Table 1. Scheme of experiments and controls

The introduced strain was previously grown in liquid LB medium till stationary phase (28°С, 18 h) and then uniformly introduced into soil to a concentration of 106 cells g-1 soil. The control of the bacteria strain growth was accomplished weekly during 67 days. A composite soil sample was collected from three separate sub-samples from the vial and analyzed for bacterial quantities. Approximately one g of the composite soil sample was suspended in 10 ml of 0.85% NaCl on "Vortex", soil particles were precipitated, and 1 ml of supernatant was used for making dilutions (10×-10000×). Volume of 0.1 ml of the

g C-CO2 g-1 dw h-1

Rate g C-CO

2

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

g


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

The Waste Oil Resulting from Crude Oil Microbial Biodegradation in Soil 77

T im e , d

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

introduced into agricultural soil caused an exponential increase in the CO2 emission rate

T im e , d 012345678

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

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

**E x p . 2**

indicating microbial growth after lag-phase (Fig. 2).

microbiota with strain *P. aureofaciens* BS1393(pBS216)

Two to 3 days (Exp. 2) and 5 to 6 days (Exp. 1) days after the start of exposure,,

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0

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

**E x p . 1**

the crude oil

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 experiments were calculated.

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 level of 104 cells g-1 DS.


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

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 soil introduced into arable soil.
