Bioremediation of soil polluted with spent lubricating oil using bacteria isolated from soil in Abuja Metropolis

BIOMED Natural and Applied Science, 2022; 2(3):33-40,

Abstract

Background: The presence of spent hydrocarbon in soil is a serious problem to the environment hence study on bioremediation of soil polluted with auto-mechanic oil in Abuja Metropolis was carried out. Methods: A total of twenty (20) soil samples were collected, bacteria were isolated from the contaminated soil and identified using standard microbiological methods. The spent hydrocarbon utilization was determined using Atomic Adsorption UV Spectrometer. Results: The total viable count of the bacteria was 1.07 x 106 from Apo Mechanic village, 1.10 x 106 from Utako Mechanic workshops, 0.40 x 106 from Gwarinpa Mechanic workshops and 2.04 x 106 from Area one Mechanic workshops. The percentage occurrence of bacteria from Apo Mechanic village was Enterobacter species 40.0%, Pseudomonas synxantha 60.0%, Bacillus zanthoxyli 40.0% and Proteus vulgaris 20.0%. Utako Mechanic workshops were Enterobacter kobei 20.0%, Pseudomonas synxantha 40.0%, Bacillus zanthoxyli 20.0% and Proteus vulgaris 40.0%. Gwarinpa Mechanic workshops were Pseudomonas synxantha 20.0% and Bacillus zanthoxyli 20.0%. Area one Mechanic workshops were Enterobacter kobei 40.0% and Pseudomonas synxantha 40.0%. The effect of days on utilization of spent hydrocarbon showed that Pseudomonas synxantha had highest utilized of spent hydrocarbon 19.55mg/ml after 21 days. The effect of pH on utilization of spent hydrocarbon show that at pH 7.5, Enterobacter kobei, Bacillus zanthoxyli and Proteus vulgaris species had the highest utilization of spent hydrocarbon ranging from 5 9.33mg/ml-12.70mg/ml. Effect of temperature on utilization of spent hydrocarbon showed that at 28OC Enterobacter kobei, Pseudomonas synxantha, Bacillus zanthxyli and Proteus vulgaris had the highest utilization of spent hydrocarbon ranging from 5.51mg/ml- 11.11mg/ml. the bacteria isolated from the contaminated soil have the ability to utilized the hydrocarbon if the soil is amended with some mineral element as shown in this study. Conclusion: In conclusion bacteria isolates effectively bioremediated the automechanic oil polluted soil with a reduction of hydrocarbon pollutants.

Corresponding Author(s)

Correspondence; Toyosi Michelle Adebayo; Email: tma2035@gmail.com

Citations

Makut, M.D., Adebayo, T.M., Owuna, J.E.C(2022). Bioremediation of soil polluted with spent lubricating oil using bacteria isolated from soil in Abuja Metropolis. BIOMED Natural and Applied Science, 2(3):33-40, https://doi.org/10.53858/bnas02033340

1.0 Introduction.                                                                                   

Engine oil could simply be defined as a thick mineral liquid applied to a machine or engine to reduce friction between the moving parts of the machine [1]. Oil released into the environment is a well-recognized problem in today’s world. Oil spills affect many species of plants and animals in the environment, as well as humans [2]. Spent/Used engine oil is defined as used lubricating oils removed from the crankcase of internal combustion engines [3].

Spent engine oil is a common and toxic environmental contaminant not naturally found in the environment [4], large amount of them are liberated into the environment when the motor oil is changed and disposed into the soil which is a common practice by motor mechanics and generator mechanics including small scale engine oil sellers along the road [5].  Engine oils are altered during use by vehicles, motorbikes, generators and other machinery because of the breakdown of additives, contamination with the products of combustion and the addition of metals from the wear and tear of the engine.

Bioremediation is, therefore, the application of naturally occurring process by which microorganisms transform environmental contaminants into harmless end products [5]. Bioremediation makes use of indigenous oil consuming microorganisms, called petrophiles by enhancing and fertilizing them in their natural habitats [6]. Petrophiles are very unique organisms that can naturally degrade large hydrocarbons and utilize them as a food source [7]. Microbial remediation of a hydrocarbon contaminated site is accomplished with the help of a diverse group of microorganisms, particularly the indigenous bacteria present in soil. These microorganisms can degrade a wide range of target constituents present in oily sludge [6] there by, can be used in cleaning up contaminated sites.

