Identification of Catabolic Genes Involved in the Degradation of Used Engine Lubricant- Contaminated Soil by Lysinibacillus odysseyi and Bacillus sp. (in: firmicutes)
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This study aimed to identify selected catabolic genes in two indigenous hydrocarbon-degrading bacteria involved in the biodegradation of used engine lubricant-contaminated soil. Used engine lubricant-contaminated and uncontaminated soil samples were collected, and their physicochemical characteristics were evaluated. The results indicated that water holding capacity, potassium, nitrogen, and phosphate were higher in used lubricant-uncontaminated soil than in used lubricant-contaminated soil but vice versa for pH, total carbon, cation exchange, organic carbon, nickel, and lead levels. Culturable heterotrophic and hydrocarbon-utilizing bacterial counts were carried out on both Nutrient agar and Mineral Salt Medium (MSM) amended with used engine lubricant. The counts for heterotrophic bacteria (3.8×108±0.21 cfu/gm) were higher than that of hydrocarbon-utilizing bacteria (2.2×108±0.15 cfu/gm). All isolated bacteria (16) underwent screening for hydrocarbon degradation potential. Lysinibacillus odysseyi and Bacillus sp (in: firmicutes) emerged as the best oil degraders. Further screening of the chromosomal and plasmid DNA of the two bacteria was done to determine the presence and location of some selected catabolic genes (NidA, AlkB, NahH, NahAC, and Alma). The presence of Alkane monooxygenase (AlkB) and Naphthalene dioxygenase (NahAC) genes was confirmed in both isolates, while Pyrene dioxygenase (NidA) was confirmed in Bacillus sp. (in: firmicutes) only. The location of AlkB was confirmed to be both plasmid and chromosome, while NidA and NahAC genes were confirmed to be the plasmid. In conclusion, the soil contaminated with used engine lubricant contained indigenous bacteria, Lysinibacillus odysseyi, and Bacillus sp. (in: firmicutes), the two bacteria with the highest degradation potential, contained catabolic genes, monooxygenase (AlkB), NidA, and dioxygenase (NahAC). Therefore, they are effectively used as engine lubricant degraders, and the possibility of horizontal gene transfer can be used in important industrial applications and is recommended for the bioremediation of petroleum compounds.
Introduction
The careless disposal of used engine lubricants in mechanic workshops has become a primary cause of environmental pollution (Muzeet al., 2020). The use of petroleum fuels, including petrol, diesel, engines, and grease oil in motor repair workshops causes heavy and extensive spillage of these products into the environment. Waste generated in mechanic workshops during service, repair, and cleaning of vehicle parts is usually not well disposed of. The mechanics usually dispose of, or spill spent motor oils, lubricant oils, and other solvents that contain hydrocarbons from petroleum around their mechanic shades. The major pollutants found in the soil of most mechanic workshops are made up of these waste lubricants (Iwegbue, 2007; Stephenet al., 2021). In addition to products of decomposition and heavy metals from engine part wear, the used engine lubricant contains a variety of compounds, such as additives, PHs, and chlorinated biphenyls (Wanget al., 2000; Kidman & Boehlecke, 2011). These environmental pollutants often bioaccumulate in the soil and eventually find their way into plant tissues, nearby surfaces, and groundwater, posing a huge threat to the health and livelihood of communities using such waters. Air, water, and soil exposed to hydrocarbons are severely contaminated, whether by intentional discharge or from human activities (Okwuteet al., 2015; Stanleyet al., 2017).
Depending on the kind and concentration of toxic components present, different remediation procedures are utilized for polluted soils, such as chemical oxidation, soil stabilization, physical approaches, and bioremediation. However, researchers have given bioremediation the most attention of all the remediation techniques because it is not only generally safe but also an affordable choice for treating contaminated sites (Erdogan & Karaca, 2011). According to various experts, several variables determine how quickly oil degrades (Jahangeer & Kumar, 2013; Varjani & Upasani, 2017). The presence of sufficient metabolically capable bacteria is a crucial requirement for bioremediation.
