In situ biosurfactant production and hydrocarbon removal by Pseudomonas putida CB-100 in bioaugmented and biostimulated oil-contaminated soil

Brazilian Journal of Microbiology 44, 2, 595-605 (2013) ISSN 1678-4405 Copyright © 2013, Sociedade Brasileira de Microbiologia www.sbmicrobiologia.org.br Research Paper In situ biosurfactant production and hydrocarbon removal by Pseudomonas putida CB-100 in bioaugmented and biostimulated oil-contaminated soil Martínez-Toledo Ángeles1,2, Rodríguez-Vázquez Refugio2 1 Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, SLP, Mexico. 2 Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN, Col. San Pedro Zacatenco, Mexico, D.F., Mexico Submitted: June 29, 2011; Approved: June 05, 2012. Abstract In situ biosurfactant (rhamnolipid) production by Pseudomonas putida CB-100 was achieved during a bioaugmented and biostimulated treatment to remove hydrocarbons from aged contaminated soil from oil well drilling operations. Rhamnolipid production and contaminant removal were determined for several treatments of irradiated and non-irradiated soils: nutrient addition (nitrogen and phosphorus), P. putida addition, and addition of both (P. putida and nutrients). The results were compared against a control treatment that consisted of adding only sterilized water to the soils. In treatment with native microorganisms (non-irradiated soils) supplemented with P. putida, the removal of total petroleum hydrocarbons (TPH) was 40.6%, the rhamnolipid production was 1.54 mg/kg, and a surface tension of 64 mN/m was observed as well as a negative correlation (R = -0.54; p < 0.019) between TPH concentration (mg/kg) and surface tension (mN/m), When both bacteria and nutrients were involved, TPH levels were lowered to 33.7%, and biosurfactant production and surface tension were 2.03 mg/kg and 67.3 mN/m, respectively. In irradiated soil treated with P. putida, TPH removal was 24.5% with rhamnolipid generation of 1.79 mg/kg and 65.6 mN/m of surface tension, and a correlation between bacterial growth and biosurfactant production (R = -0.64; p < 0.009) was observed. When the nutrients and P. putida were added, TPH removal was 61.1%, 1.85 mg/kg of biosurfactants were produced, and the surface tension was 55.6 mN/m. In summary, in irradiated and non-irradiated soils, in situ rhamnolipid production by P. putida enhanced TPH decontamination of the soil. Key words: bioremediation, irradiated soil, total petroleum hydrocarbons, rhamnolipids, P. putida. Introduction At present, Mexico has severe water, soil, and air pollution problems due to contamination by both organic and inorganic compounds. Total petroleum hydrocarbons (TPH) are considered to be high-priority compounds that should be removed from polluted areas in Mexico (SEMARNAT, 2008). TPH are a mix of pollutants, and in soil, they represent a risk to the health of people and ecosystems (DOF, 2005). Some of these pollutants have low solubility in water and are, therefore, difficult to remove from polluted environments. For such pollutants, the use of synthetic surface agents has been suggested. These agents enhance the pollutants solubility’s (Volkering et al., 1995), leading to their desorption from the soil, thereby making them more accessible to microbial degradation. Certain microorganisms are able to produce extracellular substances (biosurfactants) with similar characteristics to those of synthetic surfactants. Because they have the advantage of being highly biodegradable, these microbial compounds are attractive agents to use in the bioremediation field (Maier and Soberón-Chávez, 2000). Pseudomonas putida can produce rhamnolipids (Amézcua-Vega et al., 2004) and are able to remove phe- Send correspondence to R. Rodríguez-Vázquez. Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN, Av. Instituto Politécnico Nacional #2508, Col. San Pedro Zacatenco, C. P. 07360, Mexico, D.F., Mexico. E-mail: . 596 nanthrene in liquid culture medium under certain culture conditions (Martínez-Toledo et al., 2006). The aim of this work was to measure biosurfactant production by P. putida in a biostimulated and bioaugmented soil sample and to assess its ability to remove TPH from polluted soils. Materials and Methods Soil sampling and processing: The soil used in this study came from the Petrochemical Complex located in Poza Rica, Veracruz, Mexico, 20°30’6” N and 97°28’33” W. The complex is located in the hydrological region RH27 “Tuxpan Nautla,” which includes the river basins of Nautla, Tecolutla, Cazones, and Tuxpan, as well as the Tamihua lagoons. According to the FAO/UNESCO/IRIS, sedimentary rocks are the predominant geological formation of the soil and Vertisols-Gleyic is the dominant type of soil. The area has a warm and wet climate with an annual mean temperature of 24.4 °C, annual precipitation of 1,472.4 mm, and a relative humidity between 76 and 80% (INEGI, 1997). Waste from petroleum drilling operations, maintenance, and duct repair has been accumulating in this soil. For this study, the soil was sampled (Figure 1) at a depth of 50 cm at thirteen points (S.1 - S.13) and down to 2 m at six points (P.M.1 - P.M.6). The bulk samples were homogenized manually and sieved (2-cm mesh), then kept at 4 °C in polypropylene containers holding 200 kg. From these containers, 200 kg was taken and air-dried at room temperature, homogenized manually again, and 500 g was taken as a representative sample as reported elsewhere (Cline, 1994). Each sample was crushed with a mortar and pestle, passed through a standard sieve of 2-mm mesh size, and kept at 4 °C in glass amber bottles until its characterization and analysis. Gamma irradiation of the soil was performed at the Gamma Irradiation Department of the National Insti- Figure 1 - Soil sampling map. Ángeles and Refugio tute of Nuclear Research located in the state of Mexico. The soil was irradiated at 25 kGy in 2-kg polyethylene bags in 5-kg containers (ININ, 2010). Physical and chemical characteristics of the soil: Humidity was measured by the gravimetric method (Gardner and Klute, 982). The method used for total nitrogen (N) determination (Kjeldahl method) was that reported by Muñoz-Iniestra et al. (2000). Available phosphorus (P) was determined according to Bray and Kurtz in1945 with modifications (Roldán-Martín et al., 2006). Organic matter was determined through the method of oxidation with chromic and sulfuric acids developed by Walkley and Black in 1947 (Muñoz-Iniestra et al., 2000). The pH was determined in a 1:10 (w/v) suspension of the soil in distilled water and measured with a Jenway Mod. 3020 potentiometer using epoxy electrodes (Muñoz-Iniestra et al., 2000). After soil characterization, nutrients were balanced based on the biostimulation experiment. The nutrient sources were NH4Cl (20 mg/g) and NaNO3 (320 mg/g) for N and KH2PO4 (3.62 mg/g) and K2HPO4 (46.66 mg/g) for P, and these were added to obtain a carbon:nitrogen:phosphorus (C:N:P) ratio of 100:10:1 in the s (...truncated)