.
