Bioremediation Potentials and Emulsification Activity of Indigenous Bacteria Isolated from Crude Oil Polluted Soil in Ohaji-Egbema, Nigeria

Crude oil pollution is a global problem because of its hazardous effect to aquatic and terrestrial life, as well as having adverse effect on soil productivity. Bioremediation technology has been established as a successful alternative to physicochemical technique in cleaning up of contaminated site. This study was aimed at determining the bioremediation potentials and emulsification activity of indigenous bacteria isolated from crude oil polluted soil. Soil samples were randomly collected from Water-Smith and Base-Camp refineries in Ohaji-Egbema Local Government Area of Imo State, Nigeria. Bacteria isolation and identification were done using standard methods. The isolates were screened for crude oil biodegradation potential, and the extent of crude oil removal was determined using gravimetric method. Emulsification activity of the isolates was also determined using emulsification index (E24). Data were analyzed using analysis of variance (ANOVA) at P < 0.05. Bacteria isolates such as Arthrobacter sp., Enterobacter sp., Bacillus sp. and Pseudomonas sp. were identified in this study. Pseudomonas sp. achieved the highest extent of crude oil removal within the experimental period. Moreover, highest E24 of 81.80±2.8 and 84.00±4.0 was recorded in the media containing Bacillus sp. and Pseudomonas sp., respectively at 0.75mg/l crude oil concentration. Among the isolates, Pseudomonas sp. has greater potential for effective bioremediation of crude oil polluted soil as well as biosurfactant production due to its higher biodegradation efficiency and emulsification activity.


Introduction
Crude oil, commonly referred to as petroleum, is a liquid found within the earth comprised of hydrocarbons, organic compounds and small amount of metals [1]. Crude oil which is abundantly located in the Niger Delta region of Nigeria is spilled on the environment due to pipeline rupture, oil well blowout, oil tanker wreckages, oil bunkering to mention but a few. Crude oil spill can also occur at different stages of production and transportation either for export or refining processes.
The petroleum hydrocarbons are hazardous to various forms of terrestrial and aquatic life and are also carcinogenic, mutagenic and potentially immunotoxigenic [2,3]. The economic life of the people in the affected area is disrupted. Farmlands, navigational activities, availability of potable water and fishing activities are jeopardized. Previous studies on crude oil pollution in soil had revealed its adverse effects on soil productivity [4,5].
The traditional physical and chemical treatments to clean up the petroleum hydrocarbons are expensive and appear ineffective. This is because they do not lead to complete mineralization, and awfully can produce toxic byproducts or residues [2,3]. In contrast, bioremediation involving microbial agents, such as protozoa, bacteria, fungi, plants; offers successful alternatives to clean up the petroleum pollution [2]. Bioremediation technology is safe, economical, more efficient and reliable method that is harmless and eco-friendly [6,7].
The effect of exogenously added microbial biosurfactants in enhancing the bioremediation of crude oil-polluted soils by indigenous microbes have been reported [8,9]. Biosurfactant producing microorganisms are commonly found in different environments, such as soil or water samples that are contaminated with hydrophobic organic compounds (i.e., oil contaminated soils) like refinery wastes [8,10]. Moreover, biosurfactants 2 Bioremediation Potentials and Emulsification Activity of Indigenous Bacteria Isolated from Crude Oil Polluted Soil in Ohaji-Egbema, Nigeria have a wide array of applications such as; emulsifiers, de-emulsifiers, wetting agents, spreading agents, foaming agents, functional food ingredients, as well as detergents [11]. Due to their unique properties and vast array of applications, sourcing of new biosurfactant producing microbes is currently in great demand [9]. Biosurfactants emulsify oil thereby enhancing its removal from the environment. This study was therefore undertaking with a view to determine the bioremediation potentials and emulsification activity of indigenous bacteria isolated from crude oil polluted soil.

Collection of Samples
Crude oil polluted soil samples were randomly collected using hand auger at a depth of 3 -5 cm, from four different locations at the Water-Smith and Base-Camp refineries in Umu-Akpu, Ohaji and Awaraka Communities, both in OhajiEgbema Local Government Area of Imo State. Crude oil used in this study was also collected from the refineries. Samples were placed in sterile bottles and transported immediately in cold storage container to the laboratory.

