ISOLATION AND IDENTIFICATION OF MICROORGANISMS FROM ENGINE OILS

ABSTRACT

This study investigated the isolation of microorganisms from used and unused engine oil samples. The major bacterial isolates from the engine oil samples belong to Bacillus sp., Staphylococcus aureus, Escherichia coli and Pseudomonas sp. respectively, whereas the fungal isolates includes Aspergillus niger, Aspergillus flavus, and Penicillium sp. The total heterotrophic plate count (THPC) for the used and unused engine oils ranged from 1.4 × 105cfu/ml to 4.4 × 105cfu/ml. The total coliform plate count (TCPC) of the used and unused engine oils ranged from 1.4 × 105cfu/ml to 4.1 × 105cfu/ml. The total fungal plate count (TFPC) of the used and unused engine oils ranged from 1.5 × 105cfu/ml to 4.0 × 105cfu/ml.It was observed that Pseudomonas sp. is the most frequently occurring bacteria isolate with a percentage occurrence of (35.0%) whereas Escherichia coli had the least percentage occurrence of (10.0%). Aspergillus niger is the most frequently occurring fungal isolate with a percentage occurrence of (41.7%) whereas Aspergillus flavus has the least percentage occurrence of (25.0%). Finding feasibility in utilization of engine oil by microbial groups revealed that the isolates utilized these oily materials. The isolates showed different degrees of turbidity with the highest turbidity showed in the used engine oil sample for some of the tested strains. Engine oils are prone to deterioration when they are exposed to microbial activities, especially when they are in-use as shown from the study. Therefore microbial growths are proxies for deterioration in engine oils. The introduction of preservatives, additives and biocides in the engine oil could prevent microorganisms from infesting the engine oil thereby increasing its shelf life and preventing the engine parts from touching each other.

TABLE OF CONTENTS

Title Page                                                                                                                      i

Certification                                                                                                               iii

Dedication                                                                                                                  iv

Acknowledgement                                                                                                      v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              ix

Abstract                                                                                                                      x

1.0       CHAPTER ONE                                                                                                       1

1.1       Introduction                                                                                                                1

1.2       Types of Engine Oil                                                                                                   2

1.3       Advantages of Engine Oil                                                                                          4

1.4       Disadvantages of Engine Oil                                                                                      5

1.5       Factors Influencing Engine Oil Biodeterioration                                                       7

1.6       Aims and Objectives                                                                                                  11

2.0       CHAPTER TWO                                                                                                      12

2.1       Literature Review                                                                                                       12

2.1.1    Assessment of Bacterial and Fungal Deteriogens of Selected Lubricating

Oils Used In Industrial Generators                                                                             12

2.1.2    Biodegradation of Used Engine Oil Using Mixed and Isolated Cultures                      14

2.1.3    A Comparative Study of Oil Degradation with Used and Unused Engine

Oil by Microbes Isolated from Water Sample of Mechanic Workshops                 16

2.2       Microorganisms Responsible For Engine Oil Biodeterioration                                    18

2.2.1    Aerobic microorganisms                                                                                            18

2.2.2    Anaerobic microorganisms                                                                                        19

3.0       CHAPTER THREE                                                                                                  20

3.1       Materials and Method                                                                                                 20

3.2       Study Area                                                                                                                  20

3.3       Collection of Samples                                                                                                20

3.4       Sterilization of Materials                                                                                            20

3.5       Preparation of Culture Media                                                                                     21

3.6       Culturing of Used and Unused Engine Oil Samples                                                  21

3.7       Inoculation and Isolation                                                                                            21

3.8       Purification of Isolates                                                                                               21

3.9       Identification of Bacterial Isolates                                                                             22

3.9.1    Gram Staining                                                                                                            22

3.9.2    Biochemical Test                                                                                                        22

3.9.2.1 Indole test                                                                                                                   22

3.9.2.2 Methyl red (MR)                                                                                                        23

3.9.2.3 Voges proskauer (VP)                                                                                                23

3.9.2.4 Hydrogen sulphide test (H₂S)                                                                                     23

3.9.2.5 Citrate test                                                                                                                   23

3.9.2.6 Urease test                                                                                                                  24

3.9.2.7 Catalase test                                                                                                                24

3.9.2.8 Coagulase test                                                                                                             24

3.9.2.9 Sugar fermentation test                                                                                               24

3.9.2.10 Starch test                                                                                                                 25

3.10     Identification of Fungal Isolates                                                                                 25