Many studies have been reported on bioremediation of hydrocarbon pollutants degrading bacteria from auto mechanic workshops’ soils but just a few had reported on bioremediation of soil contaminated with used motor oil. Little work has been reported in literature to determine the bioremediation potential of the isolated bacterial consortium by measuring the percentage oil loss and their enzyme activities as a monitoring tool for bioremediation process which is the major focus of this work [7]. Therefore, this study is aimed at bioremediation of soil polluted with auto-mechanic oil using bacteria isolated from soil in Abuja metropolis.

2.0 Materials and Methods

2.1 Chemicals and Reagents

The chemicals and reagents that were used in this study include: England lactophenol cotton blue stain, HCL, NaOH, NH4Cl, KH2PO4, MgSO4,7H2O, ethanol, Xylene solution, Creatinine, Potassium hydroxide and Kovac’s reagent, from BDH Chemicals Ltd, England; Ethidium bromide, Iodine solution, EDTA and Glycerol from Sigma chemical Ltd, England; and Agarose gel from Agarose from Schwarz/ Mann Biotech. Nutrient agar and basal medium (minimal salt medium –MSM)

2.2 Study Area

The study area was carried out in Federal Capital Territory Abuja, Nigeria. Abuja is located in the center of the country. Federal Capital Territory is located between latitude 9o4’N of the equator and longitude 7o29’E and situated on an altitude of 360m above sea level.

2.3 Collection of Samples

The study sites were 15 different mechanic workshops situated in different locations in Area One, Utako, Gwarinpa and Apo mechanic village. Apart from visual observation, the attendants at the auto-mechanic workshops were asked questions pertaining to the sites with heavy oil  spillage. The sites with the oil spillage had a characteristic black color and the surfaces were hard.  They also had no grasses growing on them.  Samples were collected at different sites with a shovel to the depth of 0-5cm in triplicates using random sampling technique from each mechanic workshop, augured, and bulked together and put in well labeled black polythene bags and transported to the laboratory for analysis. The samples were subjected to microbiological analyses. Used motor engine oil was also collected from one of the mechanic workshops as the fresh engine oil sample.

2.4 Isolation of bacteria

 The isolation of bacteria was carried out using a technique described by Barathi and Vasudvan [9]. The bacteria were isolated using Nutrient agar and basal medium containing 0.3% yeast extract supplemented with 10% V/V oil substrates. The oil substrates were represent equivalent mixture of Gasoline, Engine oil, Diesel oil and spent engine oil. One (1) gram of the hydrocarbon contaminated soil sample was suspension in a test tube containing 9 ml of sterile water to make the soil suspension.  Ten-fold serial dilution was made by transferring one ml of the soil suspension to another test tube containing 9 ml of sterile distilled water. These steps were repeated seven times to obtain a dilution of 10-7. From the fourth test tubes, 0.2ml of the aliquot were spread on Nutrient agar and basal medium and incubated at 28oC for 24hours [10]. 

2.4.1 Total Viable Count

The mean total bacteria present in the samples were determined at weekly interval using spread plate method with Nutrient agar (NA) medium. After tenfold dilution using phosphate buffer dilution saline and 0.1ml of appropriate dilution was plated in triplicates, to be incubated for 24-48 hours at room temperature after which the colonies were counted [11].

2.4.2 Identification of Bacteria isolated from contamination with hydrocarbon soil

Colonies was chosen from each of the cultured plate on the bases of their colonial and morphological similarities. Pure bacterial colonies was identified using Gram staining reaction and biochemical tests according to the methods Cheesbrough.

2.5 Molecular Identification

2.5.1 DNA extraction

Five milliliters of an overnight culture of the bacterial isolate in Luria Bertani (LB) broth were spun at 14000rpm for 3 min. The cells were re-suspended in 500µl of normal saline and heated at 950C for 20 min. The heated bacterial suspension was cooled on ice and spun for 3 min at 14000rpm. The supernatant containing the DNA was transferred to a 1.5ml microcentrifuge tube and stored at -20oC for other downstream reactions.

