SURFACE ACTIVE AGENTS AND MICROBIOLOGICAL CULTURES TREATMENT OF HYDROCARBONS CONTAMINATED SOIL Jonas Plauska, Lina Ragelienė, Lina Kubilė Department of Biochemistry and Department of Environmental Sciences Vytautas Magnus University, Vileikos str. 8, Kaunas, Lithuania Introduction Soil cleaning Soil cleaning was performed by mixing contaminated soil with synthetic surfactant solution and bacterial isolates and shaking by horizontal shaker. Cleaning parameters for synthetic surfactant‘s were as follows: temperature 21 ◦C, time 20 min, shaking speed 200 rpm. Parameters for bacterial treatment were the same as synthetic surfactant, except temperature (37 ◦C) and time (till the stationary optical density and 1 hour after reaching stationary optical density). After cleaning, wastewater was decanted. Hydrocarbons determination Rinsed soil was extracted with 50ml n-hexane (with n- decane and n-tetracontane mixture, to identify oil hydrocarbons in C10 - C40 range and for better peak integration). Concentration of oil hydrocarbons was determinated using „Shimadzu Ultra 2010“ GC/MS, using this parameters: GC column (Rxi®-5ms, length 30 m, diameter 0,25 mm, USA), injection volume 1μl, detector temperature 300 ◦C, flowing gas – helium. Letter S shows integration start, E shows integration end. (Fig. 2.) Environmental pollution – is the contamination of air, water or soil by the discharge of harmful substances. One of the most important contamination in terrestrial ecosystems is oil hydrocarbons pollution. Surfactants give a possibility to eliminate oil products from contaminated soil. Because of surfactant molecular structure, the oil molecule can be wrapped by surfactant molecule and then eliminated from soil. After that, micelized oil molecule can be decomposed by soil bacterias and get to the natural material circuit. Second way to clean polluted soil is using bacterial isolates which produces biosurfactants. To achieve the best synthetic surfactants and microorganisms usage results for this purpose, we must select suitable surfactants or biosurfactants with most effective concentration. The concentration of hydrocarbons removed from contaminated soil indicates that the highest amount of hydrocarbons was removed by washing it with anionic and non-ionic surfactant mixture (Fig. 5.). Aim The aim of this research work was to determine most effective surfactant type and surfactant concentration for oil contaminated soil cleaning. Fig. 5. Hydrocarbons removed from contaminated soil with SAA solutions mixture Materials and Method When contaminated soil was treated with Pseudomonas Aeruginosa DS10-126 bacterial isolates, which produces bioSAA rhamnolipid, oil hydrocarbons concentration was reduced more than 87% in comparison with contaminated soil. Pseudomonas Aeruginosa PAO1 isolates also produced bioSAA but cleaned 67 % of oil hydrocarbons from soil in comparison with contaminated soil (Fig. 6.). Preparation of soil Soil for cleaning was prepared by mixing three different types of soil (black-soil, clay and gravel) at equal parts and then contaminated by diesel – motor oil (1:1) mixture. This procedure resulted 60000 mg/kg soil contamination with hydrocarbons. Surfactant and biosurfactant solutions preparation Two types of synthetic surfactant (anionic sodium dodecylsulphate (SDS) and non-ionic polyoxyethylene (20) oleyl ether (Brij 98)) was used for this research. Their concentrations were selected considering to critical micelle concentration (CMC): below the CMC and approximately CMC. Another washing solution was prepared by mixing both „SDS“ and „Brij 98“ at concentrations below the CMC. As a control group was selected distilled water. (Table 1.) Fig. 2. Chromatogram of diesel and motor-oil contaminated soil Results Oil hydrocarbons determination in cleaned soil by GC/MS showed that both synthetic and biosurfactant reduced hydrocarbons concentration in soil. Soil cleaning with anionic surfactant demonstrated in Fig. 3. Oil hydrocarbons removal is about 54% in comparison with contaminated soil. Surfactant Type Concentration SDS (CMC = 8.1 mM) Anionic surfactant 1,7 mM (below CMC) 3,4 mM (below CMC) 8,1 mM „Brij 98“ (CMC = 0.025mM) Non-ionic surfactant 0,0025 mM (below CMC) 0,025 mM Mixture (SDS + “Brij 98“) Mixture of anionic and non-ionic surfactant SDS 1,7 mM and “Brij 98“ 0,0025 mM H2O Control - Fig. 6. Hydrocarbons removed from contaminated soil with Pseudomonas Aeruginosa DS10-126 isolates and Pseudomonas Aeruginosa PAO1 isolates. Conclusions The most effective concentration of synthetic anionic surfactant sodium dodecylsulphate for cleaning hydrocarbons contaminated soil is below the critical micelle concentration, but for non-ionic surfactant, concentration should be close to critical micelle concentration, because of it’s more lipophillic molecular structure. The highest amount of oil hydrocarbons was removed from contaminated soil using anionic surfactant sodium dodecylsulphate and non-ionic surfactant polyoxyethylene (20) oleyl ether mixture comparing to experiments, then the soil was cleaned with the anionic or non-ionic surfactant solutions at the same conditions. Biosurfactants produced by Pseudomonas Aeruginosa DS10-126 isolates, in comparison with all synthetic SAA and bacterial isolates used in this research, the most effectively removed oil hydrocarbons from contaminated soil. Hydrocarbons concentration was reduced 87,2 % in comparison with contaminated soil. Table 1. Selected surfactant‘s, type, concentrations. bioSAA was prepared by cultivating Pseudomonas Aeruginosa DS10-126 bacterial isolates, which produces rhamnolipid. As control group was selected Pseudomonas Aeruginosa PAO1 bacterial isolates. For different bioSAA concentrations simulation was selected two bacteria cultivation types: Cultivation till the stationary optical density (620 nm); Cultivation 1 hour after reaching stationary optical density (620 nm). Pseudomonas Aeruginosa DS10-126 growth kinetics demonstrated in Fig.1. Fig. 3. The concentration of hydrocarbons removed from contaminated with anionic SDS soil Soil cleaning with non-ionic surfactant showed better results (Fig. 4.) comparing with anionic surfactant. Oil hydrocarbons removal is more than 80% in comparison with contaminated soil. Fig. 4. The concentration of hydrocarbons removed from contaminated with non-ionic Brij 98 soil Fig. 1. Psedomonas Aeruginosa DS10-126 growth kinetics curve