Isolation and Identification of Phenanthrene-Degrading Bacteria from Crude Oil-Contaminated Soils around Assaluyeh Refinery:
Growth Evaluation and HPLC-Based Degradation Analysis
Atefeh Ranjbar1, Ali Mohammadzadeh2
1Department of Biochemistry, Faculty of Science, Payame Noor University, Bushehr, Iran.
2School of Chemistry and Lifescience, University of Bradford, Bradford BD7 1DP, UK.
*Corresponding Author E-mail: alimohammadzadeh8888@gmail.com, a.mohammadzadeh92@bradford.ac.uk, Atefehranjbar68@gmail.com
ABSTRACT:
Polycyclic Aromatic Hydrocarbons (PAHs) are toxic organic compounds that pose significant environmental contamination risks. This study aimed to screen and molecularly identifies bacteria capable of degrading phenanthrene, a representative PAH, from petroleum-contaminated soils. Samples were collected from the Arak and Bushehr oil refineries for bacterial isolation. Initial identification was based on morphological and biochemical characteristics. Ten bacterial strains were assessed for their growth on phenanthrene as the sole carbon source. The most efficient strains were further identified through 16S rRNA gene sequencing. Growth dynamics in mineral media containing phenanthrene were monitored using a spectrophotometer, while residual phenanthrene levels were quantified via High-Performance Liquid Chromatography (HPLC). The results revealed that Bacillus thuringiensis, Aeromonas caviae, and Salmonella enterica exhibited phenanthrene degradation efficiencies of 55.82%, 51.1%, and 44.7%, respectively, after 72 hours. These bacteria demonstrated both resistance to and utilization of phenanthrene as a carbon and energy source, underscoring their potential for bioremediation applications at the pilot scale.
KEYWORDS: Polycyclic aromatic hydrocarbons, Phenanthrene, Biodegradation, Degrading bacteria, Bioremediation.
1. INTRODUCTION:
Polycyclic aromatic hydrocarbons (PAHs) are organic molecules characterized by multiple fused benzene rings arranged in linear, angular, or clustered configurations. These compounds are hydrophobic, exhibiting low solubility in water and a high affinity for organic phases due to their elevated octanol-water partition coefficients. PAHs are introduced into the environment through diverse sources such as the incomplete combustion of fossil fuels, accidental releases of petroleum products, runoff from asphalt surfaces, coal processing, and natural geological mechanisms1,2. While lower molecular weight PAHs are predominantly toxic, those with higher molecular weights are more commonly associated with genotoxic effects. Phenanthrene, a three-ring PAH, and pyrene, containing four rings, are among the most prevalent in nature. Although they are not directly genotoxic, their molecular structures are frequently found in more hazardous PAHs, making them useful markers for assessing contamination3.
When released into the environment, PAHs undergo various natural attenuation processes such as photolysis, chemical and photo-oxidation, volatilization, bioaccumulation, and adsorption onto soil particles4,5. However, microbial degradation is widely regarded as the most effective mechanism for the removal of these pollutants. PAHs with fewer aromatic rings, such as benzene, naphthalene, and phenanthrene, are generally more susceptible to microbial breakdown, whereas higher molecular weight PAHs, like pyrene, are more resistant6,7. Additionally, conventional physical and chemical remediation techniques are often limited in terrestrial environments, highlighting the importance of microbial interventions.
To effectively degrade PAHs, microorganisms must possess the enzymatic capacity to metabolize these compounds as their sole carbon and energy sources. Research has identified various bacterial strains capable of degrading PAHs, including Corynebacterium and Pseudomonas, which can utilize compounds such as phenanthrene, pyrene, and naphthalene in contaminated ecosystems8,9,10. Incorporating these strains into remediation strategies has been shown to enhance microbial populations and improve the degradation efficiency in hydrocarbon-contaminated environments.
Assaluyeh, located in southern Iran, is a major industrial hub, primarily known for its extensive oil and gas activities. It houses the South Pars Gas Field, one of the largest natural gas reserves in the world, and numerous petrochemical plants and refineries operate in the region. Studies in industrialized regions, including Assaluyeh, indicate elevated levels of PAHs in air, water, and soil, posing risks to ecosystems and human health. PAHs are hydrophobic and tend to accumulate in soils and sediments, where they can persist for extended periods due to their resistance to degradation. The accumulation of PAHs in Assaluyeh’s coastal and terrestrial environments threatens biodiversity and may impact the livelihoods of communities relying on agriculture and fisheries.
This study focuses on isolating and identifying bacteria with the capability to degrade phenanthrene, a representative PAH, from petroleum-contaminated soils sourced from Assaluyeh Oil Refineries. The work involves initial screening based on morphological and biochemical traits, molecular identification through 16S rRNA sequencing, and the quantification of phenanthrene degradation using chromatographic methods. The findings aim to provide insights into the potential use of these bacterial strains for bioremediation applications, offering a sustainable approach to mitigating PAH pollution.
The incubator (Gold, Parsian Teb, Iran) was used for bacterial growth. Sterilization steps were carried out using an autoclave (75 Lit, Reyhan Teb, Iran). High-performance liquid chromatography (HPLC) (P 2.1L, Knauer, Germany) with a C18 column was employed to determine the phenanthrene concentration.
