Isolation And Identification Of Crude Oil Degrading Bacteria From Soil Polluted With Automobile Lubricants

INTRODUCTION AND LITERATURE REVIEW

1. 1. INTRODUCTION

Crude oil is composed of several compounds, such as aliphatic, aromatic and polyaromatic hydrocarbon, and also Sulphur, oxygen and nitrogen containing components. These compounds are toxic and can cause cancer. Oil waste management in oil refineries needs to be focused on administering waste and selecting appropriate strategies in order to prevent soil damage. Hydrocarbon components have been known to belong to the family of carcinogens and neurotoxic organic pollutants (Nilanjana and Preethy, 2011).

Petroleum products serve as source of energy but can also pose a serious damage to the environment, all petroleum products come from crude oil. Since  petroleum products are widely used, oil spill is inevitable  especially in crude oil producing developing countries. High concentration of these pollutants, due to their toxic and carcinogenic nature, and affect cell metabolism (Tanti et al., 2009). Hydrocarbons are bio persistent, bio accumulative and can cause harmful effects on aquatic fauna and flora as well as humans (Benson et al.,2007). Release of hydrocarbons either accidentally or due to human activities is the main cause of pollution of soil and aquatic environment.

Pollution of the soil environment with petroleum hydrocarbon and its derivatives and the consequent ecological and environmental impacts is a global concern. Therefore, soil as an essential component of our natural environment must be protected and sustained in order to achieve sustainable ecosystem and improve the livelihood of people in the risk area of crude oil pollution (Adenipekun., 2008; Etuk et al., 2013).

Multiple techniques have been developed to resolve the problem of petroleum pollution. Physicochemical methods used to reduce hydrocarbon pollution are more expensive than biological methods (Esin et al., 2011). Several physicochemical methods such as incineration, thermal desorption, coker, solvent extraction, excavation, volatilization and dispersion have been applied to return polluted sites to their pre-contamination status.

Each of these methods has limitations including ineffectiveness, cost of application, and release of toxic compounds. For instance, incineration is a very effective soil treatment method, but it is very expensive, leads to loss of nutritional value and destruction of soil structure (Jain er al., 2011). Incineration may also lead to inferno with its attendant hazards and and the smoke generated remains another form environmental pollution. Apart from physicochemical methods of contaminant removal from the environment, biological methods are also employed in the bioremediation of polluted soil (Jain et al., 2011).

A biological approach in handling these problems has emerged and this is known as bioremediation. This involves the use of microorganisms that use hydrocarbon as carbon and energy source to remediate crude oil polluted soils. This approach is safer because it does not lead to the release of toxic compounds to the environment; it is also cost effective; completely destroys the contaminant and applicable in large areas (Bento et al., 2015). The main reason for this concept is that majority of these molecules in crude oil and refined products biodegradable (Smith et al., 2012). Microorganisms involved in bioremediation include fungi and bacteria. (Das and Chandran, 2011). The success of bioremediation is affected by diverse physical and chemical parameter like the characteristics of the site and the parameters that affect microbial biodegradation of pollutants (Jain et al., 2011).

Several chemical and environmental factors like temperature, nutrient, oxygen, biodegradability, photo-oxidation, bioavailability, PH, soil acidity and alkalinity influence the biodegradation of hydrocarbons (Trindade et al., 2005). Greater degradation of oil pollutants is carried out in situ by a consortium of microorganisms and more 200 species of bacteria, fungi and even algae can biodegrade hydrocarbons (Onifade and Abubkar, 2007).

AIM OF THE STUDY

The aim of this study was to isolate and identify crude oil degrading bacteria and determine best crude oil degraders.

OBJECTIVE OF THE STUDY

• To isolate crude oil degrading bacteria.
• To identify the isolated crude oil degrading bacteria.
• To determine the rate of crude oil utilization by the isolates using spectrophotometer and select the best degraders.

