ISOLATION AND IDENTIFICATIN OF BIODEGRADING FUNGAL SPECIES FROM PETROLEUM CONTAMINATED SOIL IN RURAL AREAS OF UMUAHIA

ABSTRACT

This study evaluated the isolation and identification of biodegrading fungal species. The sites (Umudike mechanic workshop and Imo lane mechanic workshop) used for this study are designated areas for automobile repairs, petrol/diesel pump sites and generator repairs. Samples collected were cultured on Sabouraud Dextrose Agar using both streak and pour plate techniques. Two (2) fungal species, namely Penicillium spp, and Rhizopus spp were isolated and identified based on Morphological and cultural features from engine oil–contaminated soil in this study as responsible for the biodegradation of the engine oil. The presence of these fungi in soil samples indicated that the isolates were able to exist in the oil contaminated environment while those that could not survive in this environment being eliminated by the unfavorable conditions caused by the oil. The results from the Zone Diameter Produced on Modified 1% Condemned Oil Agar showed that Penicillium spp had higher ability to degrade the engine oil as the day of incubation increased.  Rhizopus spp also exhibited the ability to utilize the engine oil as its sole source of energy at day 6 but had lesser zone of inhibition compared to Penicillium spp. Therefore, it was seen that the rate of engine oil degradation by Penicillium spp. was faster as the days increased compared to the rate of degradation of engine oil by Rhizopus spp and mixture of Penicillium spp. and Rhizopus spp. Therefore, the study has also shown that fungi play a role in the biodegradation of oil contaminating soils in the environment. Studies have indicated that biodegradation by fungi could be due to their massive growth and enzyme production responses during their growth phases. Thus in future they can be effectively utilized for the degradation of oil and petroleum products as well as for biodegradation of soil already polluted with oil.

TABLE OF CONTENTS

Title Page                                                                                                                                  i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgements                                                                                                                v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  ix

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aim of Study                                                                                                              4

1.2       Specific Objectives                                                                                                     4

CHAPTER TWO

2.0       Literature Review                                                                                                       5

2.1       Oil Pollution                                                                                                               5

2.2       Bioremediation Strategies Oil Contaminated Surfaces                                              6

2.3       Micro Organisms Important for Petroleum Product Biodegradation                         8

2.4       Factors Affecting Biodegradation of Hydrocarbon                                                    8

2.4.1    Temperature                                                                                                                9

2.4.2    Nutrients                                                                                                                     9

2.4.3    Oxygen                                                                                                                       9

2.4.4    pH                                                                                                                               10

2.5       Mechanism of Hydrocarbon Degradation                                                                  10

2.5.1    Degradation Pathway for Aliphatic Compounds                                                        10

2.5.2    Degradation Pathway for Aromatic Compounds                                                       10

2.6       Isolation and Identification of Some Micro Organisms from Polluted Soil with

            Diesel around Powered Generators                                                                            11

2.7       Isolation of Oil Degrading Microorganisms, Identification of Catechol

            Gene and Its Biogas Production                                                                                 12

2.8       Isolation Identification and Characterization of Potential Oil Degrading

            Bacteria from Oil Contaminated Sites                                                                       14

2.8.1    Enrichment of Microorganisms and Screening of Microorganisms                                    14

CHAPTER THREE

3.0       Materials and Methods                                                                                               16

3.1       Study Area                                                                                                                  16

3.2       Source of Sample/Collection                                                                                     16

3.3       Sterilization of Materials                                                                                            16

3.4       Media Preparation                                                                                                      16

3.5       Fungal Evaluation                                                                                                      17

3.6       Sample Preparation and Isolation of Fungi                                                                17

3.6.1    Sample Inoculation/Serial Dilution                                                                            17

3.6.2    Isolation and Culturing of Fungi                                                                                17

3.6.3    Isolation of Fungi                                                                                                       18

3.6.4    Purification of Fungi Isolates                                                                                     18

3.6.5    Test for Biodegradation of Fungi spp From Contaminated Soil of Spent Oil         18

3.7       Identification of the Fungi Isolates                                                                             19

3.8       Morphological Identification                                                                                     19

3.8.2    Colonial Morphology                                                                                                 19

3.8.1    Cultural Characteristics                                                                                              19

CHAPTER FOUR

4.0       Results                                                                                                                        20

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendations                                                         23

5.1       Discussion                                                                                                                   23

5.2       Conclusion                                                                                                                  25

5.3       Recommendation                                                                                                       25

            References

LIST OF TABLES

CHAPTER ONE

1.0     INTRODUCTION

In Nigeria, it is common among motor mechanics to dispose spent engine oil into gutters, water drains and soil (Okonokhua et al., 2007). Used engine oil is defined as used lubricating oils obtained after servicing and subsequently draining from automobile and generator engines. Spent oils contain high percentage of aromatic and aliphatic hydrocarbons, nitrogen and sulphur compounds and metals (Mg, Ca, Zn, Pb) than fresh oils. These metals are introduced into the oil as a result of wear and tear of the engine (Mohd et al., 2011). Used engine oil causes great damage to soil and soil microflora. It creates unsatisfactory condition for life in the soil due to poor aeration, immobilization of soil nutrients and lowering of soil pH (Ugoh and Moneke, 2011). It has been shown that marked changes in properties occur in soil contaminated with hydrocarbon; this affects the physical, chemical and microbiological properties of the soil (Okonokhua et al., 2007). At low concentrations, some of these heavy metals are essential micronutrients for plants, but they can cause metabolic disorders and growth inhibition when the concentration is high. Therefore, there is the need for bioremediation of hydrocarbon contaminated soil.

