Experimental investigation of a pilot-scale concerning ex-situ 1 bioremediation of petroleum hydrocarbons contaminated soils

The soil samples were taken from the site of a former oil products depot from an industrial area (Romania). The 12 soil samples taken were analyzed from a physical and chemical point of view: texture, ph, soil micronutrient 13 content, metals concentration and petroleum hydrocarbon concentration (PHCs). The soil contaminated with TPH 14 (4280 mgkg -1 ) was disposed in the form of a pile (LxWxH:3000x1400x500 mm). Experiments a pilot-scale were 15 conducted over 12 weeks at constant pH (7.5–8), temperature (22–32 o C), nutrient contents C/N/P ratio 100:10:1, 16 soil aeration time (8 hour/day) and moisture (30%). Samples were taken every two weeks for the monitoring of 17 the TPH and the microorganisms content. During experiment every two weeks were added microoganisms 18 (Pseudomonas and Bacillus ). Results of the analyzes regarding the concentration of PHCs were revelead a linear 19 decrease of the concentration of PHCs after only two weeks of treatment. This decrease in concentration was also 20 achieved in the following weeks. Following the analysis performed on the model at the pilot scale regarding the 21 depollution process, it can be concluded that a soil contaminated with petroleum hydrocarbons can be efficiently 22 depolluted by performing an aeration of 8 h/day, adding microorganisms Pseudomonas and Bacillus to ensure the 23 conditions for increasing in the total number of germs (colony forming units–CFU) from 151x10 5 to 213x10 7 24 CFU/gram of soil, after 12 weeks of soil treatment - the depollution efficiency achieved 83%.

2 through pipes) can be introduced to improve soil ventilation in order to assure the oxygen supply needed for the 51 bio-reactions taking place in the pile of polluted soil 21 .

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In order to evaluate the use of the biopile method in bioremediation of soils polluted with oil, Iturbe et colab.

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conducted several studies: 1) in a former station of storage and distribution of oil, after 66 days of treatment they 54 achieved a remediation efficiency of 85.2% 22 ; 2) at an oil plant in northern Mexico, after 22 weeks of treatment 55 they achieved a remediation efficiency of 80% 23 .

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In a study conducted on soil contaminated with hydrocarbons (2000, 4000 and 6000 mgkg -1 ), three biopile piles of 0.6 m 3 were made, they were bio-stimulated with nutritive substances and aerated, obtaining an efficiency 58 of 66-75% 24 . 59 Gogoi et al. (2003) depolluted soil contaminated with HTP (44.000 mgkg -1 ). Soil was placed in cells (500 kg 60 of soil/cell), amended with nutrients and inoculated with a microbial consortium isolated from hydrocarbon-61 contaminated soils. The system was aerated one hour per day at a rate of 100 m 3 /h. At the end of the 365 days of 62 operation, remediation in the cells was 75% with a degradation rate of 90 mgkg -1 /day 25 .

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Studies have shown that using indigenous microbial strains is preferred in bioremediation processes 26,27 , and 64 adding nutritive substances may increase the efficiency of removal 28,29 .
3 -The soil pH was determined in 1/2.5 (w/v) soil/water extract using a HANNA pH-meter; 107 -Nitrogen was determined by Kjeldhal 39 ; 108 -For determining total potassium and phosphorus content 3 g of soil with 100 μm granulation was used over 109 which was added 7 mL of 12 M HCl and 21 mL of 15.8 M HNO3 and the mixture was refluxed for 2 hours, 110 filtered and diluted up to 100 mL with 2% (v/v) HNO3 40 ;

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-Mobile phosphorus and potassium were determined by ICP-OES after extraction of 5 g soil in 100 ml 112 ammonium acetate-lactate mixture (pH=3.75) for 4 hours according to Egnèr-Riehm-Domingo method;

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-The organic carbon was determined by Walkley-Black method by oxidising the organic matter from 0.2 g soil 114 with 5-10 ml of 1.6% (w/v) sulfochromic mixture on a hot plate for 20 min. The excess of chromic acid was

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HNO3 and the mixture was mineralized for 3 hours, filtered and diluted up to 100 mL; 120 -Hydrophysical indices: withering coefficient, field capacity and useful capacity were determined taking into 121 account soil moisture that was determined by the gravimetric method 39,41 ;

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After extraction, the supernatant was separated and the soil residue. Polar compounds (water, vegetable oils 125 and animal fats) was removed applied by passing the extract through a 10 cm long and 0.6 cm with column 126 packed with 0.150-0.250 mm grain-size magnesium silicate for column chromatography (Florisil). The Where: C-is the concentration of TPH in soil (mgkg -1 ); c-is the concentration of TPH in the extract (mgmL -1 ),

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Df-is the dilution factor; Cf-is the concentration factor; V is the volume of the extract (mL); w is the weight of the 137 sample (kg).

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Bacterial strains were incubated and shaken (120 rpm) at 25°C for 24h. Inoculation was made with 100 μL of 145 culture from liquid culture medium. The development of microorganisms was observed at 600 nm were using a 146 UV spectrophotometer (Lambda 25, Perkin-Elmer). These cultures were morphologically and tinctorially 147 characterized using the Gram staining technique 38 .

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The amount of soil microflora in the samples taken from the experimental groups was established by the numerical 149 determination of the microorganisms existent in the PHC polluted soils. For the numerical determination of microorganisms and placed in a pile (Lxlxh: 3000x1400x500 mm) in order to be subjected to the proposed diameter, which favors the aeration process. In this layer of gravel was introduced a part of the pipe system through 160 which aeration and wetting are carried out.

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The system for introducing water and the nutrients and microorganisms solution (Pseudomonas and Bacillus) 162 consists of a tank with a capacity of 100 L, a self-priming pump with a flow of 50 L/min, corrugated absorption 163 and discharge hoses and a blower. The discharge hose is connected to the distribution network that consists of an 164 Ø180 mm PVC pipe branched into 5 perforated Ø50 mm PVC pipes placed horizontally in the middle of the pile.

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The temperature at which the pilot scale experiment was carried out was between 25-32°C. Temperature was 173 measured and monitored throughout the experiment with the WTW Multiline IDS-3430 Multiparameter meter.

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Soil pH was monitored weekly using the HI 3512-02 pH meter.

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Soil aeration was achieved through the aeration system consisting of a blower and an air distribution network.

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The air distribution network consists of 5 perforated PVC pipes with a diameter of 50 mm each. These pipes are     (Table 1).

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The average concentration of PHC in the tested soil was of 4280 ± 400 mgkg -1 . This value was considered the 222 initial content of PHC for the amount of TPH in the pile of soil. The initial concentration of PHC exceeded more 223 than 2 times the intervention threshold for less sensitive soil uses (2000 mgkg -1 ) established by Romanian 224 legislation (Order no. 756/1997) 43 , thus requiring remediation.

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In the test soil two bacterial strains with bioremediation potential were isolated using a selective enrichment

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During the experiment there was an increase in the number of microorganisms from 102x10 5 -209x10 5 to 328 127x10 7 -238x10 7 CFU /g of soil, which led to a significant decrease of TPH.

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Results obtained on the pilot scale model showed a depollution efficiency between 64-94%, the average