Material was obtained from the sediment delta at the dam “Presa Agua de Minas”, in the Texistepec Mining Unit in Texistepec Municipality, in southern Veracruz State, Mexico, in March 2004. In the area where material was collected, an oily crust of approx. 10 cm had already been removed manually as part of the remediation of the site. Material was collected with a straight shovel from the surface to a depth of 90–100 cm, obtaining roughly one-half cubic meter of the material. This material was placed in hard plastic containers and transported to the remediation patio of the Bioremediation Laboratory in the Juarez Autonomous University of Tabasco (Universidad Juárez Autónoma de Tabasco, UJAT), where it was thoroughly mixed.
The material was treated by three methods:
1) bioremediation – land farm with biostimulation only,
2) bioremediation – land farm with bioaugmentation,
3) chemico-biological stabilization, and a control (without treatment). Each treatment and control included three replicates. For each replicate, the material (after being mixed) was placed in cells 40 cm x 40 cm x 20 cm depth. The cells were formed of cinderblock and situated on a concrete slab. All tests were run in the open air in a tropical environment: ~ 1800 mm annual average precipitation, ~ 29 ºC average annual temperature (West et al., 1987). The three treatments are described below:
Biostimulation: addition of inorganic nutrients (Grow-Feed 20-30-10 commercial fertilizer) sufficient to provide 100 ppm of nitrogen, an organic amendment (0.74% of chopped water lily, dry weight basis), daily aeration and periodic moisture addition (to maintain approx. 15 -22% moisture). The actual moisture maintained in the treatment cells was 19.3% ± 1.51%. Temperature was measured daily and overly high temperature was controlled by spraying a small quantity of water on the treatment cells. The average temperature in the cells during the study was 32 ºC ± 2.7 ºC.
Bioaugmentation: fertilizer and organic amendment addition as in the biostimulation treatment, followed by the addition of autochthonous microorganisms, aeration, and moisture control (to maintain approx. 15- 22% moisture). Moisture and temperature were controlled as described for biostimulation, with the actual moisture maintained at 19.4% ± 2.21%, and the average temperature was the same as that in the biostimulation treatment (32 ºC ± 2.7 ºC). Chemico-biological stabilization: addition of 4% (w/ w) hydrated lime, mixing, followed by adding 4% (w/w) organic amendment (cachasse: sugar cane process waste) three days latter (Adams, 2004b). Fifteen days later, humidicola grass (Brachiaria humidicola) seeds were planted on the surface of the treatment cells.
Preparation of autochthonous microorganisms for bioaugmentation
Contaminated soil from the sediment delta was used as a source of microorganisms to prepare an undefined mixed culture. A 1/10 dilution of field moist sediment in deionized water was prepared, mixed thoroughly, and allowed to settle for 20 min. One milliliter of the supernatant was collected, taking care to avoid floating particles, and used to inoculate 100 mL of mineral nutrient which consisted of 1 g/L of soluble commercial fertilizer (Grow-Feed 20-30-10) in deionized water. As the C source, a drop of oil recovered from the contaminated sediments was added to the culture flask on the first day of incubation, followed by a drop of spent lubricant (motor oil) on the second day (the oil from the contaminated sediment was extremely viscous and produced a relatively low level of growth by itself). The culture flasks was placed on a New Brunswick Scientific model 01071-0000 orbital shaker (~180 RPM) to aerate the culture, at room temperature (~28 ºC). After three days, a notable increase in turbidity was observed, and the culture was used to inoculate a second culture flask using 1 mL of the first culture to inoculate 100 mL in the second culture. This process was repeated (three sequential cultures in all) to ensure that the selected microorganisms were actually using the added hydrocarbons as the C source and not alternative C sources from the original dilution of the sediment. In the third sequential culture, growth was measured with a Spectronic 20 (Bausch and Lomb) spectophotometer at 600 nm using uninoculated growth medium as a blank. This third culture was started using oil from the site as a C source and after roughly three days a drop of spent lubricant was added. After another three days, another drop of spent lubricant was added. After 12 days, this culture showed an increase in absorbance of > 0.2 O.D. and was used to inoculate the final culture used for bioaugmentation in the treatment cells.
One and one-quarter milliliter of the third sequential culture was added to each of twelve 250 mL flasks, each containing 125 mL of mineral growth medium (1 g/L of Grow Feed fertilizer). One drop of oil from the site was added to each flask to start the culture. After three days and again after another three days, one drop of spent lubricant was added to each flask. After 12 days, the cultures were harvested, pooled and divided into three 500 mL portions.
Each portion was added to a bioaugmentation treatment cell, applying the culture with a manual spray bottle, mixing the solution into the soil with a garden shovel. The final inoculation was estimated to be ~ 6 x 106 bacteria/g of dry soil considering the turbidity of the final culture and the application rate.
Initially and on a monthly basis, core samples were taken from each treatment cell, from the surface to 20 cm depth and analyzed for a variety of parameters. pH was measured with a Corning brand, model 240 pH meter with a Sensorex brand, model 5200c pH electrode (SECOFI, 1980).
Total Petroleum Hydrocarbons (TPH) were measured by EPA method 418.1 (EPA, 1997), using perchloroethylene as an extraction solvent with an Infracal TOG/TPH analyzer (Wilks Enterprise). Acute toxicity was measured with the Microtox system using a Microbics model 500 analyzer (SECOFI, 1996). Toxicity Characteristic Leaching Proceedure (TCLP) leachates were prepared (SEDESOL, 1993) and TPH in the TCLP leachates was measured by extraction in perchloroethylene, solvent evaporation, re-dissolution in methanol, and measurement of hydrocarbons in the methanol solution using EPA method 9074 (EPA, 1997; Adams and Ramírez, 1999). For this TPH analysis in leachate a Petroflag soil analyzer (Dexsil Corporation) was used. Samples for carcinogenic Polyaromatic Hydrocarbon (PAH) analyses were sent to a commercial laboratory accredited by the Mexican Accreditation Entity (EMA for its abbreviation in Spanish) and were analyzed by EPA method 8270 D (EPA, 1997) by gas chromatography/mass spectrometry (GC/MS) using a Hewlit Packard HP5890 GS coupled to a Hewlit Packard HP5971 MS detector, or equivalent.