Kerosene Biodegradation by Highly Efficient Indigenous Bacteria Isolated From Hydrocarbon-Contaminated Sites

Abstract

Kerosene is widely used in Ethiopia as a household fuel (for lighting and heating), as a solvent in paint and grease, and as a lubricant in glass cutting. It causes environmental pollution and escorts to loss of ecological functioning and health problems. Therefore, this research was designed to isolate, identify, and characterize indigenous kerosene-degrading bacteria that are effective in cleaning ecological units that have been contaminated by kerosene. Soil samples were collected from hydrocarbon-contaminated sites (flower farms, garages, and old-aged asphalt roads) and spread-plated on mineral salt medium (Bushnell Hass Mineral Salts Agar Medium: BHMS), which consists of kerosene as the only carbon source. Seven kerosene-degrading bacterial species were isolated, 2 from flower farms, 3 from garage areas, and 2 from asphalt areas. Three genera from hydrocarbon-contaminated sites were identified, including Pseudomonas, Bacillus, and Acinetobacter using biochemical characterization and the Biolog database. Growth studies in the presence of various concentrations of kerosene (1% and 3% v/v) showed that the bacterial isolates could metabolize kerosene as energy and biomass. Thereby, a gravimetric study was performed on bacterial strains that proliferated well on a BHMS medium with kerosene. Remarkably, bacterial isolates were able to degrade 5% kerosene from 57.2% to 91% in 15 days. Moreover, 2 of the most potent isolates, AUG2 and AUG1, resulted in 85% and 91% kerosene degradation, respectively, when allowed to grow on a medium containing kerosene. In addition, 16S rRNA gene analysis indicated that strain AAUG1 belonged to Bacillus tequilensis, whereas isolate AAUG showed the highest similarity to Bacillus subtilis. Therefore, these indigenous bacterial isolates have the potential to be applied for kerosene removal from hydrocarbon-contaminated sites and the development of remediation approaches.

Background

Globally, environmental pollution associated with the use of petroleum hydrocarbons (saturates, aromatics, resins, asphaltenes, etc.) and their products is an alarming issue.^1,2 Among these, kerosene, which is derived from fractional distillation (150°C-275°C) of petroleum oil, is extensively used for household activities (cooking and lighting) in developing countries,³ besides its use as a solvent for painting and greases, oil glazing agent, lubricant (in the glass cutting process), jet fuel, insecticide, and pesticide. Despite its versatile use, it might be discharged and/or spilled into the environment (soil, water, or air) knowingly or unknowingly via anthropological activities or natural processes.⁴ This has a detrimental impact on ecosystem functioning and exhibits moderate to high acute toxicity, affecting the health of biota and fauna.⁵ Studies showed that soil pollution with kerosene also affects agricultural productivity by adversely affecting soil fertility.⁶

Many studies have been dedicated to maintaining ecosystem health and functioning by reclaiming polluted lands and utilizing several restoration technologies. Consequently, mechanical, chemical, and biological approaches have been used to reduce and/or eliminate pollution from petroleum compounds.^7,8 The 2 aforementioned methods are, however, not cost-effective for operation, not safe for the environment^9,10 and cause secondary pollution after treatment.¹¹ The biological method (bioremediation, biodegradation), on the other hand, is a cost-effective, simple, and eco-friendly approach that uses biological agents such as bacteria, plants, fungi, and microalgae to reduce, detoxify, and/or mineralize contaminants to harmless compounds (H₂O, CO₂, and O₂), which are then transformed into energy and biomass.¹²

