BIODEGRADATION OF POLYAROMATIC HYDROCARBONS BY BACTERIA ISOLATED FROM THE INTESTINAL GUT LINING OF COW
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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 Samples were withdrawn at intervals of three days and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
XX
XXI
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.
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
XX
XXI
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.
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