Sample Site Description
OhajiEgbeme is an oil-rich Local Government Area of Imo State, Nigeria. It is located in the South-western part of Imo State, shares common boundaries with Owerri in the East, Oguta LGA in the North and Ogba/Egbema/Ndoni in Rivers State in the South-west [12].

Isolation of Crude Oil Degrading Bacteria
Crude oil degrading bacteria were isolated from the soil samples using mineral salt agar, with 1% (v/v) crude oil as carbon source. One (1) gram of the homogenized soil samples were measured into 9 ml of sterile distilled water in a test tube and swirled gently. 1 ml of the sample was pipetted and serially diluted up to 10 -3 dilutions. 0.1 ml of the sample from the 10 -2 and 10 -3 dilutions were transferred onto the surface of a freshly prepared mineral salt agar, using the spread plate technique [3]. The plates were incubated at 30 o C for 48 hours. After 48 hours' incubation, pure cultures were obtained by sub culturing onto fresh nutrient agar plates. The pure cultures were stored on nutrient agar slant for further use.

Inoculum Development
Mineral salt broth containing 0.3 ml of crude oil was dispensed in 30 ml quantities into four 250 ml Erlenmeyer flasks. A loopful of the isolates were inoculated into the medium and incubated in an Orbital Shaker at 120 rpm at 30 o C for 24 hours. After incubation, 10 ml of the culture broth were aseptically withdrawn and the pH, total viable count (TVC) and optical density were measured at 550nm.

Crude Oil Degrading Potentials of the Isolates
One (1.0) milliliter of the inoculums was each added into four 250 ml Erlenmeyer flasks containing 1 ml of crude oil in 100 ml of mineral salt broth. Control flask without inoculum was also prepared. The flasks were incubated in a rotary shaker at 30 o C for 15 days, at 120 rpm. At 5 days interval, samples were withdrawn from the flasks and the pH, total viable count (TVC) and optical density (OD) were determined at 550 nm [13].

Estimation of Crude Oil Removal by the Isolates
The extent of removal of crude oil by the isolates was determined at the end of the incubation period. The residual oil in the flasks was extracted in a pre-weighed beaker with toluene, in a separating funnel. Extraction was repeated twice to ensure complete extraction. After extraction, toluene was evaporated in a hot air oven at 70 o C; the beaker was cooled in a desiccator and weighed. Then the percentage oil removal was calculated [14].

Emulsification Activity
Emulsification activity was carried out as an emulsification index (E24). The bacterial isolates were grown in mineral salt broth supplemented with 0.10, 0.25, 0.50, 0.75, and 1.00 mg/l of crude oil as sole carbon source, and incubated for 48 hours at 30 o C at 120 rpm. One hundred (100) ml culture contained in 250 ml Erlenmeyer flask was centrifuged at 1000 rpm for 20 minutes to obtain a cell free broth supernatant. The supernatant was carefully pipetted and filtered through a filter paper to remove any suspended hydrocarbons.
Emulsification activity was carried out by mixing equal volume of crude oil and cell free broth supernatant in a test tube and vortexed at high speed for 2 minutes. The mixture was allowed to stand for 24 hours. E24 was calculated by dividing the height of the emulsion layer by the mixture total height and multiplying by 100 [15].

Identification of the Isolates
The cultural and microscopic characteristics of the pure isolates were noted. Biochemical tests (such as oxidase, catalase, indole, sugar fermentation, methyl red, vogesproskauer, etc) were also carried out, with reference to the Bergey's Manual of Systematic Bacteriology.

Statistical Analysis
Data were analyzed and presented as mean ± standard Universal Journal of Microbiology Research 8(1): 1-6, 2020 3 deviation (SD) of three replicates. Analysis of variance (ANOVA) was used to test significance of variations within and among the groups. When significant difference was indicated by ANOVA, the least significant difference (LSD) and Duncan multiple range test was used for pair-wise separation of the means. A statistical package for social sciences (SPSS) software was used for statistical analysis in this study and test for significance between means was implied at P = 0.05 level.

