ISOLATION AND IDENTIFICATION OF BACTERIAL STRAINS FROM A MIXED WASTEWATER TREATMENT SYSTEM USED TO TREAT PETROCHEMICAL EFFLUENTS Juan L. García Rojas (*) Instituto Mexicano de Tecnología del Agua :Biólogo, Universidad Nacional Autónoma de México, Maestría en Ciencias (Biotecnología) Universidad Nacional Autónoma de México.,: Investigador, Laboratorio de Calidad del Agua Trabajando en tecnología enzimática y microbiología del agua. Gabriela. Moeller-Chávez, Instituto Mexicano de Tecnología del Agua. Esperanza. Ramírez-Camperos, Instituto Mexicano de Tecnología del Agua Petia. Mijailova-Nacheva, Instituto Mexicano de Tecnología del Agua. Luciano Sandoval-Yoval, Instituto Mexicano de Tecnología del Agua. Lina Cardoso-Vigueros Instituto Mexicano de Tecnología del Agua. Marco Antonio. Garzon-Zuñiga Instituto Mexicano de Tecnología del Agua Miguel Angel Córdova Rodríguez Instituto Mexicano de Tecnología del Agua (*)Instituto Mexicano de Tecnología del Agua, Paseo Cuauhnáhuac 8532, C.P. 62550, Jiutepec, Mor. México. Tel. (777) 3-19-4000 ext. 357 Tel. y Fax (777) 3-19-4281, e-mail:
[email protected] ABSTRACT Petrochemical industry generates highly toxic effluents with a large variety of chemicals, being most of them recalcitrant and toxic such as: organic chlorinated compounds (mainly 1,2 dichloroethane) and volatile and semivolatile organic compounds. The most common wastewater treatment systems used in Mexico to treat this kind of effluents are activated sludge and aerated lagoons. Treatibility studies in batch aerated reactors were made to define the best operating conditions that could treat efficiently a mixed effluent from several petrochemical industries. Operating conditions evaluated were F/M relationship in a range of 0.2 – 0.7, kg -1 3 -1 COD/kg VSS*day , volumetric organic load, HRT 2-4 days, kg COD ≥ 0.25 m day . Organic matter removal achieved was more than 80 %. Some of the persistent molecules identified after treatment were: 1,1,2 trichloroethene, 1,2 dichloroethane, ethilbenzene and 1,4 dicholorobenzene. This highly toxic compounds are removed by aeration and the residual concentration degradated by the microbial biomass produced for each condition. Mixed liquor volatile suspended solids (MLVSS) were in the range of 2000 mg/L. During the experiment, microbial activity was followed and a selection of bacterial biomass was observed in the reactors; the main bacterial strains isolated were: Pseudomonas aeruginosa, Pseudomonas fluorescens, Pantoea sp., Chryseomonas luteola, Proteus peenneri and Serratia sp. This native microorganisms were acclimated to this kind of residues and are potentially suitable to biodegradation bioremediation in the wastewater petrochemical industry. Key words: Wastewater, treatment, bacterial isolation INTRODUCTION The petrochemical industry is normally found in tropics, where a great amount of wastewaters are released on waters and soils. Although these wastewaters are treated to reduce contaminant organic charge, they still contain high contaminant concentrations that are discharged on the swampy zones. The wastewaters released by oil-processing and petrochemical enterprises contain large amounts of toxic derivatives, such as polycyclic and aromatic hydrocarbons, phenols, sulfides, and heavy metals. The major problems faced by oil refineries are the safe disposal of oily sludge generated during the processing of crude oil. Improper disposal of oily sludge leads to environmental pollution, particularly soil contamination, and represents a serious threat
to groundwater. Many of the constituents of oily sludge are carcinogenic and potent immunotoxicants (Sanjeet-Mishra et. al, 2001; Propst, et. al., 1999). In these areas the wastewaters remain for large periods where they make possible the development of a great quantity of native strains acclimated to the toxic derivatives, being potentially capable of degrading hidrocarbures (King, et al, 1992). Some reports in the tropical regions of México have shown the presence of biodegrading bacteria. Adams, et. al (1995) show the aerobial microbial activity on soils representative of the petrochemical zone of Tabasco and Chiapas, Mexico; Mayo (1999) determined the microbial activity as aerobic to anaerobic on swamping oily zones; Ferrer (1997) isolated native bacterias in swampy oily zones of Mecoacán, Tabasco, México. Among the many techniques employed to decontaminate the affected sites, in situ bioremediation using indigenous microorganisms is by far the most widely used (Barbeau et. al., 1997; Dibble and Bartha, 1979; Eriksson et. al., 1999; Forsyth et. al., 1995; MacNaughton et. al., 1999; Venosa, et. al., 1996). This approach to reclaiming contaminated land reduces the threat to groundwater and enhances the rate of biodegradation. Many microbial strains, each capable of degrading a specific compound, are available commercially for bioremediation. However, indigenous bacteria in the soil can degrade a wide range of target constituents of the oily sludge, but their population and efficiency is affected when any toxic contaminant is present at high concentrations. In the present work, we selected toxic-resistant microorganisms from batch aerated reactors containing wastewaters of an oil refinery. Among the microorganisms selected, three of them have been previously reported as capable of biodegradating compounds derivated of wastewater treatment systems in oilprocessing and petrochemical enterprises. MATERIALS AND METHODS Batch aerated reactors. Ten batch aerated reactors with fifteen liters, containing activated sludge, were displayed with wastewater of an oil refinery and monitored collecting three samples of each one of them, at different times and different porcentual concentrations.