According to published study findings, numerous microbial populations found in various environments have proven to be capable of breaking down both low and high-molecular-weight (chrysene, pyrene, anthracene, acenaphthene, and naphthalene) hydrocarbons as energy sources using various enzyme-mediated mechanisms (catabolic genes). Since hydrocarbons are extremely complex and diverse compounds, a specific enzyme is responsible for initiating the breakdown of each component. Naphthalene, for example, is broken down by the NahAC gene (Duarteet al., 2017), AlkB for breaking down alkanes (Williamset al., 2022), and PhnAC for breaking down phenanthrene (Yinet al., 2020). Distinct genes code for distinct degradation pathways, according to multiple perspectives that have described the processes (physiological and enzyme-mediated) used by microorganisms in the breakdown of petroleum and petroleum products (Austin & Groves, 2011; Ukiweet al., 2013). An improved bioremediation procedure may be achieved by understanding the type, location, and presence of these genes on specific hydrocarbon-utilizing microorganisms. Therefore, this study seeks to screen the biodegradation capacity and the presence/location of catabolic genes of two autochthonous hydrocarbon-utilizing bacteria isolated from used engine lubricant-polluted soil.
Materials and Methods
Soil Sample Collection and Physicochemical Analysis
Soil polluted with used engine lubricant was obtained from a mechanic workshop. In contrast, engine lubricant-uncontaminated soil was obtained from the University of Abuja Biology Department Garden both in Gwagwalada, Federal Capital Territory, Nigeria, with the use of a soil boring machine at depths 0.5 m, 1 m, 1.5 m, and 2 m from various points. Within two hours of collection, soil samples were aggregated for uniformity and transported in an ice box to the laboratory for analysis. Preliminary physicochemical characteristics (water holding capacity, pH, total carbon, cation exchange, total organic carbon, nickel, potassium, nitrogen content, phosphate, and lead) of both used engine oil-contaminated and uncontaminated soils were assessed via the use of prescribed techniques outlined by the American Public Health Association (APHA) (2008) and American Society for Testing and Materials (ASTM) (2007).
Culturable Heterotrophic Bacteria and Hydrocarbon-Utilizing Bacteria Counts
For culturable heterotrophic bacteria count, 1 g of used engine lubricant-polluted soil (wet weight) in 0.85% normal saline was homogenized using a vortexing machine. Plate count agar was used for the suspensions at ten-fold decimal dilutions, and they were incubated for 24 hours at 30°C. To conduct oil-degrading bacterial counts, 1 g of soil contaminated by spent oil was added into 100 ml of Bushnell-Haas Mineral Salts (BHMS) that had been augmented with 0.5% (v/v) of used motor oil as the only carbon supplier for bacteria growth. The medium was then grown in a shaker incubator for five days at 37°C and 130 rpm. Subsequently, serial dilution was performed using the method reported by Corteset al. (2012). For additional examination, discrete microbial populations were counted and replated on Nutrient agar.
Screening Isolates for Used Engine Lubricant Degradation Potential
To screen for the used engine lubricant degradation capability of the isolates and to select the two bacteria with the highest hydrocarbon degradation potential, screening by turbidometry was conducted. Using a standard loop, culture at 18 hours post inoculation of each representative bacterial isolate was picked and inoculated into Bushnell-Haas Mineral Salt (BHMS) Medium enriched with 1% used engine lubricant. The culture was grown in a rotary shaker incubator set at 150 revolutions per minute (RPM) at 30°C for 21 days to screen the isolates for their capacity to degrade oil under aerobic conditions. Optical density readings were recorded on days 0, 7, 14, and 21, respectively, with a standard spectrophotometer at 600 nm optical density using water as a control.
