**3. Results**

### **3.1. Nutrient and pH control**

The use of salt bridges permitted a better regulation of pH levels in the soil, especially with the use of the phosphate buffer bridge (Fig. 3) and this did affect bacterial counts (Table 2). The introduction of phosphate in the soil benefits biodegradation due to the fact that this nutrient is necessary, especially in Patagonian soils, where the concentration of nutrients is very low. On the other hand, the NaCl bridge introduced chlorine ions into the soil, the accumulation of which results in toxic effects on the bacteria [32]. This inconvenience was observed in the electrokinetic cell and arose as a consequence of electromigration produced by the applied electric field. At the end of experiment, the concentration of K+ and Cl ions following their respective migration to the cathode and anode was measured. Chlorine concentrations of 1207 mg / kg were found at the cathode whereas 836 mg / kg was observed at the anode. TheK+ ions were found at a concentration of 50 and 42 mg / kg in the cathode and the anode, respectively. For experiment II, with electrode buried in the soil, significant changes in soil pH were observed throughout the cell (Fig. 3).

**Figure 3. Effect of EKB on soil pH over time.** Readings were taken from samples taken throughout the cell as follows: **a.** from the cathode, **b.** from the centre of the cell and **c.** from the anode. Data are shown for each of the reactor cell designs: I - Cells using a NaCl bridge, II – Cells in which the electrodes were placed directly into the soil and III-Cells using a PO4-3 bridge.

In all three experiments, a substantial loss of moisture was observed in the electrokinetic cells. Therefore, weekly addition of water was required in order to keep the parameter between 12 and 15%. The region near the anode tended to dry, this is why the addition of water was required in all EK cells. In addition, the electrode in the soil generated a greater change in the pH and this impacted in the bacterial communities (Table 2) as well as in the moisture values.


**Table 2.** Obligate and Facultative Aerobic Bacteria Isolated from the EK Reactor

The results shown in Fig. 3 and Table 2 indicated that the best bridge to work with was the phosphate salt (Exp III), which could introduce nutrients to the soil and this produced an increase of bioremediation of hydrocarbons. Therefore, subsequent experiments were carried out in cells in which a phosphate bridge was placed. The nutrients introduction in Patagonian soil (Table 1), is necessary because of the soil properties [32]. In spite of this, the applied current still moves the ions. This soil needs nutrients C:N:P in the ratio 100:1:0.5 for bioremediation [32], but the anions are moved by the current, the phosphate bridges provide phosphate ions for biodegradation, and these ions accumulate in the area of the cathode.

The soil moisture contents were higher in EK remediated cells than those of the control cells due to the supply of electrolyte. However, EK remediation showed the reduced soil moisture content compared to the original soil, and the soil close to the cathode had higher moisture content than other soil, indicating the influence of electroosmosis.

Because of the electrical charge of the ions, the migration occurred and it was modified nutrient the bioavailability of phosphates. Nitrates have a relatively high mobility [33] and as shown in Table 3, the nitrates moved towards the anode. A high concentration of phosphate is seen in the cathode, probably as a result of the bridges. In accordance with previous findings [34] the values of phosphates were modified (Table 3).


**Table 3.** Electrically-Induced Migration of Nutrients in The EK Reactor.

#### **3.2. Soil hydrocarbon content**

36 New Technologies in the Oil and Gas Industry

Sherlock (Microbial ID, version 6.0).

**3.1. Nutrient and pH control** 

**2.5. Statistical analysis** 

**3. Results** 

**PH**

0 10 20 30

**a**

**Days**

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

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

The use of salt bridges permitted a better regulation of pH levels in the soil, especially with the use of the phosphate buffer bridge (Fig. 3) and this did affect bacterial counts (Table 2). The introduction of phosphate in the soil benefits biodegradation due to the fact that this nutrient is necessary, especially in Patagonian soils, where the concentration of nutrients is very low. On the other hand, the NaCl bridge introduced chlorine ions into the soil, the accumulation of which results in toxic effects on the bacteria [32]. This inconvenience was observed in the electrokinetic cell and arose as a consequence of electromigration produced by the applied electric field. At the end of experiment, the concentration of K+ and Cl-

following their respective migration to the cathode and anode was measured. Chlorine concentrations of 1207 mg / kg were found at the cathode whereas 836 mg / kg was observed at the anode. TheK+ ions were found at a concentration of 50 and 42 mg / kg in the cathode and the anode, respectively. For experiment II, with electrode buried in the soil, significant

