1. Introduction
Inadvertent disposal of petroleum products in the terrestrial environment presents a potential public health threat to human and animal populations. Due to the mobility of their toxicity, mutagenicity, and carcinogenic effects, most especially soil pollution which can lead to low output of farm products [1]. These hydrocarbons and their derivatives are capable of disrupting the inherent actions of the reproductive hormones and the ability to affect the neuroendocrine system of humans [2]. Several years of industrialization have led many nations to be dependent on petroleum hydrocarbons as the sole source of energy, which has caused significant damage to the ecological environment globally [3]. Inadvertent disposal of used engine oil, crude oil from multinationals and artisanal refining of hydrocarbons in Port Harcourt, Rivers State has been recognised as the major anthropogenic source of hydrocarbon pollution of soil and water. This has resulted in a high amount of hydrocarbons and their derivatives to run through and contaminate the ocean, farmlands and pipe-borne water. Hydrocarbons released in soil affect the biotic and abiotic components of soil. Therefore, it is essential to have vigorous effective measures for dealing with hydrocarbon contamination problems [4]. Bioremediation has emerged as the most auspicious treatment option for decontaminating polluted soil and water since its fruitful application to clean up the Exxon Valdez in 1989. It is a cost effective act that utilizes ubiquitous organisms carrying out natural attenuation in a polluted environment, most especially bacteria to cause an acceleration of the natural biodegradation process under suitable environmental conditions with nutrient availability [5].
This process proceeds from the ability of the microbes to carry out various energy-dependent processes which involve oxidation-reduction, accumulation, and precipitation, of petroleum hydrocarbon compounds of interest present in the contaminated environment [6]. This study was carried out with the aim of isolating and characterizing hydrocarbonoclastic bacteria species from soil contaminated with artisanal refined hydrocarbon oil and their possible application in bioremediation/biodegradation.
2. Materials and Methods
2.1. Sample Location/Collection
Top soil samples (0 - 15 cm) were collected from different points around the polluted site at Trans Amadi Port Harcourt, Rivers State. Samples were collected in sterile polythene bags and transported to the National Agency for Food and Drug Administration and Control Port Harcourt Area Laboratory for further analysis.
2.2. Isolation of Potential Hydrocarbon-Clastic Bacteria
Detection and enumeration of hydrocarbon degrading bacteria was done by the technique adopted by [1].
2.3. Determination of Physiochemical Parameters of Soil Samples
Soil samples pre-treatment was done according to US-EPA, (Method 3050B) [7]. Physiochemical parameters of soil such as pH, conductivity, temperature, moisture content, total nitrogen, and phosphorus were determined. Heavy metals were analysed with atomic absorption spectrophotometer.
2.4. Chromatographic Analysis
Total petroleum hydrocarbons (TPH) and polycyclic aromatic hydrocarbons (PAHs) residual were analysed using gas chromatography flame ionization detector (GC/FID) at Anal Concept Ltd.
2.5. Total Heterotrophic Bacteria Count
Heterotrophic bacteria counts were carried out after enrichment procedure on standard plate count agar (SPCA), via serial dilution with ringer’s solution. Aliquot (0.1 ml) of the diluents were plated out on Standard plate count agar and incubated at 30˚C for 24 hours. Colony forming units were afterwards counted
2.6. Hydrocarbon Utilization Bacteria Count
Hydrocarbon utilization bacteria counts were enumerated by a method adopted by [8]. The method involved dilution of appropriate homogenate samples and plating out on BHM. Hydrocarbons were incorporated through the vapour phase to potential hydrocarbon utilizers by placing sterile Whatman filter papers impregnated with 5 ml of crude oil on the lids of the inverted plates and incubated for 7 - 14 days at 30˚C.
2.7. Isolates Identification
Discrete colonies that are capable of degrading crude oil were identified with different techniques including morphological characters, biochemical test and microscopically as described in Bergey’s manual of determinative bacteriology [9].
2.8. Degradative Screening
Representative hydrocarbon utilizing bacteria isolates were screened for crude oil degradation abilities under aerobic conditions by inoculating a loop full of 24 hours old culture of each hydrocarbon utilizing bacterium into BHM broth containing 1% (v/v) crude oil. Biodegradation was recorded with the discolouration of DCPIP oxidation reduction reagent after 14 days’ incubation at 30˚C [5].
3. Results
Bioprospecting of culturable hydrocarbon degrading bacteria has intensified in decays due to the need to remediate and degrade xenobiotic.
Evaluation of soil in this study showed that the soil is chronically polluted with hydrocarbons. Physiochemical parameters of the soil are given in Table 1.
Table 2, and Table 3 presents the chromatographic quantification for total petroleum hydrocarbons (TPH) and polycyclic aromatic hydrocarbons (PAHs) for TSAS1, TSAS2, and TSAS3 polluted soil samples respectively.
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Table 1. Physiochemical characterization of the soil.
* = Variable limits in soil; - = No of Nigerian Midstream and Downstream Petroleum; Regulatory Authority and the Nigeria Upstream Regulatory Commission (NPRA and NURC) Intervention limit.
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Table 2. GC/FID Chromatographic quantifications result of TPH in soil.
Totals: 1487.24181.
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Table 3. GC/FID Chromatographic quantification result of PAHs in the soil.
Totals: 12.85287.
