Determination of Degrading Ability of Fungi Isolated From Hydrocarbon Polluted Soil on Crude Oil Using Gas Chromatography
Chapter One
Objective of the study
The specific objectives of the study are to :
- Isolate and identify fungi from crude oil-contaminated soil sample.
- Screen the isolates for bio-degradative abilities.
- Assessment of the degrading abilities of the fungi isolates by gas chromatography
CHAPTER TWO
LITERATURE REVIEW
Bio-degradation
Bio-degradation is defined as the biologically catalyzed reduction in complexity of chemical compounds (Davis,2009.). Indeed, biodegradation is the process by which organic substances are broken down into smaller compounds by living microbial organism . When biodegradation is complete, the process is called “mineralization”. However, in most cases the term biodegradation is generally used to describe almost any biologically mediated change in a substrate. So, understanding the process of biodegradation requires an understanding of the microorganisms that make the process work. The microbial organisms transform the substance through metabolic or enzymatic processes. It is based on two processes: growth and cometabolism. In growth, an organic pollutant is used as sole source of carbon and energy. This process results in a complete degradation (mineralization) of organic pollutants. Co-metabolism is defined as the metabolism of an organic compound in the presence of a growth substrate that is used as the primary carbon and energy source.(Atlas, 2000.) Several microorganisms, including fungi, bacteria and yeasts are involved in biodegradation process. Algae and protozoa reports are scanty regarding their involvement in biodegradation . Bio-degradation processes vary greatly, but frequently the final product of the degradation is carbon dioxide. Organic material can be degraded aerobically, with oxygen, or anaerobically, without oxygen.
Bio-degradable matter is generally organic material such as plant and animal matter and other substances originating from living organisms, or artificial materials that are similar enough to plant and animal matter to be put to use by microorganisms. Some microorganisms have the astonishing, naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds including hydrocarbons (e.g. oil). Bio-degradation is nature’s way of recycling wastes, or breaking down organic matter into nutrients that can be used and reused by other organisms. In the microbiological sense, “bio-degradation” means that the decaying of all organic materials is carried out by a huge assortment of life forms comprising mainly bacteria and fungi, and other organisms. This pivotal, natural, biologically mediated process is the one that transforms hazardous toxic chemicals into non-toxic or less toxic substances. In a very broad sense, in nature, there is no waste because almost everything gets recycled.
The term bio-degradation is often used in relation to ecology, waste management and mostly associated with environmental remediation (bioremediation). Bioremediation process can be divided into three phases or levels. First, through natural attenuation, contaminants are reduced by native microorganisms without any human augmentation. Second, bio-stimulation is employed where nutrients and oxygen are applied to the systems to improve their effectiveness and to accelerate biodegradation. Finally, during bio-augmentation, microorganisms are added to the systems. These supplemental organisms should be more efficient than native flora to degrade the target contaminant. A feasible remedial technology requires microorganisms being capable of quick adaptation and efficient uses of pollutants of interest in a particular case in a reasonable period of time. Many factors influence microorganisms to use pollutants as substrates or co-metabolize them, like, the genetic potential and certain environmental factors such as temperature, pH, and available nitrogen and phosphorus sources, then, seem to determine the rate and the extent of degradation. Therefore, applications of genetically engineered microorganisms (GEM) in bioremediation have received a great deal of attention. These GEM have higher degradative capacity and have been demonstrated successfully for the degradation of various pollutants under defined conditions. However, ecological and environmental concerns and regulatory constraints are major obstacles for testing GEM in the field.
Bio-remediation and bio-degradation
The application of bio-remediation as a biotechnological process involving microorganisms has become a crescent study field in microbiology, because of its increasing potential of solving the dangers of many pollutants through biodegradation. Microorganisms might be considered excellent pollutant removal tools in soil, water, and sediments, mostly due to their advantage over other bioremediation procedures.
Moreover, bio-remediation using biodegradation represents a high impact strategy, but still a low cost way tool of removing pollutants, hence a very viable process to be applied. The principles of bioremediation are based on natural attenuation, bio-augmentation and bio-stimulation. The simplest method of bioremediation is natural attenuation, in which soils are only monitored for variations in pollution concentrations to ensure that the pollutant transformation is active. Bio-augmentation is usually applied in cases where natural active microbial communities are present in low quantities or even absent, wherein the addition of contaminant degrading organisms can accelerate the transformation rates. In such cases, the adaptation of exogenous strains that exert highly efficient activities for pollutant transformation to new environments is a key challenge in implementation. The capacity of a microbial population to degrade pollutants can be enhanced also by stimulation of the indigenous microorganisms by addition of nutrients or electron acceptors.
