Soil and contaminant

Sandy soil was obtained from a beach in the Galician coast, Northwestern Spain, which was mixed with car engine burned oil from a local automotive workshop (Vigo, Spain).

Biosurfactant extraction from corn steep liquors

Biosurfactant (BS) in the corn steep liquors (CSL) was extracted with chloroform in three stages (^{Vecino et al., 2015}): 1) a liquid-liquid extraction at 65 °C using a CSL/chloroform ratio of 1:2 (v:v) for 1 h, 2) the mixture remained 24 h in a separating funnel to allow its equilibrium, we obtained aqueous phase and the organic phase containing the biosurfactant, and 3) we obtained a concentrated biosurfactant in chloroform extract by distilling and after diluting in water to obtain the cleaning solution.

Evaluation of the biosurfactant capacity to form emulsions with burnt engine oil

The emulsifying ability of the biosurfactant obtained from corn steep liquors to form emulsions was calculated as a function of two variables, EV (relative emulsion volume, %) (Equation 1) and ES (biosurfactant stability, %, to maintain emulsion when mixed with oil and water) (Equation 2). Variables that measure the emulsifying ability of the biosurfactant (EV and ES) were assessed 30 d with the ^{Neufeld and Zajíc (1984)} and the ^{Das et al. (1998)} equations. For this purpose, a 2 mL sample of burned oil was a dissolved in BS to the CMC (200 mg L^{- 1}), stirred 2 min in a vortex, stabilized for 72 h and then quantified the EV and ES. The EV calculation is based on the percentage of emulsion formed between the oil phase and the aqueous phase, respect to the total volume. For it, height of emulsion and both phases total height values are taken, as well as the cross section of tube (equation 1). ES is the relationship between EV at a determinate time and EV at time cero (equation 2):

Oil displacement test

The test of the oil displacement by the biosurfactant indicates it ability to solubilize oil. To perform this test, 20 mL of water were placed in a Petri dish, then, 40 µL of the engine burned oil were added and 80 µL of the biosurfactante dissolution were added over the oil, above the CMC. As a control, two other experiments were performed. On them, water or a sodium dodecyl sulphate solution (SDS) was added (detergent used for cleaning oil tanks, obtained by chemical synthesis) at its CMC (2365 mg L^-1). The cleaning solution that displaces the largest oil quantity over the water and shows a greater diameter of displacement will have greater capacity as a cleaning solution.

Surface tension evaluation

The surface tension of the biosurfactant solution, as well as the water made control, were assessed before and after the sand washing with a surface tensiometer (KRUSS K6, Spain) equipped with a 1.9 cm platinum ring (Du Noüy ring method). The measurements were carried out by triplicate at room temperature.

Contaminated soil washing test at laboratory scale

For these tests, 5 g of sandy soil we weighed. These were contaminated with burnt car engine oil with 0.25 g (50,000 ppm) oil content. Laboratory scale washing experiments were performed in batch, in 250 mL Erlenmeyer flasks, at room temperature in an orbital shaker at 150 ppm. The solid/liquid ratio established between the contaminated soil and the cleaning solutions was between 1:10 and 1:30 (w:v). Cleaning solutions were prepared with the biosurfactant twice below the CMC (BS/2) or the CMC (BS). Cleaning time was set at 6 h.

After washing the sand, the cleaning solution was separated by filtration, using a porous plate funnel, and then quantifying the extracted oil in the cleaning solution by gravimetric analysis. For this, 1 mL cleaning solution sample remained 48 h in an oven at 100 °C.

The decontaminated and filtered sand then passed to an extraction process in a Soxhlet device using acetone (Nahita, Heating mantles, Mod 655. Navarra-Spain) to quantify the oil that remained in the sand, which must be complementary to the oil extracted in the cleaning solutions.

Process to extract remaining oil in sandy soil

For the extraction of the oil remaining in the sand from the Soxhlet, the treated sand was introduced into a cellulose cartridge, and acetone was placed inside a heating mantle at 65 °C (temperature higher than the acetone´s boiling point, 56 °C), which allowed its evaporation. The Soxhlet device includes a condensation system that drags as it falls the remaining oil in the sand. This device worked for 1 h, then the Soxhlet cartridge was removed, and an acetone sample containing the residual oil from the sand was assessed in a double beam spectrophotometer at 328 nm (Jasco Mod. V-650, Spain). The relationship between the oil concentration in the acetone and the absorbance measured at 328 nm was linear (Figure 1), in this way the equation for oil concentration was obtained in relation to absorbance.

Figure 1 Calibration line established for the quantification of residual oil extracted with acetone via Soxhlet. 

Calculation of the extraction process efficiency

Extraction efficiency (EE) is defined according to equation 3 as the percentage of the oil removed from the sand:

where A₀ and A₁ represent the initial and final grams of oil present in the sand, respectively.

