ASSAY FOR CATECHOL- 2, 3- DIOXYGENASE ACTIVITY IN BACTERIA ISOLATED FROM CRUDE OIL CONTAMINATED SOIL IN ILAJE, ONDO STATE, NIGERIA.

Abstract

The present research was undertaken to assess the activity of catechol - 2, 3- dioxygenase from bacteria found in crude oil contaminated soil in Ilaje, Ondo State. Enrichment technique was employed for the isolation of the total heterotrophic crude oil degrading bacteria. The isolated bacteria were identified using conventional microbiological methods. They were screened for their degradative ability based on their growth in enrichment medium (Bushnell Haas). The physicochemical properties of the soil samples were also determined using standard chemical methods. Metabolic activity of monoculture and the mixed culture of the best degrading bacteria (selected) were measured as CO2 evolved in biometric flasks during the mineralization of crude oil, at 3 days interval, over a period of 28 days at 28±2oC. Concentrations of protein released into the basal medium by the selected bacterial isolates were determined using Lowry’s method. Specific activity of catechol 2, 3- dioxygenase was determined using standard enzymological procedure. Effects of pH at range of 3 -12 and temperature range of 30-60oC was investigated using standard buffers and thermometer. Stability of the enzyme was evaluated at pH 5 - 8, and 40 – 60oC using conventional assay techniques. Bacteria isolated from the soil sample were Bacillus cereus, Bacillus amyloliqufaciens, Bacillus firmus, Acinetobacter calcoaceticus, Pseudomonas veronii, Pseudomonas fluorescens, Pseudomonas species, Pseudomonas aeruginosa, Achromobacter xylosoxidans and Achromobacter species. Pseudomonas aeruginosa had the best oil degradative ability among the screened isolates followed by Bacillus cereus, Acinetobacter calcoaceticus and Achromobacter spp. The physicochemical characteristics of the crude oil contaminated soil in Ayetoro showed a relative low pH (6.7), moisture (30.10 %), nitrogen (0.48 mg/kg), phosphorus (3.66 mg/kg), calcium (3.85 mg/kg), magnesium (2.0 mg/kg), sodium (0.78 mg/kg), potassium (1.15 mg/kg) and organic matter contents (5.80 %). In contrast the total organic carbon content (2.79%) which was higher than that (0.05%) unpolluted soil sample at Mahin. Metabolic activity of each bacterial isolate showed that the test microbes all experienced lag phase while they had their optimum content of CO2 produced within the first three weeks of their incubation. The amounts of CO2 evolved by the isolates correspond to their biodegradation efficiency. Pseudomonas aeruginosa had higher optimum activity than the other bacterial isolates. Catechol 2,3-dioxygenase showed optimum activity within the temperature and pH of 30oC to 50oC and 6.0 to 8.0, respectively. The stability expressed by catechol 2, 3-dioxygenase from each isolate occurred at 40oC and pH of 6.0 to 7.0. This project has revealed the various bacteria that are associated with crude oil polluted soil in Ilaje. Conclusively among the bacterial isolates, Pseudomonas aeruginosa followed by Bacillus cereus, Acinetobacter calcoaceticus and Achromobacter spp were the best degrading bacteria. These promising microbes can therefore, be used in bioremediation of crude oil polluted environment.