2.5.2 DNA quantification

The extracted genomic DNA was quantified using the Nanodrop 1000 spectrophotometer. The software of the equipment was lunched by double clicking on the Nanodrop icon. The equipment was initialized with 2µl of sterile distilled water and blanked using normal saline. Two microliters of the extracted DNA were loaded onto the lower pedestal, the upper pedestal was brought down to contact the extracted DNA on the lower pedestal.

2.5.3 16S rRNA Amplification

The 16s rRNA region of the rRNA genes of the isolates were amplified using the 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R: 5′-CGGTTACCTTGTTACGACTT-3′ primers on ABI 9700 Applied Biosystems thermal cycler at a final volume of 25 microlitres for 35 cycles. The PCR mix included: the X2 Dream taq Master mix supplied by Inqaba, South Africa (Taq polymerase, DNTPs, MgCl), the primers at a concentration of 0.4M and the extracted DNA as template. The PCR conditions were as follows: Initial denaturation, 95°C for 5 minutes; denaturation, 95°C for 30 seconds; annealing, 52°C for 30 seconds; extension, 72ºC for 30 seconds for 35 cycles and final extension, 72°C for 5 minutes [12].

2.5.4 Agarose Gel Electrophoresis of the 16SrRNA gene from the bacteria isolates

The agarose gel electrophoretic separation of digested 16S rRNA gene was carried out as described previously, 8µl of PCR products stained with ethidium bromide was loaded into 1.0% (wt/vol) agarose gel wells with a molecular marker run concurrently at 120 V for 30 min. The DNA bands were visualized and photographed under UV light 595nm.

2.6 Effect of pH on utilization of spent hydrocarbon substrate from contaminated soil

The effect of pH was carried out following a method described by Makut and Majekodunmi, [13]. One hundred (100) ml of the Basal medium substrate amended was transfered into different conical flasks. The pH ranges was adjusted to, 4.5, 5.5 6.5 and 7.5 for degradation media using 1.0 N HCl to adjusting the pH of the media before autoclaving.

2.7 Effect of Temperature on utilization of spent hydrocarbon substrate from contaminated soil 

Effect of temperatures was carried out following a method described by previous study. One hundred (100) ml of the degradation media was transfer into different conical flasks and the degradation media was incubated at 28oC, 32oC, 35oC and 38oC

2.8 Total utilization of spent hydrocarbon

Total utilized spent hydrocarbon content of the degradation media samples were determined using UV Visible spectrophotometer methods according to the toluene extraction method Makut and Majekodunmi, [13]. Fifteen miligram (15ml) of each of the sample was transfer into a test tube, and then 1ml of 60ug/ml of 1-chlorooctadecane surrogate standard was added. Then 30 milliliters of dichloromethane (extraction solvent) was added to extract oil in the soil. After shaking vigorously in water bath for 5hrs, the mixture were allowed to stand for 60 minutes and then filtered through Whatman No.1 filter paper fitted with cotton wool and sodium sulphate into a clean beaker washed with methylene chloride. Residue were then washed with 20ml extracting solvent and then filtered through funnel. The extracted oil will be transferred to vial and placed on a UV Visible spectrophotometer chamber for analysis. The amount of waste lubricating oil degraded was calculated by subtracting the weight of residual waste lubricating oil from weight of the initial waste lubricating oil, divided by the weight of the initial waste lubricating oil and then multiplied by 100.

HR (%) = (Weight of oil before remediation) – (Weight of oil after remediation)×100

                 Weight of oil before remediation

Where HR = Hydrocarbon utilization

3.0 Results

3.1 Total mean viable counts

The total mean viable count of bacteria isolated from contaminated soil of hydrocarbon in Mechanic workshops is as given in Table1. The total viable count of the bacteria was 1.07 x 106 from Apo Mechanic village, 1.10 x 106 from Utako Mechanic workshops, 0.40 x 106 Gwarinpa Mechanic workshops and 2.04 x 106 from Area one Mechanic workshops respectively.