Bacterial species are currently preferred for bioremediation over other microorganisms because they have a diversified population, effective degradative genes, and a wide range of enzymes.^1,13 Additionally, bacteria also produce surface active biomolecules (biosurfactants) with diverse chemical structures (peptides, proteins, fatty acids, moieties, glycolipids, and polysaccharides) to enhance the bioavailability of hydrocarbons and their derivative contaminants and diminish interfacial and surface interaction for an effective emulsification process.^14,15 Bacteria can degrade the pollutants aerobically and/or anaerobically, but aerobic degradation is more efficient since aerobic bacteria can produce monooxygenase and dioxygenase enzymes to mineralize and completely convert hydrocarbon pollutants for metabolic uses.¹ A large group of bacterial species (Gram-positive and Gram-negative) has been identified as potential kerosene degraders, including Pseudomonas spp., Bacillus spp., Acinetobacter spp., Escherichia spp., Proteus spp., Serratia spp., Streptomyces spp., Arthrobacter spp., Gordonia spp., Brevibacerium spp., Burkholderia spp., Mycobacterium spp., Nocardia spp., Xanthomonas spp., Alcaligenes spp., Flavobacterium spp., Micrococcus spp., and Corneybacterium spp.^13,16-18

Effective biodegradation is influenced by biological characteristics and environmental parameters that exist within polluted sites.¹⁹ These include the physicochemical nature of hydrocarbon pollutants,²⁰ microbial population quality (size, single strain, and consortia),²¹ presence of xenobiotic pollutants such as heavy metals,¹ and environmental parameters in polluted sites such as nutrients availability, range of temperature and pH, soil moisture, and oxygen.^22,23 Thus, identifying potential indigenous kerosene-degrading bacteria is essential to remediate the kerosene-based polluted sites successfully. Following this, the objective of this study was to exploit, isolate, identify, and characterize the indigenous bacterial species from flower farms and hydrocarbon-contaminated soil environments in order to assess kerosene degradation. Therefore, native kerosene-degrading bacteria were isolated, characterized, and identified for their efficiency in kerosene degradation.

Materials and Methods

Sampling sites and collection

Soil samples that were presumably exposed to hydrocarbon contaminants were collected at various locations, including the Gallica flower farm (located 22 km west of Addis Ababa), where there is indirect contamination with kerosene from the use of pesticides, insecticides, and fungicides and from areas having direct contamination with petroleum oil containing kerosene such as in garage areas (Amanuel and Akaki, Addis Ababa region), and near an asphalt road (Amanuel, Addis Ababa region). Using a simple random spatial sampling technique, about 10 g of topsoil samples (5-10 cm) were collected from each site in triplicates. The soil samples were labeled as AAUA (samples from Akaki/Amanuel garages), AAUAs (samples from Amanuel old aged asphalt roads), and AAUG (samples from Gallica flower farms). The samples were then transferred into labeled sterile polyethylene bags and transported with a cold transportation ice box to the Microbial Biotechnology Laboratory of the Addis Ababa Science and Technology University, Ethiopia.

Enrichment of kerosene-degrading bacteria

To enhance the growth of kerosene-degrading bacteria from hydrocarbon-contaminated sites, enrichment techniques were performed using the methods described in Borah and Yadav.²⁴ For this, the triplicate soil samples from each site were sieved using a sterile 2 mm mesh sieve and homogenized manually. About 1 g of a homogenized soil sample from each site was added to 50 ml of sterilized saline solution (0.9% NaCl) and vortexed well. From this dilution, 1 ml of the suspension was transferred into 50 ml of modified basal salt medium (BSM). The composition of the BSM supplemented with 0.5% (v/v) of kerosene was as follows(g/l): KH₂PO₄ (1 g), Na₂HPO₄ (1.388 g), KNO₃ (0.5 g), MgSO₄ (0.1 g), CaCl₂ (0.01 g), (NH₄)₂NO₄ (2.5 g), FeCl₃ (0.05 g), and 100 ml trace mineral solution (0.01 g of ZnSO₄·7H₂O, MnCl₂·4H₂O, H₃BO₄, BaCl₂, CoCl₂·6H₂O, Fe₂SO₄·2H₂O, CuCl₂·2H₂O, NaMoO₄·2H₂O, KI, NiSO₄·6H₂O, and (NH₄)₆MoO₄). Kerosene was procured at a nearby oil filling station (Jemal Ali, Tulu Dimitu Total Madeya) and sterilized using a 0.45 µm membrane filter in 100 ml Erlenmeyer flasks. The flasks were incubated in a shaker cultivation cabinet (Intelligent thermostatic, ZHP-Y2102L series) with 150 rpm at 30°C, for 7 days. To refresh the culture, 10% (v/v) of the enriched culture was transferred to the enrichment media 3 times.