3.11     Growth of the Mixed Culture of the Bacterial and Fungal isolates in the

Engine Oil                                                                                                                   25

4.0       CHAPTER FOUR                                                                                                    27

4.1       Results                                                                                                                        27

5.0       CHAPTER FIVE                                                                                                      42

5.1       Discussion, Conclusion and Recommendation                                                          42

5.1.1    Discussion                                                                                                                   42

5.1.2    Conclusion                                                                                                                  46

5.1.3    Recommendation                                                                                                       46

References                                                                                                                  47

Appendix                                                                                                                    51

LIST OF TABLES

LIST OF FIGURES

1.0                                                                   CHAPTER ONE

1.1       INTRODUCTION

Engine oil is a substance comprising base oils enhanced with particularly antiwear additive plus detergents, dispersants and, for multi-grade oils viscosity index improvers. Engine oil is used for lubrication of internal combustion engines. The main function of Engine oil is to reduce friction and wear on moving parts and to clean the engine from sludge (one of the functions of dispersants) and varnish (detergents). It also neutralizes acids that originate from fuel and from oxidation of the lubricant (detergents), improves sealing of piston rings, and cools the engine by carrying heat away from moving parts (Miller et al., 2005).

Unused engine oil is a complex mixture of hydrocarbons and other organic compounds, including some organo metallic constituents that are used to lubricate the parts of an automobiles engine, in order to keep everything running smoothly while used engine oil contains more metals and heavy polycyclic aromatic hydrocarbons (PAHs) that would contribute to chronic hazards including mutagenicity and carcinogenicity. In addition, polycyclic aromatic hydrocarbons (PAHs) have a widespread occurrence in various ecosystems that contribute to the persistence of these compounds in the environment (Van-Hamme et al., 2003). The illegal dumping of used motor oil is an environmental hazard with global ramifications (Blodgette, 2001).

Engine oil is a lubricant used in internal combustion engines, which power engine-generators, and many other machines. In engines, there are parts which move against each other, and the friction wastes otherwise useful power by converting the kinetic energy to heat. It also wears away those parts, which could lead to lower efficiency and degradation of the engine. This increases fuel consumption and decreases power output and can lead to engine failure.

Engine oil creates a separating film between surfaces of adjacent moving parts to minimize direct contact between  them, decreasing heat caused by friction and  reducing wear, thus protecting the engine. In use, engine oil transfers heat through conduction as it flows through the engine. In an engine with a recirculating oil pump, this heat is transferred by means of airflow over the exterior surface of the [oil pan], airflow through an oil cooler and through oil gases evacuated by the Positive Crankcase Ventilation (PCV) system. While modern recirculating pumps are typically provided in passenger cars and other engines similar or larger in size, total loss oiling is a design option that remains popular in small and miniature engines.

Coating metal parts with oil also keeps them from being exposed to oxygen, inhibiting oxidation at elevated operating temperatures preventing rust or corrosion. Corrosion inhibitors may also be added to the motor oil. Many motor oils also have detergents and dispersants added to help keep the engine clean and minimize oil sludge build-up. The oil is able to trap soot from combustion in itself, rather than leaving it deposited on the internal surfaces. It is a combination of this, and some singeing that turns used oil black after some running. Engine oil may also serve as a cooling agent. In some constructions, oil is sprayed through a nozzle inside the crankcase onto the piston to provide cooling of specific parts that undergo high-temperature strain. On the other hand, the thermal capacity of the oil pool has to be filled, i.e. the oil has to reach its designed temperature range before it can protect the engine under high load.

1.2       TYPES OF ENGINE OIL

Determining the type of engine oil for your generator whether synthetic, synthetic blend, high-mileage or conventional oil, depends on several factors. Some are external factors, such as the climate you live in, your driving habits, or even the age of your engine. Other factors are fixed based on your generator's engine type and the manufacturer's specifications.

·       Full Synthetic Engine Oil

Full synthetic oil is ideal for generators that demand peak level performance and high levels of lubrication. Full synthetic oil provides higher viscosity levels, resistance to oxidation and thermal breakdown, and helps fight against oil sludge. Plus, it helps improve fuel efficiency and can even increase a generator’s horsepower by reducing engine drag (Miller et al., 2005). Because synthetic generator oil can cost two to four times more than regular oil, talk to your technician about whether it’s the right oil for your generator. If you live in a climate with super cold winters or very hot summers, synthetic oil may be the best type of oil for your generator. Older engines could also benefit from synthetic oil, as it can help prevent harmful sludge build-up that some older engines seem to be prone to.