Table 1: Total mean viable count of bacteria isolated from contaminated soil of hydrocarbon in Mechanic workshops

Sample locationTotal viable count (cfu/g) x106
Apo Mechanic village1.07 x 106
Utako1.10 x 106
Gwarinpa0.40 x 106
Area one2.04 x 106

Table 2: Cultural, Morphology and Biochemical characteristics of Bacterial Isolates

CulturalGramsCatNitCoaGelUreGluSucInference
Brown on MaC and Greenish Colonies on NA+++  +Pseudomonas synxantha
Purple on MaC and yellowish on NA+++++Enterobacter kobei
Milkfish and dried on NA+++Bacillus zanthoxyli
Mucoid and brownish on NA++Proteus vulgaris

KEY: Cat-catalase, Nit- nitrate, Coa-coagulase, Gel-gelatin, Ure- urea, Glu- glucose, Suc-Sucurcose

Plate 1: Agarose gel electrophoresis of the 16SrRNA gene of bacteriaisolates. L represents the molecular ladder, Lane 1 = Enterobacter kobei strain DSM 13645, Lane 2 = Pseudomonas synxantha,.strain KENGFT3, Lane 3 = Bacillus zanthoxyli, Lane 4 = Proteus vulgaris strain FDAARGOS_556.

3.2 The cultural, morphology and biochemical characteristics of the bacteria isolate

The cultural, morphology and biochemical characteristics are as shown in Table 2. The occurrence of bacteria isolated from contaminated soil of hydrocarbon in mechanic workshops in Abuja is as given in Table 3. Out of 5 samples collected from each location. Location A; Enterobacter kobei had 40.0%, Pseudomonas synxantha 60.0%, Bacillus zanthoxyli 40.0% and Proteus vulgaris 20.0%. From location B; Enterobacter kobei had 20.0%, Pseudomonas synxantha 40.0%, Bacillus zanthoxyli 20.0% and Proteus vulgaris 40.0%. From location C; Pseudomonas sp had 20.0%, Bacillus zanthoxyli 20.0% and Proteus vulgaris 0.00%. From location D; Enterobacter kobei had 40.0% and Pseudomonas synxantha 40.0%.

Table 3: Occurrence of Bacteria Isolated from contaminated soil of hydrocarbon in mechanic workshops in Abuja in percentages

Bacterial No samples A  B  C
Enterobacter kobei52(40.0)1(20.0)0(00)2(40.0)
Pseudomonas synxantha53(60.0)2(40.0)1(20.0)2(40.0)
Bacillus zanthoxyli52(40.0)1(20.0)1(20.0)0(00)
Proteus vulgaris51(20.0)2(40.0)0(00)0(00)

Key: location A= Apo Mechanic village, B= Utako, C= Gwarinpa, D= Area one. Data are mean ± SEM of triplicate determination

3.3 Effect of time on microbial on utilization of spent oil

The effect of days on utilization of spent oil is as given in Table 4. After 7days Enterobacter kobei were able to utilized 2.20mg/ml of the spent oil, after 14 day it had 5.70mg/ml utilization, 21days it utilized 9.90mg/ml and after 30days it had 5.70mg/ml utilization from the initial concentration of the spent oil. Pseudomonas synxantha had 5.10mg/ml utilization after 7days, 9.21mg/ml after 14days, 19.55mg/ml after 21days and 15.41mg/ml after 30 days.Bacillus zanthoxyli had 4.11mg/ml after 7days, 7.40mg/ml after 14days, 14.55mg/ml after 21days and 12.01mg/ml after 30days and Proteus vulgaris had 1.21mg/ml after 7days, 3.22mg/ml after14days, 11. 00 mg/ml after 21days and 6. 20 mg/ml after 30days. The control showed no reduction.