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Figure 1. Reactors used for the bacterial isolation of wastewaters from an oil refinery Bacterial isolation The screening for microorganisms capable of biodegradating compounds derivated of wastewater treatment systems in oil-processing was made by collecting 50 mL of each reactor system an placing them in centrifuge tubes, then 30 ml were transfer to baffled flasks with 150 mL of mineral medium and 50 mL of wastewater. The flasks were incubated at 35 ° C for 24 h. Triplicate plates were seeded in standard plate count agar with 5 mL of the mixed wastewater to conserve the selective pressure of the primary conditions. In all cases, the incubation temperature an period were of 35°C and a maximum of 3 days, respectively. The total bacterial count was made using the growth in plate using the serial dilution method. The selection of the –1 -6 dilution to use was made on a range between 10 and 10 . Isolation and purification of bacteria. Original samples were inoculated on petri dish with standard plate count agar and incubated at 35 °C for 24 to 48 h. From the growth developed in the petri dishes, the original consortia in each condition were isolated by selective selective and propagation before the identification procedure.
Classification of microorganisms. The organisms were taxonomically identified with the commercial system API-20E. RESULTS Isolation of microorganisms. Microorganisms able to grow in the presence of wastewater of the petrochemical industry were isolated of the mixed original reactor containing the different selective pressure. Six isolates producing colonies with a variety of colors where grown in petri dishes and selected for further characterization. The API-20E showed that Pseudomonas aeruginosa could be isolated of the reactor mixed with 50 % of wastewater, Pseudomonas fluorescens was isolated from the mixed reactor with 3, 20, 40 and 50 % of wastwater. The Pantoea sp was isolated from the mixed ractor with 40 % of wastewater, Chryseomonas luteola was found in the mixed reactor with 10 and 30 % of wastewater. The strain of Proteus peenneri was isolated of the mixed reactor with 15 % of wastewater and, finally, Serratia sp was found in the mixed reactors with 5 and 15 % of wastewater. DISCUSSION Six bacterial strains, two Pseudomonas, Pantoea sp, Chryseomonas luteola, Proteus peenneri and Serratia sp, were capable of growning in the presence of wastewater of petrochemical industry. The genus Proteus and Serratia are clasified as enteric, Gram-negative, facultative bacteria and can be used in the degradation of a great number of non conventional organic molecules under aerobic conditions along with E. coli, Salmonella, Shigella and Enterobacter. Pseudomonas aeruginosa is a Gram negative, cosmopolitan bacteria, that can be found in soils, waters, vegetables and animals. It is an oportunistic pathogen that has been reported with degradation capacities of non conventional organic compounds. Chryseomonas luteola is also a Gram negative bacteria, recently was reclassified as Pseudomonas luteola and, in particular, this bacteria has been reported as usefull for the bioremediation of contaminated sites with heavy metals and fenols. Finally, Pseudomonas fluorescens and Pseudomonas fluorescens pútida are cosmopolitan bacteria, found in water and soil and also recognized in the degradation of non conventional compounds and usefull in environmental bioremediation (Loser, et. al., 1998). The bacterial isolates described here are potentially useful for removing contaminating compounds in effluents. We wish to thank the personal at PEMEX, Victor M. Guerrero Vivanco and Guillermina Vazquez Segura. REFERENCES Adams, R.H., G. Armenta y L. García. (1995). Bioremediation in the petroleum producing region of southeast Mexico: microbial activity in contaminated soils and spent drilling muds. Memories of Second InterAmerican Environmental Congress. 30 Aug. a 1 Sept. Instituto Tecnológico y de Estudios Superiores de Monterrey (ITESM), Monterrey, NL, México. Barbeau, C., L. Deschenes, D. Karamanev, Y. Comeau, and R. Samson. (1997). Bioremediation of pentachlorophenol-contaminated soil by bioaugmentation using activated soil. Appl. Microbiol. Biotechnol. vol. 48 :745-752. Dibble, J. T., and R. Bartha. (1979). The effect of environmental parameters on the biodegradation of oily sludge. Appl. Environ. Microbiol. vol. 37 :729-739. Eriksson, M., G. Dalhammar, and A.-K. Borg-Karlson. (1999). Aerobic degradation of a hydrocarbon mixture in natural uncontaminated potting soil by indigenous microorganisms at 20°C and 6°C. Appl. Microbiol. Biotechnol. vol. 51:532-535. Ferrer, M.I. (1997). Selección de tecnologías de restauración biológica para lodos de perforación en tres campos petroleros. Tesis de Maestría en Ciencias con especialidad en Ing. Ambiental, Instituto Politécnico Nacional, México, DF.
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