Effect Of Temperature On The Population Of Hydrocarbon Degrading Bacteria In Palm Oil Mill Effluent

Effect Of Temperature On The Population Of Hydrocarbon Degrading Bacteria In Palm Oil Mill Effluent

Abstract

The aim of this work is to determine the effect of temperature on the population of hydrocarbon degrading bacteria in palm oil mill effluent. This was done at the Department of Microbiology, University of Abuja, Abuja, F.C.T Abuja, Nigeria, between June 2015 and August 2015. Samples of POME were inoculated with Pseudomonas aeruginosa, Bacillus subtilis, Klebsiella and Staphylococcus aureus. Microbial growth was measured by determining the total viable counts in the medium containing POME and control. The isolates were analyzed for the population of bacteria at temperatures 40C, 350C and 400C with incubation periods after 24 hours, 7days and 14days. Analysis of variance of POME isolates shows that there is no significant difference between the time of incubation and temperature since P- Value (0.159) > α (0.05) for time and P- Value (0.145) > α (0.05) for temperature. ANOVA for control isolates shows that there is significant difference between incubation period of 24 hours, 7days and 14days since p- Value < α (0.05). The highest bacteria population was recorded after the 7th day of incubation at 350C from Pseudomonas aeruginosa 1.66 x 108 ± 0.6 to Klebsiella 1.27 x 108 ± 0.6.

Table of Contents

• Title Page
• Declaration
• Certification
• Dedication
• Acknowledgements
• Table of Contents
• List of Tables
• List of Figures
• Abstract

Chapter One

• 1.1 Introduction
• 1.2 Justification
• 1.3 Aim and Objectives

Chapter Two

• 2.1 Literature Review
• 2.2 Brief History of Oil Palm
• 2.3 Palm Oil Industry in Nigeria
• 2.4 Benefits of Palm Oil Industry
• 2.5 Palm Oil Value – Chain Analysis
• 2.6 Hydrocarbon Degrading Bacteria
• 2.7 Hydrocarbon Degrading Bacteria and Temperature
• 2.8 Characteristics of Hydrocarbon Degrading Bacteria
• 2.9 Composition of Palm Oil

Chapter Three

• 3.1 Materials and Methods
• 3.2 Sample Collection
• 3.3 Sterilization Method
• 3.4 Media Preparation
• 3.5 Inoculation of Pome With Bacteria Isolates
• 3.6 Determination of Total Viable Count
• 3.7 Statistical Analysis

Chapter Four

• 4.1 Results

Chapter Five

• 5.1 Discussion
• 5.2 Conclusion
• 5.3 Recommendation
• References
• Appendix
• 1.0 Anova Analysis
• 2.0 Materials And Equipments
• 3.0 Media Composition And Preparation
• 4.0 Abbreviations

List of Tables

• Oil Palm Hectare in Nigeria
• Number of People Dependent on Palm Oil
• Food and Non Food Uses of Palm Oil Product
• Total Viable Count of Pome Isolate at 4⁰C
• Total Viable Count of Pome Isolate at 35⁰C
• Total Viable Count of Pome Isolate at 40⁰C
• Mean of HDB at Different Temperature (CTRL)
• Anova Table (CTRL)
• Anova Comparison Table (CTRL)
• Mean of HDB at Different Temperature (POME)
• Anova Table for Pome

List of Figures

• Population HDB AT 4⁰C (CTRL)
• Population HDBAT 35⁰C (CTRL)
• Population HDB at 40⁰C (CTRL)

Chapter One

1.1 Introduction

Palm oil is one of the most important vegetable oil in the world’s oil fat market. The extraction and purification processes generate different kinds of wastes generally known as palm oil mill effluent (POME).

The biodegradation of hydrocarbons by microorganisms has become the main mechanism for eliminating palm oil and petroleum derived pollution in the environment. Palm oil mill effluent is waste water generated from palm oil milling activities which requires effective treatment before discharge into water course because of its highly polluting properties (Phaik et al., 2010). The raw effluents contain 90% – 95% water and include residual oil, soil particles and suspended solids (Phaik et al., 2010).