Screening of kerosene-degrading bacteria

An aliquot of serially diluted enriched suspensions was aseptically spread-plated on a BHMS agar medium containing 0.5% (v/v) kerosene as a growth substrate. The BHMS media contained (g/l): CaCl₂ (0.02), NH₄NO₃ (1), MgSO₄.7H2O (0.2), KH₂PO₄ (1), K₂HPO₄ (1), and 2 drops of FeCl₃ (60%). Three plates were used for each sample and incubated for up to 6 days at 30°C. Separately grown, discrete colonies were picked, and re-streaked on BHMS to obtain a pure culture. Two negative controls were used: media with kerosene as carbon source but not enriched culture, and another group of media inoculated with enriched culture but without kerosene supplement as carbon source. Then isolates obtained from the experimental group (media with kerosene and enriched culture) were labeled as AAU (Addis Ababa University) with the identification sites (A = Akaki and Amanuel garage; G = Gallica Flower farm, and As = Amanuel old aged asphalt) with their respective identification numbers and preserved in 25% v/v glycerol at −20°C (IGnIS CHEST FREEZER, C0450W) for future use and subsequently subcultured for refreshment when needed.

Identification of kerosene-degrading bacteria

Morphological and biochemical characterization

The cellular morphs (shapes) of all potential kerosene-degrading bacteria were identified using microscopic examination (Gram’s staining). In addition, the isolates were also characterized biochemically, including catalase, urease, starch, and casein hydrolysis, as described in Zhang et al.²⁵ For the catalase test, 2 to 3 drops of hydrogen peroxide (3%, v/v) were added to the overnight grown culture in the test tubes and the formation of vigorous bubbles indicated positive activity. For casein hydrolysis, isolates were grown overnight in nutrient broth and inoculated onto skim milk agar (HiMEDIA), and incubated at 30°C for 48 hours. The formation of a clear zone around the isolates against the white background indicated the casein hydrolysis activity of the isolates. In addition, the urease test was conducted by the introduction of pure bacteria into urea broth (Difco, BD, Wokingham, UK) and incubated at 30°C for 24 to 48 hours. Following this, the change of color from yellow to pink indicated that there was urease production. Finally, for the starch hydrolysis test, kerosene-degrading isolates were grown overnight in nutrient broth and inoculated onto a starch agar medium (Alpha Chemika, Mumbai, India), and incubated at 30°C for 48 hours. The plates were flooded with Gram iodine. The formation of a clear area around the isolate against the blue-black background indicated starch hydrolysis.

Biolog identification

Primary identification of kerosene-degrading isolates was done using the Biolog machine from NAHDIC (National Animal Health Diagnostic and Investigation Center, Sebeta, Ethiopia). Using the standard protocol of the manufacturer (BIOLOG Inc., Hayward, CA, USA), the isolates were grown on BUG agar (Biolog Universal Growth agar medium). For this Protocol “A” was applied which has inoculation fluid A (IF A) and a default method for the identification of diverse bacterial species. Following this protocol, the isolates with 90% to 98% cell density were suspended in an inoculating fluid 3 (IF; special gelling agent). The MicroPlates were prefilled with all essential nutrients and biochemicals for microbial growth and tetrazolium redox dyes to indicate the utilization of the carbon sources and/or resistance to inhibitory chemicals calorimetrically. Subsequently, 100 μl of each cell suspension was inoculated into the MicroPlate. Then the MicroPlates were incubated for 16 hours at 33°C. Then, using a microplate reader, the phenotypic fingerprint of wells was compared to Biolog’s bacterial species library.