1. 1. LITERATURE REVIEW
       

      1. COMPOSITION OF CRUDE OIL

Crude oil is composed of paraffinic, alicyclic and aromatic hydrocarbons (Forkan et al., 2010). Aromatic hydrocarbons are mainly benzene, toluene, ethylbenzene and xylene (BTEX). Petroleum means ‘rock oil’ in Latin and occurs as dark, sticky and viscous liquid (Varjani, 2017). The aliphatic hydrocarbons are alkanes, alkenes and alkynes. Hydrocarbons are composed mainly of carbon and hydrogen, nevertheless, crude oil also contains some amount of nitrogen, Sulphur and oxygen compounds. Crude oil composition can vary based on the site and age of oil well. About 85% components of types of crude oil can  be categorized as asphalt base, paraffin base and/or mixed base.

Crude oil is grouped into four broad fractions: Saturates (aliphatics); Aromatics; Resins; and Asphaltenes (Chandra et al., 2013). Saturates are hydrocarbons with single bond and constitute the highest percentage of crude oil constituents. They are grouped based in their chemical structures into cycloalkanes and alkanes (Abbasian et al., 2015). They are saturated or unsaturated and linear or branched structures such as steranes, terpenes, naphthenes (cycloalkanes), terpenes, isoalkanes and n-alkanes (Varjani, 2017). N-Alkanes are divided into four molecular weight groups: aliphatic hydrocarbons with medium molecular weight (C17-C18), aliphatic compounds with high molecular weight (>C28), aliphatic hydrocarbons with lower molecular weight (C8-C16) and gaseous alkanes (Abbasian et al., 2015).

Aromantics have one or more aromantic rings mainly substituted with several alkyl groups (Meckenstock et al., 2016). They are ringed hydrocarbon molecules and divided into two groups: monocyclic aromatic hydrocarbons (MAHs) which include benzenes, toluene, ethylbenzene and xylenes (BTEX) and polycyclic aromatic hydrocarbons (PAHs). PAHs that contain two or three cyclic rings are referred to as light PAHs while those that contain four cyclic or more are referred to as heavy PAHs (Varjani, 2017).

Resins and asphaltenes consists of non-hydrocarbon polar compounds and complex and mostly unknown carbon structures with addition of several oxygen, sulphur and nitrogen atoms (Chandra et al., 2013). Resins contain several polar functional groups formed with nitrogen, sulphur, oxygen and trace metals such as iron, vanadium and nickel. Resins are amorphous solids and readily dissolved in oil. Asphaltenes is a dark brown, large and complex molecule which is colloidally dissipated in aromatics and saturates (Varjani, 2017).

1.2.2. CRUDE OIL DEGRADING MICROORGANISMS

Microorganisms play very important role in preserving the biosphere and ecosystem to maintain a sustainable environment (Widdel and Rabus, 2001). Microorganisms are ubiquitous and widely distributed in fresh and marine water, soil and air (Varjani et al., 2013). They are easily isolated from hydrocarbon polluted soil, sediments or water and have degrading ability (Ron and Rosenberg, 2014). Microorganisms, such as bacteria and fungi can degrade PAHs to other less harmful organic compounds or to inorganic end products such as carbon dioxide and water (Brassington et al., 2007). Microorganisms such as algae, bacteria and fungi have been reported to degrade hydrocarbon pollutions. (Wilkes et al., 2016).

1.2.2.1. BACTERIA

Bacteria genera that are hydrocarbon degraders include Achromobacter, Stenotrophomaonas, Nocardia, Alcaligenes, Azoarcus, Acinetobacter, Cellulomonas, Corynebacterium, Vibrio, Marinobacter, Streptomyces, Flavobacterium, Staphylococcus, Brevibacterium, Micrococcus, Mycobacterium, Kliebsiella, Pseudomonas, Bacillum, Exiguobacterium, Ochrobactrum, Oleisphira, Alcanivorax (Widdel and Rabus, 2001; Varjani, 2017; Sorkhoh et al., 1990)

1.2.2.2. FUNGI AND YEASTS

Fungi of genera Aspergillus, Talaromyces, Penicillium, Sporobolomyces, Paecilomyces, Neosartoria, Fusarium, Amorphoteca, Graphium and yeast of genera Candida, Yarrowia, Pseudozyma, Saccharomyces, Pichia and Rhodotorula have been reported as hydrocarbon degraders (Wilkes et al., 2016; Xenia and Refugio, 2016).