Table 4 Effect of days on utilization of spent hydrocarbon by bacteria isolated from soil in mechanic workshops

 Concentration (mg/ml) at different days
Isolates                                    Initial                    7                    14                       21                     30
Enterobacter kobei26.85± 0.552.20 ± 0.195.70±0.2814.10 ± 0.109.90 ± 0.20
Pseudomonas synxantha26.85± 0.555.10±0.189.21±1.2819.55±1.2815.41±0.48
Bacillus zanthoxyli26.85± 0.554.11±0.087.40±2.0814.55±1.0112.01±0.66
Proteus vulgaris26.85± 0.551.21±0.053.22±0.5411.00±0.166.20±1.28
Control26.85± 0.550.00± 0.000.00±0.000.00±0.000.00±0.00

Data are mean ± SEM of triplicate determination

3.4 Effect of pH on microbial on utilization of spent oil

The effect of pH on utilization of spent oil by bacteria isolated from mechanic workshop is as shown in Table 5. Enterobacter kobei showed utilization ability of 2.10mg/ml at pH 4.5, 3.90mg/ml at pH5.5, 5.22mg/ml at pH6.5 and 12.70mg/ml at pH7.5. Pseudomonas synxantha had 4.15mg/ml at pH4.5, 5.31mg/ml at pH 5.5, 14.11mg/ml at pH6.5 and 8.11mg/ml at pH7.5.Bacillus zanthoxyli utilized 4.05mg/ml at pH4.5, 4.11mg/ml at pH5.5, 6.51mg/ml at pH6.5 and 9.33mg/ml at pH7.5 and Proteus vulgaris had 2.20mg/ml at pH4.5, 3.80mg/ml pH5.5, and 4.01 mg/ml at pH6.5 and 7.52mg/ml at pH7.5 respectively

Table 5: Effect of pH on utilization of spent hydrocarbon by bacteria isolated from soil in mechanic workshops

 Concentration (mg/ml) at different pH
IsolatesInitial4.5                                5.5                      6.5                    7.5
Enterobacter kobei26.85± 0.552.10 ± 0.113.90 ± 0.205.22 ± 0.0912.70±1.48
Pseudomonas synxantha26.85± 0.554.15±1.085.31±0.2814.11±0.188.11±0.18
Bacillus zanthoxyli26.85± 0.554.05±0.014.11±0.166.51±0.329.33±2.08
Proteus vulgaris26.85± 0.552.20±0.103.80±0.514.01±0.257.52±0.14
Control26.85± 0.550.00±0.000.00±0.000.00±0.000.00±0.00

Data are mean ± SEM of triplicate determination

3.5 Effect of temperature on microbial on utilization of spent oil

Table 6 shows the effect of temperature on spent hydrocarbon utilization by some bacteria isolated from mechanic workshops in Abuja. At temperature of 28OC Enterobacter kobei utilized 10.12mg/ml of the initial concentration of 23.85mg/l of the spent hydrocarbon, at 32OC it utilized 6.10mg/ml, 35OC it utilized 4.30mg/ml and at 38 OC it had 4.01mg/ml.Pseudomonas synxantha utilized 11.11mg/ml at 28 OC, 7.08mg/ml at 32 OC, 5.61mg/ml at 35 OC and 4.15mg/ml at 38 OC.Bacillus zanthoxyli was able to utilized 9.21mg/ml at 28 OC, 6.63mg/ml at 32 OC, 4.88mg/ml at 35 OC and 3.05mg/ml at 38 OC while Proteus vulgaris 5.51mg/ml at 28 OC, 5.00mg/ml at 32 OC, 3.00mg/ml at 35 OC and 2.21mg/ml at 38 OC respectively.

Table 6: Effect of temperature on utilization of spent hydrocarbon by bacteria isolated from soil in mechanic workshops

 Concentration (mg/ml) at varying temperature
IsolatesInitial28OC32OC35OC38OC
Enterobacter kobei26.85± 0.5510.12 ± 1.196.10±0.184.30 ± 0.064.01 ± 0.31
Pseudomonas synxantha26.85± 0.5511.11±1.117.08±0.685.61±0.244.15±1..18
Bacillus zanthoxyli26.85± 0.559.21±0.026.63±0.084.88±1.123.05±0.21
Proteus vulgaris26.85± 0.555.51±0.255.00±0.143.00±0.012.21±0.50
Control26.85± 0.550.00±0.000.00±0.000.00±0.000.00±0.00