16S rRNA sequencing and analysis

For genotyping bacterial isolates, the DNA was extracted by a freeze-thaw technique. The colonies that were plated on nutrient agar were suspended in 50 μl sterile H₂O and heated for 5 minutes at 95°C to 100°C and then centrifuged for 10 minutes at 12 000×g. Then, about 1 μl of the suspension and 20 µl of the master mix were used for PCR. The master mix consisted of 16.2 µl PCR grade H₂O, 2 µl of 10× PCR buffer (Life Technologies), 0.4 µl of 10 mM dNTPs mix (Life Technologies), 0.4 µl of 20 mg/ml BSA, 0.8 µl of 25 mM MgCl₂, 0.08 µl of 50 µM of each primer 27F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) and Dream Taq DNA polymerase (Life Technologies). PCR amplifications were conducted using a thermal cycler (Verticycler, Applied Biosystems). For this, the standard PCR reaction conditions were kept (initial denaturation at 96°C for 10 minutes, denaturation at 95°C for 30 seconds with 35 cycles, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, and final extension at 72°C for 7 minutes). For the 50 μl PCR reaction mixture, 5 μl of genomic DNA extract, Dream Taq DNA polymerase buffer (10×), 2.5 pmol of dNTPs, 20 pmol of each primer, and 2.5 U of Dream Taq DNA polymerase were used. Finally, according to the manufacturer‘s specifications, the 16S rDNA PCR amplicons were purified using the Illustra Exostar DNA purification kit (GE Health Care). Then, the 16S rRNA gene amplification was performed using the bacterial universal oligonucleotide primers 8F and 1492R using the Verticycler PCR system (Applied Biosystems) as described by Overmann and Tuschak²⁶ and purified PCR products were then submitted to partial sequencing at the sequencing facility of the Leibniz Institute DSMZ-German, Collection of Microorganisms and Cell Cultures, Germany.

Phylogenetic analysis

For phylogenetic analysis, Illumina/Solexa was used for partial 16S rRNA gene sequencing.²⁷ Following this, the raw DNA sequences (DNA chromatogram) were viewed, edited using BioEdit Programme,²⁸ and stored in FASTA format. Consequently, poor-quality sequence products were removed from the 3′ and 5′ sequence ends and assembled using MEGA X. Then, using BLASTn the sequence data were compared to the NCBI (http://www.ncbi.nlm.nih.gov) DNA sequence database and the phylogenetic analysis was performed. As a result, the kerosene-degrading bacterial phylogenetic tree was constructed using MEGA X with bootstrap values of 1000 replications with maximum likelihood method²⁹ and Kimura-2 parameter model.³⁰

The capacity of kerosene-degrading bacteria

About 100 µl of bacterial culture (10⁸ cells/ml) of each isolate was inoculated into 100 ml of BHMS broth in 250 ml flasks containing different concentrations of kerosene (1% and 3%) as substrates. Then the flasks were incubated at 30°C in a shaking incubator (150 rpm ) for several days (1, 5, 10, and 15 days).³¹ The bacterial growth (turbidity) was determined at the end of each incubation period using a UV/Vis spectrophotometer (Mecasys, Optizen POP Series) at 600 nm (OD₆₀₀) in triplicates and BHMS medium was used as a blank.

Estimation of kerosene biodegradation efficiency using gravimetric analysis

About 10⁸ cells/ml of culture were inoculated into 100 ml of BHMS in 250 ml conical flasks containing 5% kerosene, kept on a rotary shaker (150 rpm), and incubated for 10 and 15 days at 30°C. At the end of each incubation time, the remaining amount of undegraded kerosene was extracted using an equal amount of solvent (100 ml of chloroform), as described in Shabir et al.²² Therefore, to determine the residual concentration, the solvent containing kerosene was taken from the upper part of the flask and transferred into a pre-weighted Petri dish. This extraction process was repeated in triplicate (3 times) to collect and secure the residuals completely. Following this, the collected samples were exposed to evaporation in a hot air oven at 40°C overnight. Subsequently, the extracted component was determined as the percentage of kerosene degradation as per the formula shown below.³²

$$\mathrm{Percent}\mathrm{degradation}(\%)=\frac{\mathrm{Initial}\mathrm{concetration}\mathrm{of}\mathrm{kerosene}-\mathrm{Final}\mathrm{concentation}\mathrm{of}\mathrm{kerosene}}{\mathrm{Initial}\mathrm{concentration}\mathrm{of}\mathrm{kerosene}}*100$$

Data analysis

Statistical analysis software (version 9.1.3; 2003) was used to analyze quantitative data. For this, analysis of variance (ANOVA) with multiple comparison test (Duncan) was used to consider statistically significant differences with a P < .0001 of potent kerosene-degrading bacteria.