DNA Extraction and PCR-based 16S rRNA Gene Analysis
Using a Zymo research DNA prep kit, the genomic DNA of both bacteria was extracted. The bacterial identification was carried out by gene amplification of the 16S gene using RIBOS 1 (for) GGACTACAGGGTATCTAAT and RIBOS 2 (rev) AGAGTTTGATCCTGG primer. A thermal cycler (Bioer, Japan) was used to amplify them. Each 20 µL Hotstart PCR reaction mixture volume contained approximately 50 ηg of purified DNA, 0.1 µM primers, 250 µM of each dNTPs (3 mM MgCl2, 0.5 U of Taq DNA Polymerase, in 10 mM Tris-HCl (pH 9) and 50 mM KCl. The temperature profile is one cycle at 94°C for 5 minutes, 25 cycles at 94°C for 1 minute, 52°C for 1 minute, 72°C for 1 minute, and a final extension at 72°C for 7 minutes. Using 1.5% agarose gel electrophoresis, the amplified products were evaluated.
Detection of Catabolic Genes from the Isolates
Five catabolic genes targeting Pyrene dioxygenase (NidA), Alkane monooxygenase (AlkB), Catechol 2, 3 dioxygenase (NahH), Naphthalene dioxygenase (NahAC), and Flavin-binding monooxygenase (Alma) were used to screen for these enzymes in two of the bacteria with highest hydrocarbon utilizing ability to ascertain their capacities and potentials for catabolism. Also, plasmid and chromosomal DNA analyses were performed on the two isolates to determine the location of the genes. The process involved primer-specific PCR amplification of bacteria DNA sequences.
Sequencing
Using an ABI3130 XL genetic analyzer incorporating the ABI Big Dye Terminator Cycle sequencing 2kit version 3.1, amplified PCR products were purified and sequenced with universal primer set 27F and 1492R. Sequencing was performed by Inqaba Biotech, West Africa Ltd. Comparisons were made between the unique fragment’s nucleotide sequences and those found in the GenBank database using the NCBI BLAST program.
Results
Physicochemical Parameters of Used Engine Lubricant-Polluted and Used Engine Lubricant-Unpolluted Soil
The physicochemical properties of both used engine lubricant-uncontaminated and used engine lubricant-contaminated soils were determined and are shown in Fig. 1.
The results showed that the levels of water-holding capacity, Potassium, Nitrogen, and Phosphate were higher in used engine lubricant-uncontaminated soil than in used engine lubricant-contaminated soil. However, the pH, total carbon, cation exchange, Total organic carbon, nickel, and lead levels were lower in used engine lubricant-uncontaminated soil than in used engine lubricant-contaminated soil.
Culturable Heterotrophic Bacteria and Hydrocarbon-Utilising Bacteria
Culturable heterotrophic and hydrocarbon-utilizing bacteria counts were carried out by plating ten-fold serial dilution on both nutrient agar and BHMS amended with used engine lubricant as the sole carbon source. The results are presented in Table I below.
Isolated bacteria | Result (cfu/g) |
---|---|
Total heterotrophic bacteria counts | 3.8 × 108 ± 0.21 |
Total hydrocarbon-utilizing bacteria counts | 2.2 × 108 ± 0.15 |
The counts for total heterotrophic bacteria were higher than those of total hydrocarbon-utilizing bacteria.
Screening Isolates for Used Engine Lubricant Degradation Potential by Turbidometry
Screening by turbidometry was carried out to ascertain the ability of the isolates to break down used engine lubricant. The absorbance values for the sixteen bacterial isolates varied at various days (0, 7, 14, and 21), as shown in Fig. 2.
The isolate with the best potential for breaking down used engine lubricant (2.11) is Isolate OD2 (Lysinibacillus odysseyi). Isolate OD11 Bacillus sp (in: firmicutes) showed the second-best potential for used engine lubricant breakdown (1.89). With an optical density of 0.99, OD1 demonstrated the lowest potential for the breakdown of used engine lubricant. For the control, which had no bacterial isolate but contained only used engine lubricant in a mineral salt medium, the absorbance was 0.38 at day 21.