A stock solution of phenanthrene was prepared by dissolving 1g of phenanthrene in 100mL of acetone, resulting in a concentration of 100mg/L. The mineral salts medium (MSM) was composed of 1.0g of NH₄Cl, 5.0g of KH2PO₄, 0.1g of MgSO₄•7H₂O, 5mg of Fe (SO₄) ₂ (all from MERCK, Germany), 1.0mL of a trace element solution, and 2.0 mL of the phenanthrene stock solution, which served as the primary source of carbon and energy.
Soil samples were collected from the vicinity of the refinery in Assaluyeh, Bushehr. Soil samples were randomly collected from depths of 10–15cm and kept in sterile polybags. The samples were stored at 4°C before being transferred to the laboratory for further processing.
To prepare soil samples for plating, 4.5mL of physiological serum were added to each of five 5mL tubes, followed by the addition of 0.5g of soil sample to each tube. A 0.1mL aliquot of the mixture containing the physiological serum and soil sample was then inoculated onto the surface of nutrient agar plates and incubated.
After 24hours of incubation on nutrient agar plates, bacterial colonies that developed were transferred to blood agar plates using the replica plating technique. After another 24hours, phenanthrene was applied to the blood agar surface using the spray plating method. The phenanthrene solution was prepared by dissolving 0.4g of phenanthrene in 20mL of acetone, and 0.5mL of the solution was sprayed onto the agar surface. The plates were incubated for seven days. After incubation, only the bacterial colonies capable of phenanthrene degradation were observed on the agar surface11.
High-performance liquid chromatography (HPLC) was employed to determine the residual phenanthrene concentration in the mineral base medium. For this analysis, bacterial strains of Bacillus tequilensis, Salmonella enterica, and Aeromonas caviae were individually cultured in 5mL of nutrient broth medium and incubated at 30°C for 24hours. After incubation, 1 mL of the nutrient broth containing each bacterial strain was introduced into 100mL of mineral base medium in Erlenmeyer flasks containing phenanthrene at an initial concentration of 100mg/L. The flasks were then incubated on a rotary shaker for three days.
After the incubation period, the cultures were centrifuged at 6000rpm for 15minutes, and the cells were collected. This process was repeated several times, followed by three washes with basal mineral medium. The final cultures were suspended in 5mL of the same medium, and 2mL of hexane was added to each suspension in screw-cap glass tubes. The tubes were vigorously shaken, and 1mL of the upper hexane phase was transferred to clean tubes, and then evaporated using a rotary evaporator. The remaining residue was dissolved in 2mL of HPLC mobile phase and stored at 4°C for subsequent HPLC analysis12.
In this study, we isolated and identified 102 bacterial strains belonging to 10 genera: Bacillus, Pseudomonas, Aeromonas, Salmonella, Enterococcus, Micrococcus, Bacteroides, Corynebacterium, Lactobacillus, and Staphylococcus. The results indicated that the proportion of Gram-positive (G+) hydrocarbon-degrading bacteria was 73.6%, compared to 26.4% of Gram-negative (G−) strains.
The nucleotide sequences of the isolated phenanthrene-degrading bacteria were analyzed using BLAST software. The results revealed that Bacillus tequilensis, Salmonella enterica, and Aeromonas caviae exhibited the highest phenanthrene degradation potential.
The growth of different bacterial species was analyzed using a spectrophotometer at 600nm. For bacterial cultivation, 0.5g of phenanthrene was added to a nutrient broth medium and incubated for four days. The absorbance values ranged from 0.214 to 0.801, with Bacillus tequilensis, Salmonella enterica, and Aeromonas caviae exhibiting absorbance values of 0.801, 0.732, and 0.649, respectively (Figure 1).
Figure 1: Growth curve of three selected bacteria (Bacillus tequilensis, Salmonella enterica, and Aeromonas caviae).
The biodegradation performance of three bacterial species was evaluated over a period of 24 to 72hours. Bacillus tequilensis achieved the highest phenanthrene reduction of 23.78% within the first 24hours. After 72 hours, the phenanthrene concentration was reduced by 55.82%, 48.90%, and 45.70% by Bacillus tequilensis, Salmonella enterica, and Aeromonas caviae, respectively (Figure 2).
Figure 2: Phenenthrene degradation rate in presence of three selected bacteria.
Numerous bacterial species are capable of biodegrading pollutants in soil, particularly due to their presence in soil and water ecosystems 13. Environmental factors such as temperature, humidity, and oxygen availability significantly influence bacterial growth. During the spring in Assaluyeh, the humid and rainy conditions create an environment favorable for bacterial growth14-19. As a result, among the 10 tested genera, three bacterial species demonstrated the ability to degrade more than 45% of phenanthrene within 72 hours. As shown in Figures 3a–c, the chromatograms of HPLC from remaining phenanthrene percentages after 72 hours were 44.18%, 55.30%, and 48.90% for Bacillus tequilensis, Salmonella enterica, and Aeromonas caviae, respectively.
Figure 3a-c: Chromatograms of Phenenthrene degradation after 72 h presence of bacteria.
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Received on 17.01.2025 Revised on 27.02.2025 Accepted on 03.04.2025 Published on 06.05.2025 Available online from May 10, 2025 Asian Journal of Pharmaceutical Analysis. 2025; 15(2):99-102. DOI: 10.52711/2231-5675.2025.00016 ©Asian Pharma Press All Right Reserved
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