Evaluation of the emulsifying and dispersing capacity of the biosurfactant on burnt engine oil

After the extraction process, a biosurfactant extract with a yield of 8 ± 0.5 g extract kg^-1 of CSL with 50 % solids was obtained, although this yield may vary slightly between batches because the corn washing liquors are spontaneously fermented in uncontrolled conditions, if an industrial waste is assumed.

The extract was dissolved in water above its CMC (Figure 2). At the CMC (200 mg L^-1) or above it, the biosurfactant can keep the water surface tension constant at values close to 40 mN m^-1, this is the value where the biosurfactant begins to form micelles with the capacity to surround those molecules not soluble in water, which facilitates their mobilization and solubilization.

Figure 2 Aspect of the biosurfactant dissolution extracted from corn steep liquors solubilized in water. 

Once established the biosurfactant CMC the cleaning solution was prepared to CMC concentration (BS) or to half of the CMC (BS/2) and their ability to stabilize oil/water emulsions was assessed. The biosurfactant to the CMC (BS) was able to emulsify the oil with water as a whole, compared to the water/oil system in absence of the biosurfactant (Figure 3). This emulsion was stable for more than one month, period during which its stability was evaluated.

Figure 3 Water/oil system stabilized and emulsified with a biosurfactant from corn steep liquors (left), compared to an oil/water system without biosurfactant (right). 

The biosurfactant from corn steep liquor scattered the oil and displaced it to a greater extent to the edges of the plate compared with the detergent of synthetic origin, SDS, indicating a greater capacity of the biosurfactant to extract and solubilize oil (Figure 4).

Figure 4 Burnt oil motor displacement in water in presence of a biosurfactant (BS), extracted from corn steep liquor and dodecyl sulfate sodium (SDS), respect to a water control. 

Washing tests for sandy soils contaminated with burned car engine oil

The efficiency of the extraction process achieved with the biosurfactant extracted from corn steep liquor was significantly greater than that achieved with water as the control, especially when the cleaning solution was formulated with biosurfactant concentrations around the CMC (Figure 5).

Figure 5 Oil extraction percentages reached with a biosurfactant obtained from corn steep liquors after 6 h of treatment compared with water (control). 

Biosurfactant concentrations around the CMC, defined as BS, produced nearby 67 % of oil extraction, while BS/2 set the oil extraction percentage at 28 %, in these conditions although, the cleaning solution volume increased, the results were closer to those obtained with the cleaning solution consisting of the water control.

The results obtained in our study regard the BS are better than those reported by ^{Vreysen and Maes (2005)} of 50 % and 20 % extraction of diesel oil using Tergitol NP-10 surfactant. ^{Moldes et al. (2011)} used a biosurfactant produced by Lactobacillus pentosus and eliminated 58.6 % octane after 15 d of contact between the BS and contaminated soil.

This allows us to consider that the causes of the less satisfactory results below the CMC in our study could be due the low biosurfactant concentrations, which does not form micelles that surround oil molecules, or promotes mobilization and solubilization of the oil present in the sandy soil.

Figure 6 shows the surface tension values recorded in the different cleaning solutions after the treatment of the contaminated sand. Experiments that used 100 mL cleaning solution had a solid/liquid ratio of 1:20 (w:v), while with 150 mL this ratio was of 1:30 (w:v). The lowest surface tension (37 mN m^-1) was observed in the cleaning solution with the highest biosurfactant concentration (200 mg L^-1), but the BS/2 cleaning solution with 100 mg L^-1 biosurfactant has a higher surface tension (46 mN m^-1), which explains its lower cleaning capacity compared with the BS cleaning solution. The approximate surface tension of the water-based cleaning solution (water control) was 60 mN m^-1 (Figure 6).

Figure 6 Surface tension comparison between the evaluated cleaning solutions. 

^{Moya-Ramirez et al. (2014)} showed that, during the treatment of a sandy soil with 10 g L^-1 of a synthetic surfactant (G600), the increase of temperature makes the extraction process more effective. These authors reported extraction rates under 20 % at room temperature, and up to almost 40 % when the temperature increased up to 65 °C. The use of temperatures around 65 °C makes this process unviable for industrial scale, mainly in “in situ” extraction processes, since the energy cost and equipment for such a process would be high. Compared to natural detergents ^{Moya-Ramirez et al. (2014)} used a biosurfactant produced by Bacillus subtillis and obtained higher percentages than those obtained with G600 synthetic surfactant cleaning agent; although, higher biosurfactant concentrations caused lower yields. The extraction with biosurfactant used by these authors was less than 40 %, at 45 °C, and was less than that obtained with the biosurfactant evaluated in our study (67 %) at room temperature (25 °C).