·       Synthetic Blend Engine Oil

Synthetic blend oil offers the best of both worlds. It has many of the characteristics of full synthetic oil, but at a much lower price. This type of oil is a mixture of synthetic and conventional base oils, plus some additives, for extra resistance to oxidation and excellent low-temperature properties. Synthetic blends make it easy for generator users to make the switch from conventional to synthetic oil, which is why this type of engine oil is becoming increasingly popular among today’s savviest users. It’s also a great middle ground for generator users who want the added protection and performance of synthetic oil, but might not be ready to foot the bill for a total switch to full synthetic oil.

·       Conventional Engine Oil

Conventional oil is the most commonly used type of engine oil. It is ideal for light-duty, late-model generators with low to average mileage and a simple engine design.

·       High Mileage Engine Oil

High mileage engine oil is specifically designed for cars with more than 75,000 miles. This type of oil can help reduce oil consumption, minimize leaks and oil seepage, and can also help reduce smoke and emissions in older engines.

1.3       ADVANTAGES OF ENGINE OIL

When it comes to longer and healthier life of your generator engine, engine oil plays a vital role in it. High quality oil is important and a fundamental characteristic of your generator depends on it like smooth running, fuel efficiency, performance and emissions. Below are the major advantages of good engine oil (both Synthetic and Mineral) for your generator.

·       Better Lubrication

Engine Oil’s first and foremost task is to lubricate the running parts, the engine components are exposed to grueling temperatures and are tend to gradual wear and tear. However, having a good engine oil and timely change helps in appropriate lubrication of parts and in-turn smooth and quieter running of the engine.

·       Cleaner Engine

Apart from lubrication, engine oil also clean internals of your generator engine; as mentioned above, working engine parts are susceptible to wear and tear. The microscopic debris and particles are removed by the oils and stops them from building up. But over the time, engine oil will get dirty and will not work as indicated. In a nutshell, timely change is immensely important along with the oil filter. And as these oils come with extra additives, it helps in working as detergents in the cleaning activity. So, higher the quality of oil, the cleaner your engine will be.

·       Improved Fuel Efficiency and Performance

As engine oils helps in keeping the engine clean and in top nick, the generator extracts the best fuel efficiency as marketed by the respective automaker. However, the same things also apply here, regular and cheap oil might fulfill the purpose but your generator can't really benefit from the good stuff. Moreover, smoother running of the engine and cleaner internals also maximizes the mechanical output of the generator.   

·       Maximum Engine Life

Engine’s life is no where less nowadays compared to yesteryears, but they are rather plagued with wear and tear. But having a good engine oils helps in maximizing the life of your engine. As with better additives, it helps in minimizing the sludge and keeps the internals clean and well lubricated.

·       Lower Engine Emissions

Emission norms are getting strict day by day and engine oils keeps the generator running in healthy state and in-turn, a cleaner exhaust. Moreover, older generators sometimes burn the dirty oil, causing an excess of harmful engine emissions. Fresh and good engine oil is less likely to burn and has better ability to absorb particles and engine by-product emissions, making your generator pollute much less.

1.4       DISADVANTAGES OF ENGINE OIL

Due to its chemical composition, worldwide dispersion and effects on the environment, used engine oil is considered a serious environmental problem. Most current engine oil lubricants contain petroleum base stocks, which are toxic to the environment and difficult to dispose of after use. Toxic effects of used engine oil on freshwater and marine organisms vary, but significant long-term effects have been found at concentrations of 310 ppm in several freshwater fish species and as low as 1 ppm in marine life forms (Vijayendran, 2014). Engine oil can have an incredibly detrimental effect on the environment, particularly to plants that depend on healthy soil to grow. There are some ways in which engine oil affects humans and plants:

·       Contaminating water supplies, contaminating soil, and poisoning plants.

·       Used engine oil dumped on land reduces soil productivity.

·       Improperly disposed used oil ends up in landfills, sewers, backyards, or storm drains where soil, groundwater and drinking water may be contaminated (Vijayendran, 2014).