Results

Isolation of kerosene-degrading bacteria from hydrocarbon-contaminated sites

Seven kerosene-degrading bacteria were identified from hydrocarbon-contaminated sites (garages, asphalt, and flower fields) using an enrichment procedure and plate separation (Table 1). These isolates were designated as AAUG1, AAUG2, AAUA3, AAUA4, AAUA5, AAUAs6, and AAUAs7. In addition, the cell density of the kerosene-degrading bacteria varied depending on the sample location on media containing kerosene, with the total CFU ranging from 2.2 × 10³ to 5.5 × 10⁵.

Table 1.

Morphological and biochemical characteristics of isolates.

Code	Biolog ID	Gram’s stain	Shape	Catalase	Urase	Casein	Starch
AAUG1	Bacillus tequilensis	+	Bacilli	+	+	+	+
AAUG2	Bacillus subtilis	+	Bacilli	+	+	+	+
AAUA3	Pseudomonas aeruginosa	−	Bacilli	+	−	+	−
AAUA4	Pseudomonas aeruginosa	−	Bacilli	+	−	+	−
AAUA5	Pseudomonas aeruginosa	−	Bacilli	+	−	+	−
AAUAs6	Acinetobacter beijerinckii	−	Coccobacilli	+	+	+	−
AAUAs7	Pseudomonas aeruginosa	−	Bacilli	+	−	+	−

⁺for positive test.

−for negative test.

Morphological and biochemical characterization of kerosene-degrading bacteria

The purified isolates were subjected to conventional biochemical testing as well as Biolog analysis (Table 1). Accordingly, 5 isolates (71.43%) were Gram-negative bacteria, while 2 (28.57%) were Gram-positive. Microscopic examination of kerosene-degrading isolates also showed that the isolates were rod-shaped bacteria. In addition, bacterial isolates were tested using conventional biochemical assays. All isolates were identified as catalase and casein positive, while starch was only hydrolyzed by the 2 isolates. In addition, 3 isolates (AAUG1, AAUG2, and AAUAs6) were able to hydrolyze urea when cultured in urea broth.

Identification of kerosene-degrading bacteria

The GEN III MicroPlate test panel uses 94 phenotypic tests (71 biochemical assays for carbon source and 23 chemical sensitivity tests) to profile and identify a wide range of bacteria, with an accuracy range of 65.5% to 99.9% in identifying species within genera (Table 1). Three genera from hydrocarbon-contaminated sites (garages, asphalt, and flower farms) were identified, including Pseudomonas, Bacillus, and Acinetobacter (Table 1). Pseudomonas aeruginosa accounted for 57.14% of the isolates, followed by Bacillus tequilensis, Bacillus subtilis, and Acinetobacter beijerinckii, each accounting for 14.29% of the isolates. This study also showed that Pseudomonas aeruginosa was frequently isolated from hydrocarbon-contaminated sites, followed by Bacillus spp.

16S rRNA sequencing and phylogenetic analysis

To corroborate the findings of the biochemical, morphological, and Biolog identification, 16S rRNA gene sequencing for 4 potent isolates was carried out and compared to already deposited sequences data in NCBI. Thus, sequence analysis of the 16S rRNA gene showed that the isolates belong to the genera of Pseudomonas and Bacillus (Table 2). Moreover, isolates AAUG1 (MT669829.1) and AAUG2 (MT669833.1) recovered from Galica flower farm had 100% similarity with B. tequilensis strain CFR01 (MT641220.1), previously isolated from fermented foods, and Bacillus spp. C1BY-1 (MK503666.1) recovered from rhizosphere soil, respectively (Table 2). In addition, isolates AAUA5 (MT669827.1) and AAUAs7 (MT669828.1) which were recovered from a garage and asphaltene soil sample, respectively, exhibited 100% similarity to P. aeruginosa strain 1816 (MK045608.1), which had previously been identified from Kanher reservoir fish cage culture system. Consequently, our partial 16S rRNA gene sequences from the member of the Pseudomonas and Bacillus spp. were used to create the phylogenetic tree by incorporating additional isolates from NCBI (Figure 1). Thereby, isolates AAUG1 (MT669829.1) and AAUG2 (MT669833.1) were named B. tequilensis strain AAUG1 and B. subtilis strain AAUG2, respectively. The 2 Pseudomonas affiliated spp, AAUA5 (MT669827.1) and AAUAs7 (MT669828.1) were also named as P. aeruginosa AAUA5 and P. aeruginosa AAUAs7, respectively. The phylogenetic tree of the strains is shown in Figure 1.