Isolation and Identification of Crude Oil Degrading Bacteria
Four isolates were obtained in this study. They were identified as Arthrobacter sp., Enterobacter sp., Bacillus sp., and Pseudomonas sp., based on their cultural, microscopic and biochemical characteristics. Some of the isolates (such as Bacillus sp. and Pseudomonas sp.) obtained in this study were previously reported as hydrocarbon degraders [16]. Ni'matuzahroh et al. [17] reported the isolation of Propionibacterum, sp., Bacillus sp., Corynebacterium sp. and Rothia sp. from oil sludge. Figure 1 shows the variation in pH of the isolates during degradation of crude oil. There was an increase in pH of the broth cultures of the isolates from 0 to 5 days. However, from 5 to 15 days, a decrease in pH of the broth cultures was observed in all the isolates. An increase in optical density and total viable count of the broth cultures of the isolates was observed from 0 to 10 days of the study (Figures 2 and 3). However, a slight decrease in the optical density and total viable count of the broth cultures of the isolates was observed from 10 to 15 days of the study (Figures 2 and 3). The decrease in pH on the 15 th day of the study, with a concomitant decrease in optical density and total viable count, could be attributed to the mineralization of the hydrocarbons in crude oil. Sepahi et al. [18] reported that microbial degradation of hydrocarbons often leads to the production of organic acids, which probably caused the reduction in pH. The decrease in growth on the 15 th day could be attributed to the decrease in pH which may have created an unfavorable environment for the isolates. The increase in growth of the isolates from 0 to 10 days of the study could be attributed to their ability to utilize crude oil as carbon source. It could also be that the cultural condition was adequate for the growth of the organisms [3]. Obire and Nwaubeta [19] reported an initial gradual increase in bacterial population following the application of petroleum hydrocarbon, but a decline as the biodegradation progressed; which supports our findings.   Table 1 shows the extent of crude oil removal by the isolates after 15 days of the experimental period. Pseudomonas sp. showed the highest removal of 75%, followed by Bacillus sp. (58.33%), Enterobacter sp. (25%) and Arthrobacter sp. (16.67). Highest oil removal achieved by Pseudomonas sp. in this study, could be attributed to the production of biosurfactants which emulsified the oil in the growth media, thereby enhancing its removal. However, Mbachu et al. [16] reported highest used engine oil degradation of 66.67%, 65.47% and 58.33% with Bacillus sp., Acinetobacter sp. and Pseudomonas sp., respectively. Different species and different life stages of organisms have been demonstrated to have different susceptibilities to pollution [20]. Values are mean of three replicates, ± standard deviation (SD). Comparison of mean along the row, values followed by letter 'a' are not different significantly, while values followed by letter 'b' are significantly different at P < 0.05.

Emulsification Activity
The emulsification test revealed that the isolates have biosurfactant producing ability. Analysis of variance (ANOVA) showed significant differences between the emulsification indexes of the isolates at varying concentrations of crude oil. However, Duncan multiple range test and least significant difference (LSD) revealed that no obvious difference exists in the E24 of Arthrobacter sp. and Enterobacter sp. at 0.10 to 1.00mg/l crude oil concentrations (P > 0.05). Moreover, highest E24 of 81.80±2.8 and 84.00±4.0 was recorded in the media containing Bacillus sp. and Pseudomonas sp., respectively at 0.75mg/l crude oil concentration (Table 2). Highest emulsification index achieved by Pseudomonas sp. could be the reason for its highest crude oil removal observed in this study. Charan and Patel [21] reported that Pseudomonas spp. isolated from oil contaminated soil showed high potential of oil degradation and biosurfactant production, and the biosurfactant showed emulsification activity in kerosene, mannitol, glycerol and glucose. Thavasi et al. [22] reported that biosurfactant produced from a substrate can emulsify different hydrocarbons to a great extent, which confirmed its applicability against different hydrocarbon pollution. Biosurfactants have many properties including soaping, emulsification, dispersing; and have gained importance in the fields of enhanced oil recovery, environmental bioremediation, food processing and pharmaceuticals [23].

Conclusions
Among the isolates, Pseudomonas sp. has greater potential for bioremediation of crude oil polluted soil due to its high biodegradation efficiency and emulsification activity, thus could be exploited for effective clean up of crude oil polluted soil as well as biosurfactant production. Selection of Pseudomonas sp. with high biosurfactant producing ability for the scale up of bioremediation exercise is encouraged, in order to avoid the introduction to the environment of further chemicals, such as chemical surfactants, which are more toxic and non-biodegradable.