Isolation and Characterization of a Microorganism in Crude Oil Effluent : Current School News

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Isolation and Characterization of a Microorganism in Crude Oil Effluent and its Utility in Metal Ion Removal/Recovery

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Isolation and Characterization of a Microorganism in Crude Oil Effluent and its Utility in Metal Ion Removal/Recovery.

ABSTRACT

Rapid industrialization and urbanization have resulted in the generation of large quantities of aqueous effluents, many of which contain high levels of toxic heavy metals and xenobiotics that pollute groundwater and the soil of affected farmlands.

Heavy metals are not biodegradable and as such not removed from the soil but rather accumulate and persist in soil reservoirs, consequently entering the food chain and exerting toxic effects on living organisms.

Copper and lead which exert toxic effects even at very low concentrations are common constituents of the Nigerian crude oil and consequently are found in its effluent. Research has shown that the removal/recovery of these metals (through bioaccumulation/biosorption by bacteria) is an attractive alternative to traditional physicochemical techniques.

Microorganisms tolerant to metals are often isolated from areas of high metal loading, suggesting that metal tolerance or resistance is an adaptive response to excessive metal exposure.

In this study, crude oil effluent was analyzed for copper and lead contents, and both metals were found to show concentrations higher than the U.S Environmental Protection Agency (EPA) and the Compendium of Environmental Laws for African Countries (CELAC) recommended environmentally accepted standards.

Microorganisms were isolated from the effluent and from the effluent-contaminated soil from the site. The largest/most successful colony was subsequently characterized.

Through morphological and biochemical tests, it was identified as Bacillus subtilis. Four test groups of mineral salt media containing copper only (Group A), lead only (group B), copper + lead (Group C), and no lead or copper (Group D, control) were set at different pHs of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 for each group were used.

The organism was standardized and found to contain 6.0 x 108 Bacillus subtilis cells per ml of suspension. Five ml of the organism was inoculated into each experimental medium.

The absorbance change (turbidity) of the mineral salt media was measured at 540nm on the 10th, 17th, and 24th days – the evaluation criteria for microorganism growth and adaptation in the used media. The experimental media showing the highest growths for each group was analyzed for residual copper and lead.

Also, the bacterial biomass from these media was harvested and analyzed for recovered lead and copper. Results showed that Group D had the highest growth, followed by Group B, Group A, and lastly Group C.

The organism grew most at pH 7.5 – 8.0. The experimental media that showed the highest growths for each group, when analyzed for residual copper and lead had no trace of metals, implying complete biosorption by the B.subtilis. B.subtilis is therefore recommended for removal of lead at pH 7.5 – 8.0 in crude oil pollution.

TABLE OF CONTENTS

PAGE
Title Page .. .. .. .. .. .. .. .. .. .. i
Certification .. .. .. .. .. .. .. .. .. .. ii
Dedication .. .. .. .. .. .. .. .. .. .. iii
Acknowledgement .. .. .. .. .. .. .. .. .. iv
Abstract .. .. .. .. .. .. ., .. .. .. v
Table of Content .. .. .. .. .. .. .. .. .. vi
List of Figures .. .. .. .. .. .. .. .. .. .. x
List of Tables .. .. .. .. .. .. .. .. .. .. xi
List of Abbreviations .. .. .. .. .. .. .. .. .. xii