**Figure 3. Effect of EKB on soil pH over time.** Readings were taken from samples taken throughout the cell as follows: **a.** from the cathode, **b.** from the centre of the cell and **c.** from the anode. Data are shown for each of the reactor cell designs: I - Cells using a NaCl bridge, II – Cells in which the electrodes were

0 10 20 30

**PH**

0 10 20 30

**<sup>c</sup>** <sup>I</sup>

II III

**Days**

**b**

**Dyas**

In all three experiments, a substantial loss of moisture was observed in the electrokinetic cells. Therefore, weekly addition of water was required in order to keep the parameter

changes in soil pH were observed throughout the cell (Fig. 3).

**PH**

placed directly into the soil and III-Cells using a PO4-3 bridge.

ions

column temperature was raised from 170 to 270 °C at a rate of 5 °C/min.

TPH concentrations were measured by GC, TPH-IR and TPH-SE.. Analysis of the soil by TPH-IR, showed a decline in all three parts of the electrokinetics cell (Fig.4). The largest of

which occurred around the anode. After 150 days, TPH-SE showed a decrease in all parts of the cells (Fig. 4). TPH analysis showed differences in values between the hydrocarbons from cells with 0.5 V/cm and the control cell, however during the first 30 days, there was there was significant difference between the values of the cathode and the anode (P<0.05). It is at this time that nutrients are distributed in both electrodes (Table 3). Silica gel chromatography showed changes in the percentage of the fractions of the residue (Fig. 5). The aliphatic fraction showed a decrease in all three reactor treatments. The cathode showed a good degradation. The total aromatic hydrocarbons presented a better degradation in the centre of the cell. The decrease in the percentage of aliphatic and aromatic hydrocarbons was evidenced by an increase in the relative percentage of polar hydrocarbons (Fig. 5)

**Figure 4.** Effect of EKB on soil TPH levels as determined by a) TPH-SE and b)TPH-IR.

**Figure 5. Effect of EKB on soil hydrocarbon content.** Pie charts illustrate the relative percentages of soil hydrocarbon classes both before (initial) and, after EKB, at different locations within the cell (anode, centre and cathode).

The PAH contaminants were reduced throughout the cell, but degradation was greatest in the centre where pH was most favourable for microbial activity. Since PAHs are neutrally charged, electromigration does not work for migration of these hydrocarbons concentration profile across the soil specimens determined at the conclusion of experiments. The results show that the PAHs were degraded preferentially in anode and centre of the cell (Table 4). The phenanthrene, fluoranthene, pyrene, benzo pyrene, chrysene, benzo fluoranthene and anthracene concentrations are relatively higher at the cathode zone than at the anode zone. Considering the initial concentration of these PAHs in the soil, significant amounts (P<0.05) of hydrocarbon were removed by this technique.

38 New Technologies in the Oil and Gas Industry

centre and cathode).

which occurred around the anode. After 150 days, TPH-SE showed a decrease in all parts of the cells (Fig. 4). TPH analysis showed differences in values between the hydrocarbons from cells with 0.5 V/cm and the control cell, however during the first 30 days, there was there was significant difference between the values of the cathode and the anode (P<0.05). It is at this time that nutrients are distributed in both electrodes (Table 3). Silica gel chromatography showed changes in the percentage of the fractions of the residue (Fig. 5). The aliphatic fraction showed a decrease in all three reactor treatments. The cathode showed a good degradation. The total aromatic hydrocarbons presented a better degradation in the centre of the cell. The decrease in the percentage of aliphatic and aromatic hydrocarbons was evidenced by an increase in the relative percentage of polar hydrocarbons (Fig. 5)

**Figure 4.** Effect of EKB on soil TPH levels as determined by a) TPH-SE and b)TPH-IR.

**Figure 5. Effect of EKB on soil hydrocarbon content.** Pie charts illustrate the relative percentages of soil hydrocarbon classes both before (initial) and, after EKB, at different locations within the cell (anode,

The PAH contaminants were reduced throughout the cell, but degradation was greatest in the centre where pH was most favourable for microbial activity. Since PAHs are neutrally


**Table 4.** Effect of EKB on Soil Content of Hydrocarbon Compounds

#### **3.3. Bacterial counts**

Maintaining the pH values suitable for the microorganisms caused the values of the bacterial counts not to experience any modification; in all cases, the drop of a logarithm was within the error of the method (Fig. 6). The bacteria did not migrate to the area of the electrodes, as stated by other authors in the case of saturated soil [35].