Table 2.

Phylogenetic affiliation of 16S rRNA partial sequences of the selected kerosene-degrading bacterial isolates.

Isolate code	Accession number	Top-hit Taxon	Identity (%)	Taxonomy
AAUA5	MT669827.1	Pseudomonas aeruginosa	>98	Proteobacteria; Gamma proteobacteria
AAUAs7	MT669828.1	Pseudomonas aeruginosa	>99	Proteobacteria; Gamma proteobacteria
AAUG1	MT669829.1	Bacillus tequilensis	>98	Firmicutes; Bacilli
AAUG2	MT669833.1	Bacillus subtilis	>98	Firmicutes; Bacilli

Figure 1.

Phylogenetic analysis of partial 16S rRNA genes of the 2 Pseudomonas and Bacillus isolates (bolded and coded with “AAU”) and the accession numbers of 16S rRNA are followed by species names. The LC501697.1 (E.coli 25) 16S rRNA gene partial sequence was used as an out-group.

Growth of bacterial isolates at increasing kerosene concentrations

Some bacteria are capable of metabolizing kerosene as a carbon source for their growth, energy, and biomass. Thus, the effectiveness of kerosene biodegradation using potential isolates was measured by the OD value (turbidity for measuring growth) in BHMS media supplemented with kerosene (1% v/v). Subsequently, the growth patterns of the bacterial isolates; A. beijerinckii (AAUAs6), B. tequilensis (AAUG1), B. subtilis (AAUG2), and P. aeruginosa (AAUA3, AAUA4, AAUA5, and AAUAs7) displayed their growth activity within the 15th day of incubation (Figure 2). Among isolates, A. beijerinckii (AAUAs6) showed significant biodegradation potential on the 10th day of incubation with the highest OD value of 1.45 (P < .0001). On the other hand, isolates of P. aeruginosa (AAUA5, AAUA3, and AAUAs7) showed nearly the same kerosene degradation potential on the 10th day of incubation.

Figure 2.

Kerosene degradation capacity of isolates at different concentrations (1% and 3%) and incubation periods (day 1, 5, 10, and 15). Data represent the mean and standard deviation triplicate determination of optical density (OD at 600 nm). The larger error bars were due to differential responses of bacterial isolates.

The kerosene degradation capability of the isolates was also further studied by increasing the kerosene concentration to 3% (v/v) in BHMS media. Thus, the isolates exhibited increased growth activities compared to the 1% kerosene-supplemented media (Figure 2). Accordingly, the highest bacterial growth pattern by B. tequilensis (AAUG1) was obtained on the 15th day of incubation with an OD value of 2.09 (P < .0001). Likewise, B. subtilis (AAUG2) showed significant kerosene degradation potential as compared to other isolates on the 10th day of OD measurement of 2.08 (P < .0001). Other bacterial isolates (AAUA3, AAUA4, and AAUAs6) also showed a strong growth pattern when the incubation period lasted beyond 10 days, with a significant difference in the OD value. However, on the 10th day of incubation, P. aeruginosa (AAUA5 and AAUAs7) showed the highest growth pattern. Therefore, P. aeruginosa and Bacillus spp. substantially showed effective kerosene degradation capacity.