4. Discussion
The results of the physiochemical parameters of the soil in Table 1 indicate that the soil had been exposed to hydrocarbon pollution with organic and inorganic contaminants for years [10]. The pH of the soil is 6.6, which shows that the soil is slightly acidic. Soil pH is an important determinative factor that controls various physiochemical reactions that involves microbial growth and ability. It exhibits profound degradative efficiency on hydrocarbons through biotic and abiotic paths [11]. According to [12] [13], pH is an important catalogue parameter which exert a controlling influence on the biodegradation of petroleum hydrocarbon contaminants. The soil total nitrogen which comprises the concentrations of ammonia, ammonium, nitrite, nitrate, and dissolved particulate organic nitrogen ranged at 0.056%, phosphate 1.554 ppm, potassium 145.87 ppm, lead 7.02 ppm, cadmium 0.41 ppm, nickel 1.96 ppm, copper 1.14 ppm and the temperature of the soil ranged at 27.3˚C. The soil conductivity in this study measures the soluble salt content in the soil and is used as an overall indicator of the level of micro and macro nutrient availability in the soil [14]. Conductivity for the soil sample in this present study ranged at 125 μs/cm. This is lower than the figure gotten by [5], who reported 6000 μs/cm for soil sample around a crude oil polluted soil in Bie-Ama community. The identified soil pollutants in this study causes damages to humans, animals and the ecosystem. In humans the hydrocarbon residues such as PAHs are well known carcinogens, mutagens that cause alteration in genetic material (DNA), teratogens causing prenatal toxicity characterized by defect in developing embryo and congenital abnormalities [15]. The human health impact of TPH was reported by [16], while toxigenic and carcinogenic effect of several PAHs like benzo [a] anthracene, benzo [a] pyrene, benzo [b] fluoranthene, benzo [k] fluoranthene, indenol [1,2,3-cd] pyrene, anthracene, benzo [g, h, i] perylene, chrysene, fluoranthene, fluorine, phenanthrene and pyrene was reported by [15].
The presence of microbial activities in this study was determined by the enumeration of total heterotrophic bacteria and total hydrocarbon utilizing bacteria as presented in Table 1. Soil sample TSAS3 recorded highest cfu/gm for THB counts with isolates too numerous to count. This may be attributed to the pH of 6.6 that favours the growth of autochthonous soil microorganisms which agrees with the findings of [12] [13], which stated that the pH range optimal for biodegradation of hydrocarbons is 6 - 7.
Sample TSAS1 recorded the highest in HUB count with 1.1 × 105, followed by TSAS2 with 5.9 × 104, and TSAS3 with 5.4 × 104. In primary screening for microbial hydrocarbon degradation potential with 2, 6 dichlorophenol indolphenol, these microbes were tested in Bushnell Haas broth medium containing 1% crude oil and 0.1% DCPIP for 3 weeks and all the isolates showed better potential for crude oil degradation. DCPIP test was a rapid primary screening procedure that was performed to assess the indicator dye 2, 6 dichlorophenol indolphenol decolorization efficiency of the isolates for confirmation of crude oil biodegradation [17].
The residual TPH and PAHs found in the soil exceeded Nigerian Midstream and Downstream Petroleum Regulatory Authority, NPRA, and the Nigerian Upstream Regulatory Commission, NURC intervention limit of 1000 mg/kg and 40 mg/kg for TPH and PAHs [18].
The population of hydrocarbonoclastic bio-prospects investigated in this study showed that the bacteria were coliform Gram negative rods belonging to the phylum Gamma proteobacteria group, this corroborates with the study of [19], who stated that the growth of coliform bacteria on a wide range of aliphatic and aromatic hydrocarbons reflects a high potential of hydrocarbon utilization. Although Lactobacillus sp. isolates belonging to the phylum Bacillota was obtained in this study.
Several isolates such as Escherichia coli, Klebsiella sp., Lactobacillus sp., Enterobacter sp., Serratia sp., and Proteus sp., were screened in this study that had the ability to degrade crude oil presented in Table 4.
The isolation of Lactobacillus sp., andKlebsiella sp., in this study corroborates with the study of [20], and [21], who reported similar microbes for their effectiveness in the mineralization of hydrocarbons.
In the study conducted by [22] Proteus sp., was dominantly used for hydrocarbon biodegradation.
The detection and enumeration of Enterobacter sp., Escherichia coli, Serratia sp., Klebsiella sp., and Proteus sp., corroborates with the findings of [8]. The findings of [23], also reported these isolates as known crude oil degraders in a study conducted in Bodo, Ogoniland and Nembe waterside Port Harcourt.
[24] characterized Klebsiella sp. for their biosurfectant production in a hydrocarbon polluted soil in ogoni land.
In the study conductecd by [25], Escherichia coli, Enterobacter sp., Klebsiella sp., Proteus sp., and Serratia sp., was reported for their crude oil utilization ability which is in line with the findings of this current study.
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Table 4. Characterization of bacterial isolates.
R = rod, - = negative, + = positive.
5. Conclusion
The present study revealed the presence of indigenous microorganisms from petroleum hydrocarbon contaminated soil in Trans Amadi, Port Harcourt as well as known genera of hydrocarbon utilizing bacteria. There is a propensity that the increasing viability of hydrocarbon utilizing microbes in this polluted soil could influence the mechanism of biodegradation. The experimental result determined shows that indigenous petroleum hydrocarbon utilizing bacteria could be isolated and used effectively in the polluted site for hydrocarbon bioremediation/biodegradation. Furthermore, the application of metagenomics analysis, and other OMIC techniques friendly to the ecosystem should be employed to help increase our understanding of the vast microbial diversity in polluted soil.
Acknowledgements
The authorsacknowledge The National Agency for Food and Drug Administration and Control (NAFDAC) Port Harcourt Area Laboratory for their support.
Authors’ Contribution
Asime Oba conceived and carried out the work; Asime Oba, Barka John and Okeke Uchechukwu were involved in data analysis and interpretation; Asime Oba, Barka John and Okeke Uchechukwu analysed the data and took note. Asime Oba and Barka John drafted the manuscript