BIOREMEDIATION OF WASTEWATER FROM KADUNA REFINERY AND PETROCHEMICAL COMPANY (KPRC) USING IMMOBILISED BURKHOLDERIA CEPACIA AND CORYNEBACTERIUM KUTSCHERI
BIOREMEDIATION OF WASTEWATER FROM KADUNA REFINERY AND PETROCHEMICAL COMPANY (KPRC) USING IMMOBILISED BURKHOLDERIA CEPACIA AND CORYNEBACTERIUM KUTSCHERI
No Thumbnail Available
Date
2014-05
Authors
AJAO, Abdullahi Taiwo
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Refinery sludge and raw wastewater samples were collected from Kaduna Refining and
Petrochemical Company, Kaduna, Nigeria were collected and analyzed by selective enrichment
technique.This resulted in the isolation of two bacterial species, Burkholderia cepacia and
Corynebacterium kutscheri. Crude oil degradation was performed by the pure and mixed culture
of the isolates incubated in the shake flask culture. Dehydrogenase activitie (DHA), Fluorescein
diacetate (FDA) Protein Estimation, pH, Emulsification index and Optical density (OD600) were
used were used to evaluate degradation processes while CO2 production and GC-MS were used
to analyze mineralization potential.Growth, CO2 production, FDA and protein reached
maximum after 96h-192h of incubation while, DHA followed the same trend up to 162hours and
declined.This suggested that the microbial activities and their hydrolytic activities correlated to
biodegradation of Bonny light crude oil which is a function of CO2 production. The pH was
found to increase between 24h to 48h of incubation which corresponded to the period during
which maximum emulsification was recorded and subsequently decreased progressively.The
activities of all measured parameters between 24h-48h were not statistically significant (p˂
0.05). Activities of all the measured parameters in Burkholderia cepacia and Corynebacterium
kutsheri were characterized by flunctuations and also their activities not statistically different
from each other in both isolates (p˂ 0.05).This result correlated with GC-fingerprints that
showed disappearance of majority of hydrocarbons after 5days of incubation except a peak that
represented high molecular weight hydrocarbon that later broken down to low weight
hydrocarbons after 10days of incubation. Inspite of low catabolic potential of the axenic culture
of Burkholderia cepacia, it produced more of Catechol-2,3-dioxygenase that played a major role
in biodegradation of Bonny Light crude oil. Specifically, this enzyme cleaved aromatic rings of
ix
several environmental pollutants such as toluene, xylene, naphthalene and biphenyl derivatives.
Hence, the importance of Catechol-2, 3-dioxygenase and its role in the degradation of
environmental pollutants accounted for the prediction of the three-dimensional structure of
Catechol 2, 3-dioxygenase from Burkholderia cepacia. The 10ns molecular dynamics simulation
was carried out to check the stability of the modeled Catechol 2, 3- dioxygenase. The results
showed that the model was energetically stable, and it attained their equilibrium within 2000 ps
of production MD run. The docking of various petroleum hydrocarbons into the Catechol 2,3
dioxygenase revealed that the benzene, O-xylene,Toluene, Fluorene, Naphthalene, Carbazol,
Pyrene, Dibenzothiophene, Anthracene, Phenanthrene, Biphenyl made strong hydrogen bond and
Van der waals interaction with the active site residues of H150, L152, W198, H206, H220,
H252, I254, T255, Y261, E271, L276 and F309. Free energy of binding and estimated inhibition
constant of these compounds demonstrated that they were energetically stable in their binding
cavity. Chrysene shows positive energy of binding in the active site atom of Fe. Except Pyrene
all the substrates made close contact with Fe atom by the distance ranging from 1.67 to 2.43 Å.
In addition the substrates except pyrene made π-π stacking interaction with H252 by the distance
ranging from 3.40 to 3.90 Å. All these docking results revealed that, except Chrysene all other
substrate has good free energy of binding to hold enough in the active site and made strong VdW
interaction with Catechol-2,3-dioxygenase. These results suggest that, the enzyme is capable of
catalyzing the substrates. Standard methods were used to determine the physicochemical
parameters of raw refinery wastewater. The results obtained indicated that the mean values of
physicochemical parameters of raw refinery wastewater were statically significant (p˂ 0.05)..
Parameters such as pH, Total Suspended Solids, Biochemical Oxygen Demand, Chemical Oxygen
Demand, phosphate, Nitrate, sulphate, phenol, lead, were all above maximum permissible limit as
x
specified by Federal Environmental Protection Agency (FEPA) in raw wastewater. Mixed culture of
Burkholderia cepacia and Corynebacterium kutscheri isolated from refinery sludge immobilized
on agar-agar was used for bioremediation of refinery wastewater in a laboratory scale bioreactor.
Physicochemical parameters were determined before and after treatment.The results showed
that the test organisms significantly reduced all the measured parameters below permissible limit
except nitrates and sulphates (p˂ 0.05).. Each of the bacteria was screened for the presence of
plasmid DNA and for the involvement or otherwise of plasmid in the bioremediation of
wastewater.The gel electrophoresis resultof the isolated plasmid from Burkholderia cepacia and
Corynebacterium kutsheri suggested that the catabolic trait may have some relationship with
single plasmid detected in each of them. Further, investigation via curing of plasmids confirmed
that the plasmid is the ultimate source of catabolic traits. The plasmid was transferred to soil
derived mixed culture recipients in plate mating experiments and isolated transconjugant
colonies were immobilized and used for bioremediation process.The results caused the
multiplicative effect by achieving the same percentage achieved by donor cell after 20days of
treatment in10days which implied that Plasmid mediated bioaugmentation resulted in the most
rapid bioremediation of refinery wastewater. Therefore, bioaugmentation of wastewater using
transmissible catabolic plasmid will enhance efficiency of the bioremediation by spreading the
plasmid among indigenous microbial community either through horizontal gene transfer or
transformation.
Description
A DISSERTATION SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY IN PARTIAL FULFILMENT FOR THE AWARD OF Ph.D MICROBIOLOGY
DEPARTMENT OF MICROBIOLOGY, FACULTY OF SCIENCE, AHMADU BELLO UNIVERSITY, ZARIA.
Keywords
BIOREMEDIATION OF WASTEWATER,, KADUNA REFINERY AND PETROCHEMICAL COMPANY (KPRC),, IMMOBILISED BURKHOLDERIA CEPACIA ,, CORYNEBACTERIUM KUTSCHERI