The consumption of engine oil in Nigeria has been on the increase in recent years due to the upsurge in the number of vehicles, power plants, and generators that make use of these lubricants (Odjegba and Atebe, 2007). This directly affects the rate at which spent engine oil enters and pollutes the environment. The indiscriminate disposal of this waste oil increases pollution incidents in the environment (Odjegba and Atebe, 2007). Some of the pollution effects of used oil in the environment include: reduction in oxygen supply to microorganisms, pollution of ground and surface waters and accumulation of metal ions which are toxic to plant. Bioremediation process is a microorganism mediated transformation or degradation of contaminants into non-hazardous or less hazardous substances. It is an attractive approach for cleaning up of hydrocarbons from environment because it is a simple technique, easy to maintain, applicable over large areas, cost effective and leads to complete destruction of the contaminant (Achal et al., 2011). Main reason for this concept is that the majority of the molecules in engine oil are biodegradable. Many physical, chemical and environmental factors like temperature, nutrients, oxygen, biodegradability, photoxidation, bioavailability, soil moisture, soil acidity and alkalinity etc. affect the process of biodegradation of hydrocarbons (Rahman et al., 2003).

Bacteria and fungi are known to be the principal agents of biodegradation of hydrocarbons. Fungi have a higher tolerance to the toxicity of hydrocarbons due to their physiology and adaptation to such variations in the environment and have the mechanism for the elimination of spilled oil from the environment. Fungi have been found to be better degraders of petroleum than traditional bioremediation techniques with bacteria (Ojo, 2005). Fungi have also demonstrated the ability to degrade and mineralize phenols, halogenated phenolic compounds, petroleum hydrocarbons, polycyclic aromatic compounds and polychlorinatedbiphenyls. Many researchers studied the role of fungi in biodegradation process of petroleum products and the most common fungi which have been recorded as biodegraders belong to the following genera: Alternaria, Aspergillus, Candida, Cephalosporium, Cladosporium, Fusarium, Geotrichum, Gliocladium, Mucor, Pleurotus, Paecilomyces, Penicillium, Polyporus, Rhizopus, Rhodotorula, Saccharomyces, Talaromyces and Torulopsis (Adekunle and Adebambo, 2007). The advantages associated with fungal bioremediation are primarily in the versatility of the technology and its cost efficiency compared to other remediation technologies such as incineration, thermal desorption and extraction. The use of fungi is expected to be relatively economical as they can be grown on a number of inexpensive agricultural or forest wastes such as corncobs and sawdust. More so, their utilization is a gentle non-aggressive approach. Increased proliferation of automobile workshops within Nigeria has contributed markedly to the problem of soil contamination and this has resulted in the concomitant exposure of the surrounding soil within the vicinity of these workshops to high levels of spent engine oil and lubricating oils.

1.5 FACTORS INFLUENCING ENGINE OIL BIODETERIORATION

The composition of engine oil, the presence of accessible forms of nitrogen, phosphorus, potassium, magnesium, other micro-elements and water as well as other environmental conditions such as temperature, pH and oxygen modulate microbial growth and thus biofouling processes. As a general rule, all the listed chemical species are present either in the oil or in the accompanying water phase including the water dissolved in the oil.

·       Temperature

Microorganisms promoting fouling of engine oil can live in a wide range of temperatures—from 4 up to 60^oC and above (Chung et al., 2000), The species variety of Hydrocarbon oxidizing (HCO) microorganisms is highest at temperatures between 25⁰C and 30⁰C.

·       pH

At pH value from 4 up to 9, however, microorganisms tend to prefer a neutral pH (Boszczyk-Maleszak et al., 2006).

·       Water content

Microorganisms are capable of surviving at elevated temperatures and in presence of toxic substances, but are unable to live without water. It is well known that 1% water is enough for substantial microbial growth, whereas spores of microorganisms can survive in the presence of 5–80 ppm of water in fuel system. It is interesting, that for a 1lm size microorganism, 1 mm layer of water is comparable to a man standing next to 500 m of water. Therefore, a fine film of water on tank surface is enough to allow microorganisms to start growing, and the cell metabolism, once begun, causes accumulation of more water. Thus, the important factor limiting growth of microorganisms in oil is the availability of water. For example, anti-ice fuel additives such as glycols reduce the availability of water and thus inhibit growth of microorganisms. Water penetrates into fuel systems with moist air condensing on cold metal and also with watered fuel, when water is pumped as ballast into ships (Gardner and Stewart, 2002). Water is heavier than Hydrocarbon-fuel and consequently it accumulates at the bottom where the biphasic system ‘‘oil-water’’ supports a growth of microorganisms which can use oil as a carbon source (Muthukumar, 2003). In addition to the acceleration of oil biofouling, water, present in fuels, reduces their viscosity and renders pumps ineffective. Generally, it is extremely difficult to avoid the occurrence of water in tanks, as it is impossible to avoid the condensation phenomenon under conditions of changing temperatures.