**Figure 6.** Bacterial count on TSBA and MBM-PGO media.

The bacterial identification was done on the bacterial count plate. In the initial sample, the genus were *Variovorax, Escherichia, Brevundimonas, Nocardia, Bordetella Mycobacterium*, *Rhodococcus, Acromobacter, Dierzia, Gordonia* and *Stenotrophomonas* (Fig. 7) which could grow in the agar plate in a greater number than 1 x 103. The treatment with current changed the bacterial proportion and new genus present in a small number increased their number (Fig 6).

**Figure 7.** Statistic analysis of bacterial identification at the beginning and end of the experience.


**Table 5.** Shannon indices of Soil Biodiversity

#### **4. Discussion**

40 New Technologies in the Oil and Gas Industry

Maintaining the pH values suitable for the microorganisms caused the values of the bacterial counts not to experience any modification; in all cases, the drop of a logarithm was within the error of the method (Fig. 6). The bacteria did not migrate to the area of the

The bacterial identification was done on the bacterial count plate. In the initial sample, the genus were *Variovorax, Escherichia, Brevundimonas, Nocardia, Bordetella Mycobacterium*, *Rhodococcus, Acromobacter, Dierzia, Gordonia* and *Stenotrophomonas* (Fig. 7) which could grow in the agar plate in a greater number than 1 x 103. The treatment with current changed the bacterial proportion and new genus present in a small number increased their number (Fig

**Figure 7.** Statistic analysis of bacterial identification at the beginning and end of the experience.

electrodes, as stated by other authors in the case of saturated soil [35].

**Figure 6.** Bacterial count on TSBA and MBM-PGO media.

**3.3. Bacterial counts** 

6).

The efficiency and effectiveness of electrokinetic extraction can be improved by combining the technique with other remediation technologies such as bioremediation. The results demonstrate the feasibility to degrade PAHs, a particularly problematic and toxic class of contaminant. Electrokinetics appears to be promising for a range of contaminated sites including tank bottoms which are considered dangerous waste by our legislation.

The effectiveness of the contaminant reduction during electrobioremedation depended on the bioavailability, moisture and soil pH. Among the various soil parameters, the most apparent change induced by electrobioremediation was observed in soil pH and distribution of moisture content. The bacterial number in the experiment II decreased in the anode due to the low soil pH. The soil pH is a crucial factor for microbial activity as this influences the composition and physiological characteristics of enzymes such as phosphatase, glucosidase and arylsulfatase [36]. Soil pH also affects microbial cell membrane integrity and function, and the bioavailability of nutrients and contaminants. Increased biodegradation in higher pH regions, such as cathode, has been reported for one of the PCP-degrading enzymes produced by a species of *Sphingobium* UG30 [37]. In addition, a low pH has been reported as having a major negative impact on electrobioremedation by native soil microbial communities [38,39]. Accordingly, we observed a decrease in dehydrogenase activity at locations corresponding to the acid front. In fact, in the expereince II, the soil pH changes generated by electrobioremediation were a leading cause of a decreased microbial number (Table 2). However, 0.5V/cm applied soil showed more apparent decrease in culturable bacteria compared to the soil samples of extreme initial pH [19]. This soil pH change was controlled by the use of phosphate birdge and this did not change the bacterial number between current and pH but affected the biodiversity in culturable bacteria (Fig. 7 and Table 5). The stress from the growth conditions reduces the total bacterial number and is a reason for cells entering a viable but non-culturable state [40,41]. Although electric current in EK remediation can change the bacterial membrane composition and metabolic activity, many studies revealed that weak direct current treatment has no negative effect on microbial viability and activity [42, 43, 44].