CHAPTER ONE: INTRODUCTION

1.1 Introduction .. .. .. .. .. .. .. .. .. 1
1.2 Copper .. .. .. .. .. .. .. .. .. 2
1.2.1 Biological Role of Copper .. .. .. .. .. .. .. 3
1.2.2 Environmentally Acceptable limits for copper .. .. .. .. 3
1.2.3 Molecular Mechanism of Copper Toxicity .. .. .. .. .. 4
1.2.4 Copper Toxicity in Microorganisms .. .. .. .. .. .. 4
1.2.5 Copper Toxicity in Higher Organisms .. .. .. .. .. 5
1.3 LEAD .. .. .. .. .. .. .. .. .. .. 6
1.3.1 Environmentally Accepted Limits for Lead .. .. .. .. .. 6
1.3.2 Absorption .. .. .. .. .. .. .. .. .. 6
1.3.3 Distribution .. .. .. .. .. .. .. .. .. 6
1.3.4 Sources of exposure .. .. .. .. .. .. .. .. 7
1.3.5 Mechanisms of Lead Toxicity .. .. .. .. .. .. 7
1.3.6 The Haematological System .. .. .. .. .. .. .. 8
1.3.7 The Central Nervous System(CNS) .. .. .. .. .. .. 9
1.3.8 The Renal System .. .. .. .. .. .. .. .. 9
1.3.9 Symptoms .. .. .. .. .. .. .. .. .. 9
1.4 The Use of Chemicals in Heavy Metal Removal .. .. .. .. 10
1.5 The Use of Microorganisms in Heavy Metal Removal/Recovery .. .. 10
1.5.1 Microorganisms in Metal Absorption.. .. .. .. .. .. 12
1.5.1.1 Biosorption by Fungi .. .. .. .. .. .. .. .. 12
1.5.1.2 Bisorption by Algae and Moss .. .. .. .. .. .. 12
1.5.2 History of Bacterial Bisorption .. .. .. .. .. .. 13
1.5.3 Bacterial Structure and Mechanism of Bacterial Bisorption .. .. .. 13
1.5.3.1 Bacterial Structure .. .. .. .. .. .. .. .. 13
1.5.3.2 Mechanism of Bacterial Biosorption .. .. .. .. .. .. 15
1.6 Effect of pH on Bisorption .. .. .. .. .. .. .. 18
1.7 Immobilization of Microorganisms. .. .. .. .. .. .. 18
1.8 Optimization of Metal Ion Biosorption/Bioaccumulation.. .. .. .. 20
1.9 Aim of Research .. .. .. .. .. .. .. .. 21
1.10 Objectives of the Study .. .. .. .. .. .. .. 21

CHAPTER TWO MATERIALS AND METHODS

2.1 Materials .. .. .. .. .. .. .. .. .. 22
2.1.1 Crude Oil Effluent .. .. .. .. .. .. .. .. 22
2.1. 2 Soil Sample .. .. .. .. .. .. .. .. .. 22
2.1.3 Instruments/ Equipment .. .. .. .. .. .. .. 22
2.1.4. Chemicals/ Reagents .. .. .. .. .. .. .. .. 22
2.2 Method .. .. .. .. .. .. .. .. .. 23
2.2.1 Preparations of Solutions .. .. .. .. .. .. .. 23
2.2.2 Analysis of Crude Oil Effluent .. .. .. .. .. .. 24
2.2.2.1 Acidification of Sample .. .. .. .. .. .. .. 24
2.2.2.2 Preparation of Blank .. .. .. .. .. .. .. .. 24
2.2.2.3 Calibration of the Atomic Absorption Spectrophotometer (AAS) .. .. 24
2.2.2.4 Analysis of Effluent for Copper .. .. .. .. .. .. 25
2.2.2.5 Analysis of Effluent for Lead .. .. .. .. .. .. .. 25
2.2.3 Isolation of Microorganism .. .. .. .. .. .. .. 25
2.2.3.1 Microscopic Identification .. .. .. .. .. .. .. 26
2.2.3.2 Gram Staining .. .. .. .. .. .. .. .. .. 26
2.2.4 Motility Test .. .. .. .. .. .. .. .. .. 26
2.2.4.1 Preparation of Peptone Water .. .. .. .. .. .. 26
2.2.5 Catalase Test .. .. .. .. .. .. .. .. .. 27
2.2.6 Capsule Staining (The Hiss Staining Technique) .. .. .. .. 27
2.2.7 Standardization of Inoculums Using McFarland’s Standard .. .. .. 27
2.2.7.1 Preparation of 2.0 McFarland’s Standard .. .. .. .. .. 27
2.2.8 Preparation of Mineral Salt media (Broth) .. .. .. .. .. 28
2.2.8.1 Determination of Residual Copper and Lead Ions .. .. .. .. 28
2.2.9 Recovery of Accumulated/Sorbed Copper
and Lead Ions in the Microbial Biomass .. .. .. .. .. 29
2.2.9.1 Harvesting of Microbial Biomass .. .. .. .. .. .. 29
2.2.9.2 Determination of copper and lead ion concentrations in microbial biomass .. 29