Natural attenuation
Natural attenuation or bio-attenuation is the reduction of contaminant concentrations in the environment through biological processes (aerobic and anaerobic biodegradation, plant and animal uptake), physical phenomena (advection, dispersion, dilution, diffusion, volatilization, sorption/desorption), and chemical reactions (ion exchange, complexation, abiotic transformation). Terms such as intrinsic remediation or biotransformation are included within the more general natural attenuation definition. Although, one of the most important components of natural attenuation is biodegradation, the change in form of compounds carried out by living creatures such as microorganisms. Under the right conditions, microorganisms can cause or assist chemical reactions that change the form of the contaminants so that little or no health risk remains. Natural attenuation occurs at most polluted sites. However, the right conditions must exist underground to clean sites properly. If not, cleanup will not be quick enough or complete enough. Scientists monitor these conditions to make sure natural attenuation is working. This is called monitored natural attenuation or (MNA). So, Monitored natural attenuation is a technique used to monitor or test the progress of natural attenuation processes that can degrade contaminants in soil and groundwater. It may be used with other remediation processes as a finishing option or as the only remediation process if the rate of contaminant degradation is fast enough to protect human health and the environment. Natural processes can then mitigate the remaining amount of pollution; regular monitoring of the soil and groundwater can verify those reductions.
When the environment is polluted with chemicals, nature can work in four ways to clean it up
1) Tiny bugs or microbes that live in soil and groundwater use some chemicals for food. When they completely digest the chemicals, they can change them into water and harmless gases.
2) Chemicals can stick or sorb to soil, which holds them in place. This does not clean up the chemicals, but it can keep them from polluting groundwater and leaving the site.
3) As pollution moves through soil and groundwater, it can mix with clean water. This reduces or dilutes the pollution.
4) Some chemicals, like oil and solvents, can evaporate, which means they change from liquids to gases within the soil. If these gases escape to the air at the ground surface, sunlight may destroy them.
If the natural attenuation is not quick enough or complete enough, bioremediation will be enhanced either by bio-stimulation or bio-augmentation.
CHAPTER THREE
MATERIALS AND METHODS
Materials/Equipments used
Materials used include : cotton wool, aluminium foil, spatula, crude oil, soil samples, autoclave, incubator ,petri dishes, bursen burner, sugars( glucose, sucrose, maltose,lactose,galactose)
,paper tape, needle and syringe,70% ethanol, matches, testtubes, potato dextrose agar, , conical flask, measuring cylinder, glass slide, di ethyl-ether(organic solvent).
Collection of samples
Soil samples were collected from three different locations in Delta state, Nigeria. Soil samples that were not contaminated were used as control. The samples were aseptically collected using soil auger, stored in sterile aluminium foils and transported to the laboratory within 48 hours of collection. Crude oil was collected from Port Harcourt in Nigeria.
CHAPTER FOUR
Result
Microbial population of soil
The fungi population of the soil sample after 72hours is shown in table
CHAPTER FIVE
Discussion
Hydrocarbons in the environment are biodegradable primarily by bacteria and fungi. Although ubiquitous in the terrestrial and aquatic ecosystems, the fraction of the total heterotrophic community represented by the hydrocarbon utilizing bacteria and fungi is highly variable (Amadi and Braid, 2007). The results of this work indicate that many of the fungal species isolated from the soil samples were capable of degrading petroleum hydrocarbons. Bartha and Atlas (2000) listed 14 genera of fungi which had been demonstrated to contain members which utilize petroleum hydrocarbons; all of these micro organisms had been isolated from the soil. Also, Okerentugba and Ezeronye (2003) demonstrated that Penicillium spp., Aspergillus spp. and Rhizopus spp. were capable of degrading hydrocarbons especially when single cultures were used. These fungi had been isolated also from crude oil-contaminated environments in the Niger Delta area of Nigeria. Batelle (2000) showed that fungi were better degraders than traditional bioremediation techniques including bacteria.
CHAPTER SIX
CONCLUSION AND RECOMMENDATION
Conclusion
Microbial activities are very important for the renewal of our environment and maintenance of the global carbon cycle. (Ojumu, 2004). These activities are included in the term bio-degradation. Amid them substances that can be degraded or transformed by microorganisms are a huge number of synthetic compounds and other chemicals having ecotoxicological effects like hydrocarbons. Fungi are excellent hydrocarbon degraders and show tremendous diversity and adaptability in utilization of crude oil as a carbon source as seen in this study. However their abilities to degrade a specific hydrocarbon as a source of energy and/or biomass may differ. (Davies and Westlake, 1979).Therefore, it can be said that fungi can be used to degrade hydrocarbon and its derivatives.
Furthermore, due to the high level of degradation of crude oil by Aspergillus niger as seen in the gas chromatography analysis, it can be concluded that Aspergillus niger can be employed in successful large scale bioremediation of pollutants such as hydrocarbons thereby making a difference in our ability to reduce wastes, eliminate industrial pollution, and enjoy a more sustainable future.
Recommendation
The crude oil samples should be taken for further analysis such as gas chromatography and mass spectrometry.
There should be an increased focus on the use of fungi to degrade crude oil because they are very effective and prominent in the bio-degradation process and also, some studies have it that fungi are more effective than bacteria in mopping up oil spills.
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