Phylogenetic Analysis of 16S rRNA Gene Sequences for Bacterial Identification
Molecular identification by PCR revealed isolates OD02 and OD11 to be Lysinibacillus odysseyi and Bacillus sp. (in: firmicutes), respectively (Fig. 3).
PCR-based Detection of Functional Genes
The detection of five catabolic genes in Lysinibacillus odysseyi and Bacillus sp. (in: firmicutes) was done based on amplification by PCR. The presence of alkane monooxygenase (AlkB) and naphthalene dioxygenase (NahAC) genes was confirmed in both isolates. Pyrene dioxygenase (NidA) was confirmed in Bacillus sp. (in: firmicutes) only. However, no signal was detected for Catechol 2, 3 dioxygenase (NahH), and Flavin-binding monooxygenase (Alma) genes. The findings are presented in Table II and Fig. 4.
Genes | Lysinibacillus odysseyi | Bacillus sp. (in: firmicutes) | Location |
---|---|---|---|
AlkB | + | + | Plasmid/chromosome |
NahH | – | – | – |
NidA | – | + | Plasmid |
NahAC | + | + | Plasmid |
Alma | – | – | – |
Discussion
The results of the physicochemical properties presented in Fig. 1 showed a decrease in the water-retention ability of used engine lubricant-contaminated soil, which could occur due to hydrophobicity, pore blockage, surface tension, or organic matter degradation. This is in line with the findings by Ewetola (2013) and Baruahet al. (2013), who noted that contamination of soils with crude oil reduces the ability of soil to hold water. The soil pH of both used engine lubricant-contaminated soil and used engine lubricant-uncontaminated soil samples was alkaline with a slight increase from 7.03 to 7.09; the reason behind this could be the alkaline nature of spent oil or the soil’s buffering capacity that resists changes in pH. This finding is similar to that of Okafor (2023) where the pH of the used lubricant-contaminated soil was higher than that of the used lubricant-uncontaminated soil. While consistent with the findings of Egobuezeet al. (2019), the results also demonstrate an increase in the total carbon, cation exchange, and organic carbon composition of the spent lubricant-contaminated soil. The increased carbon content is likely due to the share of organic carbon in the anthropogenic hydrocarbon content of lubricant oil. The nickel level increased from 3.5633 mg/kg in uncontaminated soil to 5.0067 mg/kg in used lubricant-contaminated soil; this could be because spent oil contains nickel. While used engine lubricant-contaminated soil had lower levels of potassium and nitrogen, used engine lubricant lubricant-uncontaminated soil had greater levels; this difference might be attributed to increased microbial activity leading to the breakdown of nitrogen and potassium. The level of phosphate and lead was higher in used engine lubricant-contaminated soil. This could be because used oil contains phosphate and lead compounds or increased microbial activities, which could stimulate increased phosphate mobilization and lead solubilization from soil minerals. This result is the same as the outcome of Adekoet al. (2023) and Swapnaet al. (2021), where potassium and nitrogen levels were higher in used engine lubricant-uncontaminated soil, while phosphate and lead were higher in used engine lubricant-contaminated soil.