CHAPTER THREE RESULTS

3 Results .. .. .. .. .. .. .. .. .. 30
3.1 Analysis of Crude Oil Effluent .. .. .. .. .. .. 31
3.2 Isolation of Microorganisms .. .. .. .. .. .. .. 32
3.3 Characteristics of the Microorganism .. .. .. .. .. 33
3.4 Growth Rate of Bacillus subtilis under
Different pH Conditions .. .. .. .. .. .. .. 34
3.4.1 Growth Rate in Mineral Salt Media Contaminated
With 8.64mg/l of Copper Salt.. .. .. .. .. .. .. 34
3.4.2 Growth Rate in Mineral Salt Media Contaminated
with 24.8mg/l of Lead Salt .. .. .. .. .. .. .. 36
3.4.3 Growth Rate in Mineral Salt Media Contaminated
with a Mixture of Copper and Lead Salts .. .. .. .. .. 37
3.4.4 Growth Rate in Mineral Salt Media without
Contamination with Copper or Lead Salts (Control) .. .. .. .. 38
3.5 Residual Copper and Lead Concentrations in the
Growth Media after 38 days of Incubation .. .. .. .. .. 40
3.6 Metal Recovery by the Microbial Biomass .. .. .. .. .. 41

CHAPTER FOUR DISCUSSION

4 Discussion .. .. .. .. .. .. .. .. .. 44
REFERENCES .. .. .. .. .. .. .. .. .. 47
APPENDICES .. .. .. .. .. .. .. .. .. 55

INTRODUCTION 

Toxic heavy metals in air, soil and water are growing threats to humanity. A number of these heavy metal compounds represent an ongoing eco-toxicological threat (Sag, 2000). Heavy metals have a tendency to bioaccumulate and end up as permanent additions to the environment.

For many of the heavy metals, the amounts contributed globally from anthropogenic sources, such as industrial wastes, now exceed those from natural sources (Deans and Dixon, 1992).

The disposal of effluent on land has become a regular practice for some industries leading to subsequent pollution of groundwater and farmlands. Copper (Cu), lead (Pb), mercury (Hg), cadmium (Cd) are common heavy metal pollutants at sites in which industrial waste effluents are discharged.

One good example of such effluents includes crude oil waste effluent. Crude oil effluent is the water that is mixed with crude oil when it is mined or during refining/processing. Crude oil effluent has been associated with increased concentrations of some heavy metals.

Disposal of such effluents over time in the environment may lead to eco-toxicological hazards. This is common where mining and manufacturing operations take place, particularly those established a number of years ago. Copper and lead, which are common constituents of Nigerian crude oil are known to exert toxic effects at low concentrations (Pandey et al 2007).

REFERENCES

 Alloway, B. J. (1995). Heavy metals in soils. 2nd ed. Chapman and Hall, Glasgow, UK. Pp 374- 379.
Al-Garru, S. M. (2005). Bisorption of lead by Gram negative capulated and non-capsulated bacteria. Water Science Technology 31(3):345-349
Amanchukwu, S. C., Obafemi, A. and Okpokwasili, G. C. (1989). Hydrocarbon degradation and utilization by a palmwine yeast isolate. FEMS Microbiology Letters 57: 151-154
Andrew, R. W. J. and Jackson, J. M. (1996). Pollution and waste management In: Environment and Human Impact. Longman Publishers Ltd. Pp 281-297
ASTM D 3559 (2000). Standard Test Methods for lead in water ASTM International,West siams
ASTM D1688 (2000). Standard Test Methods for copper in water ASTM International, West Cronshohocken, PA DOI: 10.1520/D1688-07 RO3E01, www.astm.org

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