**2. Material and methods**

32 New Technologies in the Oil and Gas Industry

high humidity or saturation.

technologies can be assessed.

the ζ in most soils is negative. With increased acidity, ζ increases such that it can reach positive values [14]. The increase in ζ impacts on the electroosmotic flow. Soil characteristics as absorbency, ion exchange buffer capacity (pH) and load surface have a marked influence on the EKB. This shows successful results in clay, fine-grained and low permeability soil. Whereas sandy soils should have an impermeable structure at a reasonable depth to allow

The outer surface of bacterial cells possess numerous chemical groups which, at pH 7 or greater, result in an overall negative surface potential [21]. It is therefore possible to speculate about bacterial movement under the influence of an electrical field. Soil pH changes generated allow the bacteria to migrate by electrophoresis into one of the electrodes. As can be seen in Fig.1, the negatively charged membrane causes bacteria to migrate in the direction of anode [15]. Whilst the rate of migration under an electric field is quick (5 cm/h) in aqueous media, it is slow in soil, falling to ~0.8 cm /h. [17]. At low pH, the bacterial membrane charge is positive and the direction of movement is modified to the cathode. This amphoteric property is due to the complexity of surface charges on the bacterial membrane which arise from the combination of acidic (phosphates, carboxylic acids and sulphates) and basic (most notably amines) chemical groups found on the membrane surface. Consequently, it is difficult to predict their performance with biophysical parameters [21]. This difficulty is compounded as some microbes possess the ability to change their surface polarity, thus affording them some flexibility with respect to their relative migration [22]. Nonetheless, bacterial behaviour in the electric field will strongly depend on the field's intensity. The application of the electric field will result in the migration of negatively charged micro-organisms toward the anode and one-dimensional flow of pore fluid from the anode to cathode [22,23]. Importantly, a current of 40 mA at a density of 0.1-0.2 mA/cm2 is preferable in order to achieve a one-dimensional flow of a *Pseudomonas*-loaded pore fluid from the anode to cathode [24]. However, at a current density of 0.1-0.2 mA/cm2, the pH stabilises in the range of 2-3 at the anode destroying the acid-intolerant microbial species and in the range of 8-12 at the cathode killing the baseintolerant species. [22], thereby limiting the efficacy of the EKB process. On the other hand, the application of a recirculating buffer solution and careful regulation of electrolyte concentrations was also shown to afford some control over the pH and thereby improve distribution of a *Pseudomonas* strain which resulted in a 60% degradation of diesel over an 8 day period [24]. Maintenance of soil pH, between 5-7, is therefore necessary in order to

achieve the optimum degradation of contaminants by most native soil microbes [22].

Polycyclic aromatic hydrocarbons (PAHs) are a particularly important class of pollutant as they are generated by the incomplete combustion of carbon-based fuels and are ubiquitously found in tar, oil and coal deposits [25]. Consequently, they represent one of the most widespread and abundant class of pollutants. PAHs are of particular concern as members of this class have been identified as being mutagenic, teratogenic and carcinogenic [26]. Their abundance and relative resistance to evaporation [27] makes them ideal candidates as a model contaminant through which the effectiveness of remediation The soil used in this study was taken from a Patagonian landfill (Table 1). Samples were obtained from a depth of between 20 and 50 cm. All samples were air-dried and sieved (2 mm) prior to use in order to facilitate the even packing of the electrokinetic cells and improve the sample homogeneity.


**Table 1.** Physics-chemical Properties of Patagonian Soil Used in This Investigation.

#### **2.1. Electrokinetic reactor**

The electrokinetic reactor used in this study was similar to that used in previous electrokinetic research [28]. Electrokinetic experiments at constant potential were carried out using an experimental apparatus for the electrokinetic tests that consisted of three main parts: soil cells, electrode compartments and power supply. A schematic of the electrokinetic reactor, and the set ups of the electrokinetic cell, is shown in Fig. 2. The electrokinetic cells consisted of a glass cell (inner dimensions: length 58cm, depth 15cm and width 15cm) that was divided into three compartments: two electrodes (10cm x 15cm x 15cm) with phosphate buffers (pH 7.8 in anode and pH of 5.8 in the cathode) using platinum electrodes inside the buffers, and a soil compartment (30cm x 15cm x 15cm). The experiments were performed using three varieties of reactor cell design: (I) in the first design, the connections between compartments were made with a NaCl agar bird channel of 1cm in diameter during one

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 maintained at about 12%.

**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 electrodes were buried in the soil.

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

#### **2.2. Chemical analysis of soil samples**

#### *2.2.1. Determination of hydrocarbons via GC-analysis*

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