Assessment of the effectiveness of isolates in degrading kerosene

Bacterial isolates that demonstrated effective kerosene degradation potential at 3% kerosene (v/v) were grown with 5% kerosene (v/v), and their kerosene degradation efficiency was measured at days 10 and 15 using gravimetric analysis. The maximum degradation (91% and 85%) was recorded by B. tequilensis (AAUG1) and B. subtilis (AAUG2) respectively, on the 15th day of incubation compared to other isolates (Figure 3). Additionally, other P. aeruginosa isolates (AAUA3, AAUA4, AAUA5, and AAUAs7) also showed 75% to 83% degradation potential on the 15th day of incubation. However, the lowest degradation potential (57.2%) was shown by A. beijerinckii (AAUAs6; Figure 3).

Figure 3.

Gravimetric analysis of kerosene degradation.

Discussion

Bacteria are the most diverse and ubiquitous groups of microorganisms known to have imperative functions in the bioremediation of hydrocarbon-contaminated environments.^33,34 This work used a BHMS medium enriched with 0.5% kerosene to isolate 7 kerosene-degrading bacteria from hydrocarbon-contaminated settings. Population densities ranging from 10⁴ to 10⁷ CFU per gram of soil were observed at all sampling sites, which is within the recommended range for efficient hydrocarbon biodegradation.³⁵ Kerosene-degrading bacteria can be found abundantly in sites where hydrocarbon contaminants are prevalent, such as garages, automotive services, and asphalt sites, as a result of natural attenuation.^24,31,36 In fact, kerosene-degrading bacteria were also identified from flower farms that use a variety of agrochemicals, although this type of cultivation was only introduced to the country about a decade ago. In flower farms, Kerosene Emulsifiable Concentrate (KEC) is used as a pesticide and fungicide. In addition, various chemical solvents or diluents such as kerosene are added to an insecticide formulation in order to improve the effectiveness or physical properties of the insecticide, resulting in kerosene contamination of soil.³⁷ Multiple sample sites may be used to identify kerosene-degrading bacteria, and the chemical makeup of kerosene, which is made up of complex hydrocarbons and carbonation, allows for the growth of a diverse range of microbes with distinct metabolic pathways.³²

The kerosene degrader identified in this study belongs to the genera Pseudomonas, Bacillus, and Acinetobacter. Moreover, the isolates were identified as P. aeruginosa, A. beijerinckii, B. subtilis, and B. tequilensis based on 16S rRNA sequencing and the Biolog system and identification protocol. When these isolates are compared to numerous other known hydrocarbon-degrading organisms, they are classified into Phyla Proteobacteria (AAUA5 and AAUA7) and Firmicutes (AAUG1 and AAUG2), which are Gammaproteobacteria. These phyla possess features closely related to hydrocarbon-degrading organisms.^38,39 As a result, the current and prior studies have shown that P. aeruginosa and Bacillus spp. are easily acquired from hydrocarbon-contaminated environments, owing to their diverse metabolic capacities, which make them ubiquitous^31,40, and their potential for the production of biosurfactants, which aids their degradation efficacy.⁴¹

The kerosene-degrading bacterial isolates were also further characterized using conventional biochemical assays. Surprisingly, all of the isolates tested positive for catalase and casein, although urea and starch utilization capacities differed. Thereby, the analysis showed that the kerosene-degrading bacteria may have a cluster of biocatalysts meant for their metabolic processes for the utilization of numerous and/or definite contaminants.³² Such enzymes are essential for the hydrolysis of substrates to obtain nutrients such as carbon, nitrogen, and others. This eventually encourages growth and allows bacteria to use kerosene as a carbon source.⁴² Kerosene-degrading isolates will also produce oxygenases and dehydrogenases to break down kerosene.⁴³ Moreover, bacterial growth using kerosene as a carbon and energy source was investigated by measuring turbidity in the growth medium.⁴⁴ Thus, the growth pattern of bacterial isolates was evaluated in the presence of various concentrations of kerosene (1%, 3%, and 5% v/v) during 1, 5, 10, and 15 days of incubation. Thereby, the isolate A. beijerinckii (AAUAs6) showed a significant growth difference on 1% (v/v) kerosene on the 10th day of incubation. Consistent with this result, Acinetobacter spp. was identified as the most prevalent bacteria that utilize a wide range of hydrocarbon pollutants.⁴⁵ As a function of time, however, P. aeruginosa (AAUA4 and AAUA3) and B. tequilensis (AAUG1) showed an increased OD in the range of 1.44 to 2.09, indicating increased use of kerosene. In contrast, B. subtilis (AAUG2) and 2 other P. aeruginosa (AAUA5 and AAUAs7) isolates showed a reduced utilization of kerosene for a relatively longer culture time (15 days). This might be linked to a lower concentration of carbon and nitrogen in kerosene than the amount required for the growth.⁴⁶

