# Speciation of the Removed Pollutants in Bioremediation of Hydrocarbon-Contaminated Soil
## 2 Materials and Methods
### Diesel Oil Extraction and Gas Chromatography Analysis  
The diesel oil was extracted from each mesocosom by applying the EPA method 3546 (moisture 15-30% by mass) [11], based on microwave heating. In this methodology, 2 g of soil and 30 mL of solvent (solution acetone/_n_-hexane with a ratio of 1:1 by volume) \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Parameter/Mesocosm** & \multicolumn{2}{c}{**Natural**} & \multicolumn{2}{c}{**Biostimulation (BS)**} & \multicolumn{2}{c}{**Biostimulation + Inoculum**} \\  & **Attenuation (NA)** & & **(BS + IN)** \\ \hline Soil mass (kg) & 8.4 & 8.4 & 8.4 \\ Diameter of soil column (cm) & 19 & 19 & 19 \\ Height of soil (cm) & 20 & 20 & 20 \\ Porosity (Volume\({}_{\text{pores}}\cdot\)Volume\({}_{\text{pot}}^{-1}\)) & 0.45 & 0.45 & 0.45 \\ Quantity of inoculum (kg) & - & - & 0.65 \\ Inoculum/soil ratio (kg\({}_{\text{inoculum}}\cdot\)kg\({}_{\text{soil}}^{-1}\)) & - & - & 0.08 \\ Initial oil-degrading microorganisms & - & - & \(6.59\times 10^{7}\) \\ (CFU\(\cdot\)kg\({}_{\text{soil}}^{-1}\)) & 0.05 & 0.05 & 0.05 \\ Diesel oil content (kg\({}_{\text{soil}}\cdot\)kg\({}_{\text{soil}}^{-1}\)) & 0.33 & 0.33 & 0.33 \\ Water saturation & 0.10 & 0.10 & 0.10 \\ (Volume\({}_{\text{water}}\cdot\)Volume\({}_{\text{pores}}\)\({}^{-1}\)) & - & 135 & 135 \\ Moisture content (kgwater-kg\({}_{\text{soil}}^{-1}\)) & 0.054 & 1.45 & 1.45 \\ solution (S-m\({}^{-1}\)) & 0.05 & 0.05 & 0.05 \\ Run duration (d) & 193 & 228 & 228 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Main parameters of the mesocosms. \begin{table}
\begin{tabular}{c c} \hline \hline
**Salt** & **Concentration (g\(\cdot\)L\({}^{-1}\))** \\ \hline (NH\({}_{4}\))\({}_{2}\)HPO\({}_{4}\) & 11.62 \\ NH\({}_{4}\)NO\({}_{3}\) & 75.97 \\ KH\({}_{2}\)PO\({}_{4}\) & 0.5 \\ K\({}_{2}\)HPO\({}_{4}\)+\({}_{2}\)O & 0.5 \\ MgSO\({}_{4}\)\({}_{7}\)+H\({}_{2}\)O & 0.008 \\ CuSO\({}_{4}\)+H\({}_{2}\)O & 0.002 \\ MnSO\({}_{4}\cdot\)H\({}_{2}\)O & 0.002 \\ FeSO\({}_{4}\cdot\)7H\({}_{2}\)O & 0.002 \\ CaCl\({}_{2}\cdot\)2H\({}_{2}\)O & 0.002 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Composition of the aqueous solution to stimulate bacterial growth. were mixed, adding 2 g of anhydrous sodium sulphate to dry the soil. Then, the sample was heated at 110 \({}^{\circ}\)C and power of 1100 W for 15 min by microwaves; this temperature and power of 1100 W were kept for 10 min, and then the sample was cooled for 20 min. After the extraction, the sample was filtered through a 0.45 \(\upmu\)m filter. This procedure was replicated, thus obtaining two extracts. The diesel oil concentration was quantified by a gas chromatograph and by adopting the EPA method 8015 [12]. The gas chromatograph was equipped with a flame ionization detector and DB-5 fused silica capillary column, operated with helium as the carrier. The injector and detector were maintained at 220 \({}^{\circ}\)C and 250 \({}^{\circ}\)C, respectively. The following temperature program was used: initial temperature of 50 \({}^{\circ}\)C, ramp to 320 \({}^{\circ}\)C at 8 \({}^{\circ}\)C\(\cdot\)min\({}^{-1}\), and a constant temperature of 320 \({}^{\circ}\)C for 40 min. The characteristic peaks of the diesel oil hydrocarbons were contained between 6 and 31 min. The compounds that characterize the diesel oil were identified by standard kits, namely:  
* C7-C30 saturated \(n\)-alkanes, 1000 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) of each component in hexane;
* PAHs (acenaphthylene, anthracene, benzo(\(\alpha\))anthracene, fluorene, phenanthrene, pyrene), 500 mg\(\cdot\)L\({}^{-1}\) of each component in acetone. In each analysis, the retention times of the peaks were compared to the standard ones, and the \(n\)-alkanes from C8 to C24 and the six PAHs above-listed were identified. Table 3 shows their list and their retention time; particularly, all the compounds exhibit retention times in the range between 6 and 31 min. A calibration line of the gas chromatograph carried out with the commercial diesel oil used in the mesocosoms was exploited to measure the residual diesel oil concentration.