The microbial counting of culturable heterotrophic bacteria and culturable hydrocarbon-utilizing bacteria was done to determine the presence of microbial activity, as shown in Table I. The used engine lubricant-contaminated soil sample recorded the highest colony-forming unit (cfu/g) count for heterotrophic bacteria with a mean value of 3.8×108 cfu/g. The hydrocarbon-utilizing bacterial count was 2.2×108 cfu/g. The high numbers observed in the heterotrophic count might be a result of the diverse group of heterotrophic bacteria that can utilize a broad spectrum of nutrients. A similar observation was observed by Chikereet al. (2009) and Ibieneet al. (2011). To ascertain the degradation ability of hydrocarbon-utilizing bacterial isolates isolated from used engine lubricant-contaminated soil, a degradation assay was conducted. The values of the optical density were different for all the representative isolates on separate days. Hydrocarbon-utilizing bacterial isolates demonstrated differences in the pace at which they degraded used engine lubricant in a mineral salt medium broth, and this was quantified by visually observing turbidity in the broth and measuring the absorbance with a spectrophotometer (SP-UIV7) (600 nm wavelength). The results are shown in Fig. 2. The results at the end of day 21 demonstrated that the bacterial isolates had significantly carried out degradation. The capacity to degrade used engine lubricant revealed that these isolated bacteria from used lubricant-contaminated soil utilized the engine lubricant oil as the sole supply of carbon and energy. The cells increased in number as the experiment progressed, indicating the utilization of engine lubricant for their growth, and this was the reason for the concomitant rise in the turbidity of the mineral salt medium.
The genomic DNA of bacterial strains OD02 and OD11 was extracted from the isolates by the use of universal primers 27F and 1492R; the 16S rRNA gene fragments were amplified and sequenced. Using a BLAST search, the isolates OD02 and OD11’s 16S rRNA gene sequences were matched and contrasted with GenBank’s 16S rRNA gene data. Findings reveal that the 16S ribosomal RNA gene sequence of isolate OD02 was highly homologous to Lysinibacillus odysseyi with ascension number NR_025258.1. OD11 was closely related to Bacillus sp. (in: firmicutes) with ascension number MG705874.1, with 100% sequence similarity. A previous study by Kareemet al. (2017) found Lysinibacillus sp. and Bacillus sp. to be hydrocarbon degraders.
Polymerase chain reaction (PCR) of the isolated strains’(OD02 and OD11) genomic DNA was done to obtain the PCR of genes that encode various enzymes. The specific metabolic activities a bacterial strain can carry out can be traced through its functional genes. Five important catabolic genes known for the breakdown of PAH and aliphatic compounds were targeted. The result confirmed the presence of Alkane monooxygenase (AlkB), pyrene dioxygenase (NidA), and naphthalene dioxygenase (NahAC) genes were ascertained, and the findings are shown in Table II and Fig. 4. The AlkB gene catalyzes the terminal oxidation of n-alkanes with a chain length of C6–C12. Petroleum and petroleum products are 88% N-alkanes and can serve the role of energy supply for oil-degrading microorganisms (Van-Beilenet al., 2003; Dedovet al., 2017). Previous studies by Likhoshvayet al. (2014) and Lomakinaet al. (2014) showed the presence of alkane monooxygenase (AlkB) in bacterial populations isolated from products of petroleum hydrocarbon. Since the enzyme produced by the naphthalene dioxygenase (NahAC) gene not only breakdown naphthalene but also promotes the breakdown of other polyaromatic hydrocarbon chemicals, it is of special importance as an indicator for the degradation of polyaromatic hydrocarbons (Guoet al., 2010; Luet al., 2011). The location of AlkB was confirmed to be both the plasmid and chromosome, while the NidA and NahAC genes were confirmed to be the plasmid. These genes were all found to reside on the plasmid, which means there is a high probability of horizontal gene exchange occurring at similar sites and a likely increase in the number of bacteria that can break down used engine lubricant.
Conclusion
In conclusion, soil polluted with used engine lubricant contains bacteria that can degrade the lubricant. This research showed that Lysinibacillus odysseyi and Bacillus sp. (in: firmicutes), two indigenous bacteria with the highest degradation potential, contained catabolic genes, monooxygenase (AlkB), NidA and dioxygenase (NahAC) that break down spent engine lubricant. The presence of these functional genes confirms their ability to serve as effective bioremediation agents in the cleanup of spent engine lubricants and other petroleum products in polluted environments. The possibility of horizontal gene transfer can also be used in important industrial applications.
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