The experimental results illustrate how the application of electrokinetics to an unsaturated soil can cause major changes to the soil properties, with subsequent impact upon microbial activity and biodegradation. As expected, the lack of anodic pH control in experiment I caused progression of an acidic front through electrokinetic microcosms. However, pH control at both electrodes (experiment II) caused a large increase in moisture content in electrokinetic microcosms as the unsaturated soil absorbed water from electroosmotic flow.

In similar conditions with saturated soil this effect was not observed [18, 28, 45]. The lack of moisture change in experiment I is thought to be because the acidic pH increased the soil zeta potential, rapidly causing electroosmotic flow into the acidified region to reduce or even reverse [46]. By removing electrode fluid moisture content change was avoided but pH control had to be implemented using a regularly reversed current [47] and it may be that part of hydrocarbon fraction is eliminated in the fluids producing other contaminated residue. Changes in the concentration of pH and moisture in the soil may have mobilized pollutant fractions present in the soil in the porosity, which are often not accessible to soil microorganisms [48]. Thus, the occurrence of electroosmosis inside soil aggregates may have caused the mobilization of slowly desorbing hydrocarbons into the fast-desorbing pool, which possesses a higher bioavailability to microorganisms [49, 50]. Bioremediation in Patagonian soil with the addition of nutrients is possible; however, the remediation of PAH is problematic [29] because of their low bioavailability, which can be improved by electrobioremediation resulting in an reduction of PAH.

Maintenance of soil moisture levels proved essential, because if it decreases much it will generate problems at a metabolic level in soil bacteria. It is also necessary to apply a voltage greater than the potential in the electrodes to keep the voltage value of 0.5 V.cm-1 thus increasing the cost of treatment energy. By working with saturated soils, the electrolyte circulation volumes, generated by electroosmosis, are contaminated is a contaminated liquid residue. One of the benefits of our system is that unsaturated soil was not observed as a leachate product of the current application. The decrease in moisture of the tanks could also be due to factors, such as the heating system or for undesirable exothermic chemical reactions [5]. Soil temperature in electrokinetics cell remained at 24 ± 3 °C throughout the 150 days of runtime. A temperature range of 24 ± 3 °C can be considered an acceptable temperature for the biodegradation of hydrocarbons by native soil microbes. These results are in accordance with results from previous studies in which an applied voltage density of 0.3 V/cm was used for this purpose [16]. This indicates that the reduction of moisture in the anode is mainly due to the presence of electroosmotic flow in the soil.

Whilst the migration of bacteria towards the anode was expected (negative surface charge being responsible for this migration), bacteria were also found to migrate towards the cathode, which may be considered surprising at first glance. This bidirectional migration of bacteria may well have resulted from the competition between two phenomena: electrophoresis and electroosmosis [51, 52]. However, other phenomena may have contributed to the migration of bacteria to the cathode. The migration of ions and water in soil toward the cathode under the influence of the electric field is one such reason. This process could create favorable conditions for bacterial growth in the area near the cathode., although the net negative charge at their surface should make them move toward the anode. Kim et al. [53], concluded "Especially the number of culturable bacteria decreased significantly and only *Bacillus* and strains in *Bacillales* were found as culturable bacteria. It is thought that the main causes of changes in microbial activities were soil pH and direct electric current". The results described here suggest that if soil parameters, electric potential difference, and electrolyte are suitably controlled based on the understanding of interaction between electrokinetics, contaminants, and indigenous microbial community, the application of electrokinetics can be a promising soil remediation technology when the contaminant is hard to degrade or its degradation is really slow.

When electric current is applied, different bacterial responses to changes in the physicochemical properties, bioavailability, and toxic electrode-effect can be observed depending on the current, treatment period, cell type, and medium [14]. The soil pH had marked effects on microbial biomass, community structure, and response to substrate addition, and that low soil pH decreased microbial diversity and increased Gram-positive microbial communities such as *Bacillus* and *Arthrobacter* [38, 54] according with these authors the major presence of *Bacillus* were near the anode zone, and that bioelectrical reactors enhanced the metabolism of several strains in *Clostridium, Ralstonia, Pseudomonas*, and *Brevibacterium* [55].