The techniques of biological control over hydrocarbon based pollutants is a promising new technology, thanks to its low cost and absence of secondary contamination (Vasudevan and Rajaram, 2001; Agnieszka and Zofia, 2010). In the course of biological restoration of contaminated soil the main factors that affect the effect of remediation include pH value, the nutritional level, the hydrocarbon degrading bacteria and the temperature, among which the impact of hydrocarbon degrading bacteria on the remediation effect is critical (Venosa and Zhu, 2003; Chaillan et al., 2006). Under certain extreme environments such as in the high temperature and extreme arid regions, it is very important to select the locally specific and extremely indigenous microorganisms capable of degrading hydrocarbons.

Oil palm Elaeis guineensis cultivation and processing like other agricultural and industrial activities, also raise environmental issues. Palm oil processing is carried out using large quantities of water in mills where oil is extracted from the palm fruits. During the extraction process, about 50% of the water results in palm oil mill effluent. It is estimated that for every 1 tonne of crude palm oil produced 5 -7.5tonnes of water end up as palm oil mill effluent (POME) (Okwute and Isu, 2007; Wu et al., 2009).

In Nigeria’s palm oil industry, most of the palm oil mill effluent produced by small scale traditional operators undergo little or no treatment and are usually discharged in the surrounding environment. This could pollute streams, rivers or surrounding land (Okwute and Isu, 2007). River water consequently turns brown, smelly and slimy. Often, fish and other aquatic organisms get killed and locale people are denied of the availability of local water sources for domestic uses and fishing (Ezemonye et al., 2008).

The effect of crude oil pollution on the properties of soil has been the subject of many studies. Okolo et al., (2005) reported that oil pollution increase carbon and reduce soil nitrates and phosphorus. Similarly, Adedokun and Ataga, (2007) reported that any contact of soil with crude oil results in damage to the soil microorganisms and plants while Onuah et al., (2003) among others have shown that crude oil pollution prevents oxygen exchange between soil and the atmosphere due to hydrophobic properties of oil.

1.2 Justification

Palm oil mill effluent has been an area of interest to researchers in the field of environmental microbiology, and biotechnology. Most especially, because of the impact it has on the environment (soil and water). Among the various conditions necessary for the degradation of POME by hydrocarbon degrading bacteria, I am interested in knowing about the effect of temperature on hydrocarbon degrading bacteria, how it affects growth rate and population of bacteria, as a means of contributing to the many work that has been done.

1.3 Aim and objectives

The reason for this work is to determine the effect of temperature on the population of hydrocarbon degrading bacteria in palm oil mill effluent.

The specific objectives are:

1. Sterilization of all media and materials.
2. Inoculation of POME and the control with bacteria isolates.
3. Incubation of inoculated POME and control isolate at temperatures 40C, 350C and 400C, for 24 hours, 7 days and 14 days.
4. Determination of total viable counts through serial dilution and spread plate techniques.

Chapter Five

Discussion, Conclusion and Recommendation

5.1 Discussion

Figure 1,2 and 3 shows the population of the hydrocarbon degrading bacteria at 40C,350C, 400C after 24hours (1 day), 7 days and 14 days of incubation for the control mean values respectively. Control shows a gradual decrease in the population of each of the organism with the length of days. Because of the absence of palm oil mill effluent as the carbon source, as a result of this, nutrient composition of the mineral salt medium were used up quickly, giving a gradual decrease in population with the length of days. Figure 2shows the population of hydrocarbon degrading bacteria at 350C after 24 hours, 7 days and 14 days of incubation, at 350C population of bacteria were more than the control at 40C in all the period of incubation. This means that at 350C bacteria has optimal temperature.
The control at 400C also shows a gradual decline on the population of bacteria from 24 hours to 14 days, with increase in the population of bacteria from Klebsiella to Pseudomonas aeruginosa.