1.2.2.3. ALGAE

Marine algae such as cyanobacteria, diatoms and green algae have the ability to metabolize naphthalene into various metabolites. Prototheca zopti has been isolated and reported as a hydrocarbon degrader. Algae have potential in the consortia with bacteria, for example, Pseudomonas migulae and Sphingomonas yanoikuyae that degrades phenanthrene (Xenia and Refugio, 2016).

1.2.3 BIODEGRADING AND BIOREMEDIATION

Biodegradation is one of the mechanisms in which microorganisms are used for elimination of hydrocarbon pollutants from environment (Varjani et al., 2013 and Macaulay and Rees, 2014). In biodegradation, microorganisms acquire energy during hydrocarbon pollutant degradation (Abbasian  et al., 2015). Biodegradation processes differ widely, but often the final product of the degradation is carbon dioxide (Pramila et al., 2012). Organic compounds can be degraded aerobically, with oxygen, or anaerobically, without oxygen (Mrozik et al., 2013). The biological methods such as natural attenuation, biostimulation, bioaugmentation and bioremediation are efficient and adequate methods of remediate soil with petroleum hydrocarbons as pollutants because this methods do not negatively affect the site.

The bioremediation is one of the economical methods compared to the other methods like incineration and washing of the soil. Bioremediation is the one of the useful and inexpensive methods to achieve the optimum biodegradation condition, in which by the use of microorganisms hydrocarbons degraded which is the sources of carbon and energy for the microorganisms. Bioremediation process is the use of microorganisms to remove pollutants or make them less harmful due to their several metabolic capabilities (Shallu et al., 2014). Bioremediation is an evolving method for the biodegradation of diverse environmental pollutants including crude oil products (Varjani, 2017). It involves the use of microbes to degrade, mitigate or reduce harmful organic contaminants to compounds such as carbon dioxide, water, methane, and biomass without negatively affecting the environment (Ron and Rosenberg, 2014).

Bioremediation is a promising technology for the treatment of contaminated soil since it leads to complete mineralization and it is also cost-effective. The process of bioremediation function is basically on biodegradation, which refer to complete mineralization of organic contaminants into inorganic compounds, water, carbon dioxide and cell protein or biotransformation of complex organic contaminants to less complex organic compounds by biological agents like microorganisms (Nilanjana and Preethy, 2011). Bioremediation of crude oil pollutes sites is sometimes limited due to poor biodiversity of native microflora and/or shortage of native specialized microbes with complementary substrate specificity required for degrading different hydrocarbons in polluted sites (Ron and Rosenberg, 2014).

Bioremediation process can be divided into three phases. One, by natural attenuation, hydrocarbon pollutants are degraded by indigenous microorganisms without any human augmentation. Two, biostimulation is applied where nutrients and oxygen are applied to the system to improve their effectiveness and to enhance biodegradation. Finally, during bioaugmentation, microorganisms are added to the system. These supplemental microorganisms should be more efficient than native microbes to degrade the target pollutants (Diez, 2010).

A possible remedial technology requires microorganisms being capable of quick adaptation and efficient uses of contaminants of interest in a particular case in a period of time. (Seo et al., 2009). The major techniques to oil spill bioremediation may include, bioaugmentation, in which known oil degrading microbes are added to supplement the existing microbial pollution and biostimulation, in which the growth of nature crude oil degraders is stimulated by the adding of nutrients or other growth-limiting cosubstrates (Nilanjana and Preethy, 2011).