Engine oil could also be simply referred to as a thick mineral liquid applied in a machine or engine so as to reduce friction between the moving parts of the machine (Whitefield, 2002). Used engine oil as the name implies represent oil that has undergone destructive changes in property when subjected to oxygen, combustion gases and high temperature. The said oil also undergoes viscosity change as well as additive depletion and oxidation, these occur to degrade oil (Mark et al., 2002).

Microorganisms such as fungi are extremely diverse and can adapt to survive in inhospitable environments. Microbes are capable of breaking down many complex molecules by adaptation of their degradative enzyme system (Boonchan et al., 2000). The survival of these microbes in altered environment depends on their ability to produce endospore and vegetative cell, which can stand harsh and unfavorable environment. These changes affect plants adversely and the amount of damage depends on the size of the area involved and the degree of saturation by the pollutant (Isinguzo and Bello, 2005).

Used oil is defined by the US Environmental Protection Agency as oil that has been refined from crude oil or any synthetic oil; that has been used and as a result of such use, it has been contaminated by chemical impurities which contribute to chronic hazards; including: mutagenicity and carcinogenicity as well as environmental hazard with global ramifications (Blodgette, 2001). Bioremediation has become an alternative way to remedy oil polluted sites, where the addition of specific microorganism (bacteria, cyanobacteria, algae, fungi, protozoa) or enhancement of microorganism already present, can improve biodegradation efficiency (Hagwell et al., 1992). These microorganisms can degrade a wide range of target constituents present in oil sludge (Mishra et al., 2001). A large number of pseudomonas strains capable of degrading polycyclic aromatic hydrocarbons have been isolated from soil (Johnson et al., 1996).

Other petroleum hydrocarbon degraders include Yokenella spp., Alcaligenes spp., Roseomanas spp., Sreanotrophomanas spp., Acinetobacter spp., Flavobacter spp., cyanobacterium spp., capnocytophage spp., Moraxella spp. and Bacillus spp. (Bhattacharya et al., 2002). Other microorganism such as fungi is also capable of degrading the hydrocarbons in engine oil to a certain extent. However, they take longer period of time to grow compared to their bacterial counterparts (Prenafeta-Boldu et al., 2001). Petroleum products such as engine oil, petrol, diesel and kerosene are used daily in various forms in mechanic workshops. These products tend to harden and change the colour of the soil, which may have untold health hazard on the technicians and artisans. Their soles tend to harden, which may alter their movement.

Soil contamination is the presence of unwanted impure materials from human activities. It can also be the distortion of the soil environment by human activities. Soil is the habitat for variety of organisms, including; fungi, bacteria, protozoa, insects, nematodes, worms, and many other animals. Viruses are also present in soils. This complex biological community contributes to the formation, maintenance, and in some situations, the degradation and disappearance of soils (Prescott et al., 2005).

Biodegradation plays an important ecological role as it contributes to bioremediation. Nowadays, the role of fungi has been extensively studied to be the most potential degraders of oil and petroleum products than other traditional bioremediation techniques (Sood and Lal, 2009). Filamentous fungi are considered to be better degraders of oil and petroleum  than bacteria, because they can degrade high molecular weight polycyclic aromatic hydrocarbons whereas bacteria degrade smaller molecules (Whang et al., 2009). Another reason which enables them as good potential agents of degradation is to produce extracellular enzymes for the digestion of complex carbohydrates which further causes the degradation of hydrocarbon pollutants (Whang et al., 2009). Another advantage is that they can easily be grown in fermenters for large scale productions. Besides all these, the separation of fungal biomass is easy by filtration; due to its filamentous structure. They also have capability to under environmentally stress conditions like low pH, poor nutrition, low water activity (Whang et al., 2009) and are less sensitive towards variations in aeration and temperature. Raipur, the capital city of C.G state is now considered to be the third worst city in India on the list of top twenty polluted cities in the world, when it comes to air pollution. Central pollution control board (CPCB) recently declared it as the country’s most polluted city. Several factors are responsible to make it as the most polluted city among which higher concentration of PAHs in the air may be one. The utilization of bioremediation techniques through microorganisms, to clean up pollutants is viable and has economic values (Mnif et al., 2009). Microorganisms possess enzymatic systems for degradation and utilization of oil as a source of carbon and energy (Ilori et al., 2008). Growth and proliferation of oil utilizing microorganisms in oil contaminated soil is greatly influenced by the presence of nutrients and their hydrocarbonoclastic property (Chakraborty and Mukherji, 2010).

1.1       AIM OF STUDY

To isolate and identify biodegrading fungi species from used engine oil contaminated soil in rural areas

1.2       SPECIFIC OBJECTIVES

1.     To isolate fungi species present in used engine oil contaminated soil.

2.     To identify and characterize fungal isolates using colonial morphology and microscopic appearance.

3.     To determine the oil degrading ability of the fungi species.

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