Additionally, this work demonstrated that bacterial isolates had greater activity at a higher substrate concentration (3% kerosene) than at a concentration of 1% on the same incubation day. Thereby, B. tequilensis (AAUG1) was shown to be a more efficient kerosene degrader, whereas P. aeruginosa (AAUA3 and AAUA4) and A. beijerinckii (AAUAs6) exhibited enhanced kerosene degradation. Other isolates such as B. subtilis (AAUG2) and P. aeruginosa (AAUA5 and AAUAs7), however, showed the finest growth pattern on the 10th day of incubation. Therefore, B. tequilensis, B. subtilis, and P. aeruginosa were identified as promising kerosene biodegraders. Similarly, numerous studies also showed that Pseudomonas spp. and Bacillus spp. were potent isolates in the bioremediation of kerosene-contaminated environments.^13,47 This is because they have diverse metabolic capabilities,³⁴ cell membrane physiology,¹³ and potential for the production of bio-surfactants.⁴⁸

Upon gravimetric analysis, the potential kerosene-degrading bacterial isolates were identified. Researchers showed that the Enterobacter cloacae, Enterobacter hormaechei, and Pseudomonas stutzeri were able to degrade 67.43%, 48.48%, and 65.48% of 5% kerosene as carbon source, respectively.¹³ Additionally, Bacillus amyloliquefaciens 6A was able to degrade the aliphatic and aromatic components of kerosene oil, removing almost 64% of them from the medium over the course of 8 days of incubation.⁴⁹ However, from this study B. tequilensis (AAUG1) and B. subtilis (AAUG2) were found to be the most effective bacterial species, showing 91% and 85% degradation efficacy respectively. This is due to their diverse array of catabolic genes and enzymes, which greatly enhances their ability to degrade harmful contaminants.^13,50 The biodegradation efficacy increased with an increase in the concentration of kerosene because it is highly carbonaceous and contains some quantity of nitrogen, consequently accounting for the increased levels of carbon and nitrogen in the soil.^13,46

Altogether, both Pseudomonas and Bacillus spp. are the dominant kerosene-degrading bacteria detected in hydrocarbon-contaminated areas of garages, asphalt sites, and flower farms. Therefore, these bacteria are suitable to be applied in the bioremediation process in the field to quickly break down kerosene in many hydrocarbon-contaminated areas.

Conclusion

Bioremediation is one of the most ecologically acceptable ways of removing hydrocarbon pollutants from the environment. Bacteria, microalgae, fungi, and protozoa are among the microorganisms employed, although bacteria have a wide range of metabolic activities and are extensively used in bioremediation. Seven kerosene-degrading bacteria were isolated from garages, asphalt regions, and flower farms. Phylogenetic tree analysis and 16S rRNA sequencing revealed that the bacterial isolates AAUA4 (MT669827.1) and AAUAs7 (MT669828.1) are related to Pseudomonas spp., whereas AAUG1 (MT669829.1) and AAUG2 (MT669833.1) are related to Bacillus spp. Although the kerosene-degrading bacteria identified in this study can proliferate in 1%, 3%, and 5% (v/v) of kerosene, B. tequilensis (AAUG1) and B. subtilis (AAUG2) were shown to be the most effective strains, degrading kerosene at 91% and 85%, respectively. Therefore, bacterial isolates from hydrocarbon-contaminated locations might be explored as possible biological agents for effective kerosene biodegradation. This study also adds to existing knowledge on kerosene-degrading bacteria, and how bacterial isolates can be employed to reclaim hydrocarbon-contaminated areas.