·       Oxygen

Oxygen penetrates in storage tanks during fuel filling, ventilation of tanks, purification and processing of Hydrocarbon (HC) raw material. Oxygen can (photo) chemically reacts with Hydrocarbons (HCs) of oil and oil products with formation of coloured particles, pitches and water. Moreover, oxygen being a terminal electron acceptor for aerobic microorganisms directly contributes to microbial growth. A variety of microorganisms carrying out the decomposition of Hydrocarbons (HCs), the formation of slime, biofilms and insoluble particles, are aerobic. The rate of microbial Hydrocarbon (HC) oxidation obviously increases with an increase of aeration but, even at concentration of oxygen as low as 0.1 mg/l, conversion of Hydrocarbon (HC) still occurs (Watanabe et al., 2002). Moreover, if it would be possible to create completely anaerobic storage conditions, oil and oil products are not protected against microbial degradation since many facultative aerobic and anaerobic microorganisms continue to thrive (Watanabe et al., 2002;). For example, during the storage of engine oil, Sulphate reducing bacteria (SRB) consuming Hydrocarbon (HC) and using available sulphate as electron acceptor actively grows there.

·       Nutrients

The limiting factor of microbial growth is also an availability of mineral nutrients, for example, phosphates, which are usually present in fuels in concentrations as low as 1 mg/l. On the contrary, significant growth of microorganisms has been observed in systems containing solution of mineral salts, for example, mineralized flooding water to enhance oil extraction or seawater which is pumped into tankers as ballast. The especial danger of these waters is related to abundant presence of sulphate which triggers the growth of Sulphate reducing bacteria (SRB) enhancing biodeterioration of oil as discussed above. The corrosion caused by microorganisms in the presence of water promotes a destruction of tank walls and the influx of metal ions into oil and oil products (Gardner and Stewart, 2002; Muthukumar,, 2003). Thus, corrosion processes may supply metal ions which are required for the growth of microorganisms.

·       Chemical Composition

The chemical composition of engine oil and oil products also influences their susceptibility to biodegradation. The so called light oil, with mainly moderate chain aliphatic Hydrocarbons (HCs) and low content of aromatic Hydrocarbons (HCs), is more quickly infected by microorganisms compared to high-aromatic oils. For example, the light petroleum from Groznensky and Borislavsky oil fields (Caucasus) was heavily affected by microorganisms within 7–10 days whereas that from Anastasyevsky oilfield (Western Siberia) with an aromatic content up to 50% remained unaffected during 90 days of its storage under the same conditions. Moreover, during transport of light oils using pipelines, the intensive growth of microorganisms enhances sedimentation of paraffin on pipeline walls. Similarly, the so called sour oil having high sulphur content is more susceptible to microbial infection and degradation compared to the so called sweet oil with low sulphur content. Probably, the absence of sulphur deficiency in the former oil promotes its biofouling. Products of microorganism metabolism in diesel lead to clogging of fuel nozzles and finally to the breakage of the engine. Solid and liquid lubricants (technical vaselines, rope and gun oil) made of petroleum Hydrocarbons (HCs) are readily affected by fungi, mainly mould fungi, and bacteria during operation and storage in unprotected containers (Eisentraeger et al., 2002). The solid lubricants are usually contaminated by microorganisms only in a superficial film whereas the liquid ones are infected along the full layer. Machine oils produced from petroleum of various oilfields have a different stability to microbial infection. A variation from 10% to 100% of biofouling was recorded during the same period of supervision.

Biodegradation of various machine oils depends on Hydrocarbon (HC) structure, physical properties of processed oil and manufacture technology. A high concentration of aromatic and/or polar compounds increases the stability of machine oils. On the contrary, the machine oils produced from the sour oils are less biostable than those produced from the sweet oils, by analogy with crude oils. Generally, the biodegradability of machine oils is an inverse function of kinematical viscosity and refractive index (Haus et al., 2001, 2003).

1.6       AIMS AND OBJECTIVES

This study is therefore, aimed at isolation and identification of some possible species of microorganisms from used and unused engine oil.

The objectives are;

1.     To isolate and characterize microorganisms present on used and unused engine oil.

2.     To determine the percentage occurrence of the isolates.

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