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Assessing the Capacity of Wild and Mutant Strains of Bacillus Subtilis and Pseudomonas Putida Isolated from Refinery Effluent in the Degradation of Hydrocarbons.

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Assessing the Capacity of Wild and Mutant Strains of Bacillus Subtilis and Pseudomonas Putida Isolated from Refinery Effluent in the Degradation of Hydrocarbons.


Petroleum refinery effluents are characterized by the presence of pollutants such as hydrocarbons as such could be of serious environmental consequence if discharged into receiving sites without proper treatment to remove the pollutants.

This study assessed the capacity of wild and mutant strains of Bacillus subtilis and Pseudomonas putida isolated from refinery effluent in the degradation of hydrocarbons present in the effluent.

The physicochemical parameters of the raw and treated effluent samples collected from Kaduna Refinery and Petrochemical Company (KRPC) were determined using standard guidelines.

With the exception of turbidity (35.3 and 18.2 NTU), BOD (190 and 29.6 mg/L), COD (351.2 and 78.1 mg/L) and Oil and Grease (45.2 and 17.9 mg/L) for the raw and treated effluent respectively, and conductivity (695 µS/cm) for the raw effluent, all other parameters were within the permissible limits set by FMENV.

Six (6) and eleven (11) isolates of Bacillus subtilis and Pseudomonas putidarespectively were isolated from the effluent and screened for capacity to utilize and grow on mineral medium containing different concentrations (0.5%, 1%, 1.55% and 2%) of crude oil as the sole source of carbon.

Isolates TE8 (B. subtilis) and TEC10 (P. putida) had luxuriant growth across the first three concentrations of crude oil and medium growth on medium containing 2% crude oil.

Both isolates were treated with nitrous acid and UV- irradiation to generate mutants. Death rate of 59.67%(40.33% survival) and 66.33% (33.67% survival) were observed for Bacillus subtilis and Pseudomonas putida respectively on treatment with nitrous acid.
Also death rate of 51.67% (48.33% survival) and 40% (60%) were observed for Bacillus subtilis and Pseudomonas putida respectively on exposure to UV- irradiation.


Declaration …….. i
Certification ……… ii
Acknowledgements . iii
Dedication …….. iv
Abstract ….. v
Table of Contents …… vi
List of Figures …. x
List of Tables ….. xi
List of Appendices …. xii
List of Abbreviations, Glossary and Symbols .. xiii


1.1 Background to the Study ……. 1
1.2 Statement of Research Problem ……… 3
1.3 Justification for the Research ……….. 4
1.4 Aim and Objectives ……… 5
1.4.1 Aim ……… 5
1.4.2 Objectives …….. 5



2.1 Petroleum Hydrocarbons …… 6
2.1.2 Petroleum hydrocarbon pollution. …… 10
2.1.3 Effect of petroleum hydrocarbons on living matter. …..13
2.2 Effects of some physico-chemical parameters on water quality and Aquatic life …. 17
2.2.1 pH …….. 17
2.2.2 Temperature ………… 18
2.2.3 Total dissolved solids. 20
2.2.4 Total suspended solids ….. 20
2.2.5 Turbidity ……… 21
2.2.6 Conductivity ………. 22
2.2.7 Biological oxygen demand … 23
2.2.9 Nitrates and phosphates .. 25
2.2.10 Sulphides …. 26
2.3 Biodegradation and Bioremediation …. 27
2.3.1 Bioremediation ……. 28
2.3.2 Mechanism of Oil Biodegradation by Bacteria ………. 30
2.3.3 Enzymes Participating in Degradation of Hydrocarbons.. 33
2.3.4 Factors affecting bioremediation .. 34
2.3.5 Types of bioremediation …..37
2.3.6 Genera of bacteria involved in hydrocarbon degradation ..40
2.3.5 Recent strategies for bioremediation …… 41



3.1 Collection of Refinery effluent Samples … 43
3.2 Determination of the Physico-chemical Properties of the Raw and Treated Refinery
Effluents ….43
3.2.1 pH and temperature ….. 43
3.2.2 Electrical conductivity ….. 44
3.2.4 Total suspended solids …. 44
3.2.5 Dissolved oxygen and biochemical oxygen demand …… 45
3.2.6 Nitrates …. 45
3.2.7 Sulphates ……. 45
3.2.8 Phosphates ……… 46
3.3 Microbiological Investigations .. 47
3.3.1 Isolation of Bacillus subtilis and Pseudomonas putida from the raw refinery effluent … 47
3.4 Standardization of inoculum ….. 52
3.5 Screening the Capacity of Bacillus subtilis and Pseudomonas putida to Degrade Hydrocarbons. ……. 52
3.6 Induction of Mutation ….. 53
3.6.1 Induction of mutation using ultraviolet (UV) irradiation. ……… 53
3.6.2 Induction of mutation using nitrous acid ……. 53
3.7 Determination of Hydrocarbon Degrading Capacity of the Wild and Mutant Bacillus subtilis and Pseudomonas putida … 54

3.8 Statistical Analysis of Data .. 55


4.0 RESULTS ……. 56
4.1 Physicochemical Parameters of Raw and Treated Refinery Effluent ………………….. 56
4.2 Isolation and Characterization of Bacillus subtilis and Pseudomonas putida from the Raw and Treated Refinery Effluent ……. 56
4.3: Crude Oil Utilization by the Isolates of Bacillus subtilis and Pseudomonas putida 62
4.4: Nitrous Acid and UV- Light Modification of Selected of Bacillus subtilis and Pseudomonas putida Strains … 65
4.5 Evaluation of the Hydrocarbon Degradation potential of the Wild and Mutant Bacillus subtilis and Pseudomonas putida… 68
4.5.1 Growth Pattern …… 68
4.5.2 Oil and Grease Degradation ………. 72


5.0 DISCUSSION ….. 77


6.1 Conclusion ….. 87
6.2 Recommendations ….. 87
APPENDICIES ………………. 104


Background to the Study

Petroleum refinery and petrochemical industries play an immense role in national development and improved quality of life. However, pollution effects of the wastes from these industries are causes for worry (Nwaichi et al., 2013).

Wastewater (effluents) released from petroleum refineries are characterized by the presence of large quantities of petroleum products, polycyclic and aromatic hydrocarbons, phenols, metal derivatives, surface active substances, sulphides, naphthylenic acids and other chemicals (Musa et al., 2015.,

Most of which are known to be mutagenic, carcinogenic and growth inhibitory and by extension can have adverse effects on the ecology of the receiving sites and public health (Nwaichi et al., 2013). Petroleum refinery effluents contain organics and have a characteristic oily nature.

Hence, when discharged into water bodies cause depletion of dissolved oxygen (due to the transformation of the organic components into inorganic compounds by microorganisms), prevent reaeration thereby causing the loss of biodiversity through a decrease in amphipod population that is important in food chain and eutrophication (Nwaichi et al., 2013).


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