Total viable counts of isolates (Cfu/ml) at the various temperature and time shows that bacteria have the ability to degrade POME, as there is increase in bacteria population in the mean values of POME isolates compared to that of the control, which results from the active metabolizing activity of bacteria on palm oil mill effluent which is the carbon source.

Table 4, 5 and 6 shows the total viable count of isolates (Cfu/ml) POME at 40C, 350C and 400C, and the period of incubation within 24 hours, 7 days and 14 days.

From each of the table, the highest population of bacteriais determined. At 40C the population of hydrocarbon degrading bacteria increase gradually. Because of its low temperature, growth was not rapid after 24 hours of incubation but gradually increase at 7 days and 14 days. At 40C bacteria slowly degrade POME with the length of time, in increasing order from Bacillus subtilisto klebsiella. ZoBell (1973) and Gunkel (1967), found that hydrocarbon degradation and utilization were slower at low temperature.

At 350C,after 7 days of incubation each of bacteria has maximum growth and at 14 days, there was a reduction. This shows that 350C is the best temperature for the growth of bacteria. POME was maximally utilized at each period of incubation and thus means that POME will be fast degraded at 350C, with Klebsiellabeing the lowest Pseudomonas aeruginosa being the highest of the population from day 7 to day 14 of the incubation. This could be attributed to high metabolic activity (Sanyaolu, et al., 2012).

At 400C bacteria population was not as increased as that of incubation at 350C. After 7 days of incubation, population of bacteria was at the highest which could be as a result of active use of carbon from POME and a reduction in population after 14 days of incubation. At400C biodegradation of POME is possible, as POME is utilized as the carbon source.

Statistical analysis shows if there is significant difference in the temperature and time of incubation.
From the ANOVA CONTROL result, we reject the null hypothesis for time since P- Value (0.003) is less than the significant level α (0.05) and fail to reject the null hypothesis for temperature.

Thus, there is significant difference between the time of incubation and no significance difference between the temperatures. Since there is significantdifference between the times of incubation, hence the determination of which pair of the mean is different significantly.

The table (ANOVA CONTROL) shows which pair of the mean differs significantly at 0.05 level of significance using least significance difference (L.S.D)From the table, there is significant difference between 24hours (1 day) and 14 days, since (P- Value < 0.05) From the result of ANOVA POME, since P- Value (0.159) > α (0.05) for time and P- Value (0.145) > α (0.05) for temperature. Thus, we accept the null hypothesis and conclude that there is no significant difference between the time of incubation and temperature.

5.2 Conclusion

The study reveals that biodegradation of POME is a possibility at the various temperature over a period of 14days with the best degrading temperature at 350C and after 7 days of incubation.

The hydrocarbon degrading bacteria were able to utilize POME as the carbon source because of their ability to attach to oil surface in lipolytic activity. Microorganisms were able to grow and survive at the two extreme temperatures because of the phosphorus and nitrogen compound in the mineral salt medium with the addition of POME as the carbon source.

Values of bacteria count, which are means of triplicate readings, show the order of increase in the utilization of POME.

At 40C, the order of increased utilization of POME is from the highest Bacillus subtilisto Klebsiellathe lowest.

At 350C, Pseudomonas aeruginosato Klebsiella.

At 400C, Pseudomonas aeruginosato Bacillus subtilis.

Therefore, Bacillus subtilis utilize POME the best at 40C, while Pseudomonas aeruginosa utilize POME best at 350C and 400C.

5.3 Recommendation

For bioremediation and biodegradation purposes, the application of inoculums of hydrocarbon degrading bacteria will be of great help in areas where there is oil spillage or contamination. These organisms degrade and utilize oil which is a source of carbon containing some mineral nutrients for bacteria growth. The use of hydrocarbon degrading bacteria in the application of biodegradation is not only safe, but also economical.

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