*2.2.2. Determination of TPH content by Infrared Spectroscopy (TPH-IR)*

34 New Technologies in the Oil and Gas Industry

maintained at about 12%.

 **+**

electrodes were buried in the soil.

**2.2. Chemical analysis of soil samples** 

*2.2.1. Determination of hydrocarbons via GC-analysis* 

<sup>c</sup> **-** <sup>m</sup>

15cm

**SUELO**

month; (II) in the second design, the electrodes were buried in the soil during one month; and (III) in the third design, the connections between compartments were done with a phosphate agar bird channel of 1cm of diameter during 150 days. All experiments were run using a constant electric field of 0.5 V/cm, and a control without electrical field was also carried out. Moisture was monitored on a weekly basis by a gravimeter method, and it was

a b

**Figure 2.** Design of the EK reactor used to treat polluted soil samples. Drawing A shows Experiment I, which was carried out with an agar bird channel made of Na Cl and Experiment III, which was carried out with agar bird channel made of buffer phosphate; Drawing B shows Experiment II , where the

1

38cm

38

**SUELO**

At the end of each experiment, the soil sample was extracted from the cell and divided into 3 layers (cathode, centre and anaode), which were then divided in two samples to obtain the pH value and pollutant concentration. The pH was obtained by suspending the soil samples

Two grams of each individual sample were dissolved in 5 ml of pentane, phase separated, and percolated through 2 g of silica gel. One millilitre of the elute was carefully evaporated until dry to determine the fuel oil content of the sample. The fractions were analyzed and quantified by gas chromatography using a Varian 3800 GC, equipped with a split/splitless injector, a flame ionization detector, and a capillary column VF-5ms (30 m, 0.25 mm, 0.25 µm). The injector and detector temperatures were maintained at 200 ºC and 340 °C respectively. The Sample (1 µL) was injected in split mode and the column temperature was raised from 45 to 100 °C at a rate of 5 °C/min and a second ramp from 100 to 275 °C at a rate

in de-ionised water (1:2.5, w/w) for bacterial counts, biochemical and TPH analysis.

of 8 °C/min. The final temperature, of 275 °C, was maintained for 5 minutes.

The soil TPH concentration was determined by infrared spectroscopy as previously described Environmental Protection Agency method [EPA 418.1]. Essentially, two grams of each individual sample were dissolved in 10 ml of carbon tetrachloride, phase separated, and percolated through 2 g of silica gel and the absorbance was measured at 2930 cm-1.

### *2.2.3. Determination of TPH content by Soxhlet extraction (TPH-SE).*

TPH concentration of the samples were determined by Soxhlet extractor using trichlorinethane as the extraction solvent. The extracted hydrocarbons were quantified on a mass difference basis as previously described [29] and separated into class fractions by silica gel column chromatography as formerly reported [30]. Essentially, the aliphatic, aromatic and polar oil fractions were respectively eluted using hexane (250 mL), benzene (150 mL) or 150 mL of 1:1 (v/v) chloroform-methanol.

#### **2.3. Enumeration and isolation of aerobic bacteria.**

Culturable bacteria from each sample were counted using the standard plate dilution method. One gram of soil (wet weight) was suspended in 9 ml of physiology sterile water (pH 7.2) and vortexed for 1 min at low speed. Aliquots of 100 µl of undiluted samples, and 10-1 to 10-6 dilutions were grown on TSBA (comprised of trypticase soy broth (30 g/L) and granulated agar (15 g/L)) and MBM-PGO media (comprised: NaCl (5 g/L), K2PO4H (0.5 g/L), NH4PO4H2 (0.5 g/L), (NH4)2SO4 (1 g/L), MgSO4 (0.2 g/L), KNO3 (3 g/L), FeSO4 (0.05 g/L), suspended in distilled water), 30 L of a mixture 1:1 of petroleum-diesel oil was spread on the surface once set [29] and plates incubated at 28 ºC for up to 21 days.

#### **2.4. Chemotaxonomic analysis of soil microbe populations**

The diversity of cultured sediment bacteria was determined by fatty acid methyl ester (FAME) analysis of the samples taken from the cell. FAME analysis allowed the characterization of individual bacterial colonies. Fatty acids were extracted and compared against a database, to identify isolated bacteria. From each culture plate, containing between 30 and 300 colonies, individual colonies were randomly isolated and incubated on tryptic soy broth agar for 24h. The FAMEs were extracted and analyzed by MIDI (MIDI Newark, Del., USA) as per manufacturer's instructions.

Shannon index was calculated by Sherlock (Microbial ID, version 6.0).

#### *2.4.1. GC parameters for MIDI analysis*

The MIDI microbial identification system (Microbial ID, Inc, Newark, NJ) was applied to separate fatty acid methyl ester using a gas chromatograph (HP 6890) equipped with a split/splitless injector, a flame ionization detector, a capillary column Ultra 2 (25 m, 0.2 mm, 0.33 µm); an automatic sampler; an integrator; and a program which identifies the fatty

acids (Microbial ID 6.0 version). The injector and detector temperatures were maintained at 250 °C and 300 °C respectively. The Sample (2 µL) was injected in split mode and the column temperature was raised from 170 to 270 °C at a rate of 5 °C/min.

#### **2.5. Statistical analysis**

The mean values were compared by ANOVA test by BIOM (Applied Biostatistics Inc., NY, USA). Differences were considered significant when P<0.05. To identify possible similarity between FAME profiles, the data were subjected to analysis of variance using PAST [31] and Sherlock (Microbial ID, version 6.0).
