ABSTRACT
Polyaromatic hydrocarbons (PAHs) are environmental pollutants that can be found on many surfaces including grasses that are often consumed by ruminants. It was therefore hypothesized that bacteria in the intestine of cow may have the potential to degrade PAHs. The aim of the study was to isolate PAH(s) degrading organisms from the large intestine of cows.
Bacteria were isolated from the intestinal chyme of the large intestine of a healthy cow collected at the point of slaughter by serial dilution/direct plating and enrichment methods. Physicochemical parameters including; pH, temperature, moisture content, total solids, volatile solids and total suspended solids were analysed. The isolates were identified by standard microbiological test protocol (Gram reaction and biochemical tests) and screened for PAH degradation potential using the 2-6 dichlorophenol indophenol (DCPIP) redox dye, quantified by a UV_VIS spectrophotometer. The identity of the two isolates with the highest PAH degradation capacity after preliminary degradation tests, was confirmed following API20e tests analysis and 16S rRNA molecular analysis. The two isolates were used to inoculate carbon free Bushnell Haas medium containing the PAHs in single and combined cultures for the degradation tests. Samples were withdrawn at intervals of three days and analyzed for bacterial growth, pH, temperature, BOD, COD and changes in the concentration of the PAHs for 16 days. The optimum temperature, pH, concentration and nutrient supplementation for efficient degradation was analyzed following standard protocol and genes responsible for degradation identified.
The two test isolates selected after screening and identification were Escherichia coli and Klebsiella pneumoniae. HPLC/GCMS analyses showed that the concentration of Benzo[a]Pyrene declined by 84.8%, 91.04% and 96.44% by E. coli, K. pnuemoniae and a combination of both respectively after 16 days. The reduction in pyrene concentration was 89.36%, 90.98% and 97.76% after exposure to E. coli, K. puemoniae and a combination of both respectively while the decline of floranthene concentration stood at 86.4%, 90.3% and 92.3.7% after similar exposure to the test bacteria. ANOVA confirmed significant differences in the extent of the degradation of the PAHs by the test bacteria and their combined cultures (P<0.05). The growth of the isolates combined peaked at 1.98 log cfu/ml between days 10 and 13 during degradation of BaP. With respect to pyrene, it peaked at day thirteen with a log cfu value of 2.86 while in medium containing floranthene day 13 with a log cfu value of 3.23. The pH of medium adjusted to 7.0 declined in the three mediums with the least pH value for BaP, Pyrene and Flouranthene being 6.5, 6.6 and 6.7 respectively during degradation of the PAHs. Phthalate was the major degradation product in the course of degradation of the PAHs. The optimum temperature and pH conditions for the degradation of the PAHs was 35°C and pH 7, respectively while PAHs ≥ 1000 mg inhibited the growth of the test bacteria. Application of fertilizer (NPK) enhanced growth of the test bacteria and degradation of the PAHs. The genes associated with the degradation of PAHs in E. coli and K. pneumoniae were found to be alkane monooxygenase (alkB), Napthalene dioxygenase (NahAC) and Catechol dioxygenase (C230). It can be concluded that the intestine of Bos tarus harbor strains of bacteria that are capable of a high degree of degradation of PAHs; and that the consortium of the bacterial strains can be potentially useful for bioremediation of PAH-polluted environment.