Data are mean ± SEM of triplicate determination

4.0 Discussions

The indiscriminate disposal of waste oil effects the environment and reduction in oxygen supply to microorganisms [14]. Bioremediation process is a microorganism mediated transformation or degradation of contaminants into non-hazardous or less hazardous substances [14,15]. It is an attractive approach for cleaning up of hydrocarbons from environment because it is a simple technique, easy to maintain [16], applicable over large areas, cost effective and leads to complete destruction of the contaminant. Many physical, chemical, and environmental factors like temperature soil moisture, soil acidity and alkalinity etc affects the process of biodegradation of hydrocarbons [17]. Bacteria and fungi are known to be the principal agents of biodegradation of hydrocarbons. Fungi have a higher tolerance to the toxicity of hydrocarbons due to their physiology and adaptation to such variations in the environment and have the mechanism for the elimination of spilled oil from the environment.

In this study the total viable count observed was high but lower than the one reported by Bada et al. [18] and similar to the study reported by Gallego et al. (2010), The reasons for the significant low counts of bacteria in the contaminated soil may be due to the non-availability of appreciable quantities of nitrogen, phosphorus and oxygen in the contaminated soil which enhances the reduction of bacteria population.

The bacteria isolated from different mechanic workshops contaminated with spent hydrocarbon were Enterobacter kobei, Pseudomonas synxantha, Bacillus zanthoxyli and Proteus vulgaris this is in agreement with the study reported by Onuoha et al. [19]. This means that these bacteria exhibited responses and potential to breakdown petroleum hydrocarbon and utilize it as source of energy and carbon in their natural ability.  These bacteria species has been implicated in the hydrocarbon degradation by the authors [19]. It has also been observed that some bacteria are more abundant in areas of high concentration of hydrocarbons. These bacteria are actively oxidizing the hydrocarbons, and this is considered as another source of carbon for use in the ecosystem Bada et al. [18]

The effect of days on utilization of spent hydrocarbon as observed in this study was indifferent from studies reported by Olubunm et al. [20] who reported high utilization of spent oil at 28days but in this study it was observed that after 28days the amount of the reduction or utilization by the bacteria dropped suggesting decline in the population of the bacteria. Also it was observed that pH plays important role in the degradation or utilization of spent oil by different bacteria as observed in this study which is similar to studies reported by different authors such as Bada et al. [18]; Akpoveta et al., [21] and Yakubu (2007) who reported high utilization of spent oil at pH of 6.5 and 7.5 which is in agreement with our findings in this study.  Soil nutrient decreased after contamination with petroleum products. Akpoveta et al. [21] observed significant reduction in pH and available nutrients in soil on simulation of soil with kerosene. Increase in soil acidity as a result of petroleum hydrocarbon in soil is a concern for agricultural soil because low pH values are associated with adverse soil condition which includes reduced availability of nutrients, reduced microbial activity. The effect of temperature on utilization of spent oil from different mechanic workshop as observed in this study showed that high temperature affects the utilization of hydrocarbon. It was observed that at temperature of 28OC were was high utilization of spent hydrocarbon than other temperature tested in-vitro and this is similar to study reported by Schafer et al. [22] who reported decrease in population of bacteria in soil and water due to high temperature in area pollution or contaminated with hydrocarbon.

5.0 Conclusion

From this study it was observed that different bacteria namely Pseudomonas synxantha, Bacillus zanthoxyli, Proteus vulgaris and Enterobacter kobei isolated from soil contaminated with inMechanic workshops were able to show various ability to utilize spent hydrocarbon in varying amounts. This study however, recommended that further studies on the use of bacteria isolates from polluted soils should be carried out to enhance their potential in hydrocarbon utilization

Conflict of Interest: The author declared no conflict of interest exist

Funding: No funding was received 

Authors contribution: This work was conducted with the collaboration of all authors: Author TMA conducted the research and wrote the manuscript. Author MDM supervised the work. Author JEO CO-supervised the work. All authors read and approved the final version of the manuscript

Acknowledgement: We acknowledged all the lecturers and the technical staff of the Department of Microbiology, Nasarawa State University

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