Degradation of crude oil and pure hydrocarbon fractions by some wild bacterial and fungal species

Abstract: The use of biodegradation as a method for cleaning up soil that has been contaminated by spilt petroleum can be an effective strategy. So, this study investigated the existence of the wild microorganism in soil contaminated with oil and study their ability to degrade petroleum in vitro. Nineteen samples were collected from various locations near Taq Taq (TTOPCO) natural seeps in the Kurdistan Region of Iraq. Morphological, cultural, biochemical tests and molecular identification were used to identify the microbial communities, in addition, spore texture and the colour of the fungal isolates were investigated on the fungal isolates. Out of the19 samples, 17 indigenous bacterial strains and 5 fungal strains were successfully isolated. From the absorption spectrophotometry, Bacillus anthracis, Bacillus cereus, Achromobacter sp. and Pseudomonas aeruginosa for the bacterial isolates grew well on a minimal salt medium supplemented with 1% crude oil. Results showed that these isolates mentioned above had a strong ability to degrade crude oil by reducing the colour of 2,6-dichlorophenol indophenol (DCPIP) from deep blue to colourless. However, for the fractions of hydrocarbon, the bacterial isolates failed and did not affect the colour of any of the fractions. The results for fungi showed that Aspergillus lentulus and Rhizopus arrhizus had a strong ability to degrade both crude oil and fraction F1 by reducing the colour of DCPIP. Each fungal isolates also had a great tolerance to different concentrations of crude oil when grown on solid MSM. This study showed these microorganisms have a strong ability to degrade crude oil and can be used to clean up soil and the environment.

• The paper identifies indigenous bacterial and fungal species, including Pseudomonas aeruginosa and Rhizopus arrhizus, with significant crude oil degradation potential.
• The study employs spectrophotometry and DCPIP assays to quantify microbial growth and hydrocarbon breakdown under controlled laboratory conditions.
• The findings support practical bioremediation strategies by optimizing indigenous microbial consortia for effective treatment of oil-contaminated soils.

This paper (2301.08715) investigates the potential of indigenous bacterial and fungal species isolated from oil-contaminated soil in Iraq for the bioremediation of crude oil and its fractions. The study aims to identify these microorganisms and evaluate their ability to degrade hydrocarbons in vitro.

The researchers collected soil samples from various locations around the TaqTaq oil field in the Kurdistan Region of Iraq, including highly contaminated sites, flanking regions, and a control site. From these samples, 17 bacterial and 5 fungal strains were successfully isolated using standard microbiological techniques. Identification was performed using morphological, cultural, biochemical tests, and molecular methods including PCR amplification of the 16S rRNA gene for bacteria and the ITS region for fungi, followed by sequencing and BLAST analysis.

Key microbial species identified as having potential for hydrocarbon degradation included:

• Bacteria: Bacillus anthracis, Bacillus cereus, Achromobacter sp., and Pseudomonas aeruginosa were specifically highlighted based on their performance in degradation tests. Other identified bacteria included Aneurinibacillus migulanus, Brevibacillus borstelensis, Paenibacillus dentritiformis, Pseudomonas stutzeri, Lysinibacillus sp., Bacillus paramycoides, Caldibacillus thermoamylovorans, Bacillus pumilus, and Bacillus tropicus.
• Fungi: Aspergillus lentulus and Rhizopus arrhizus showed strong degradation abilities. Other fungal isolates were Aspergillus fellis, Aspergillus luteonubrus, and Aspergillus arizonicus.

To assess degradation ability, the isolated strains were cultured in a Minimal Salt Medium (MSM) supplemented with 1% crude oil or specific crude oil fractions (separated by distillation based on boiling point ranges: F1 (40-60°C), F2 (60-80°C), F3 (80-100°C), F4 (100-130°C)) as the sole carbon source.

Two main methods were used to evaluate degradation:

1. Spectrophotometry: Bacterial growth in liquid MSM with crude oil or fractions was monitored by measuring optical density (OD) at 600 nm over eight weeks. Increased OD indicated microbial growth, implying the utilization of the hydrocarbon substrate. Results showed significant growth for several bacterial isolates on crude oil, decreasing over time as the carbon source was depleted. Some bacterial isolates also showed growth on F2 and F4 fractions, suggesting consumption.

   # Pseudocode for Spectrophotometry Monitoring Initialize Minimal Salt Medium (MSM) Sterilize MSM and Crude Oil/Fractions separately Add 1% Crude Oil or Fraction to MSM Inoculate with standardized bacterial suspension (OD 600nm = 1) Incubate at 30°C, 150 rpm shaking Measure OD at 600nm at intervals (24h, 48h, 1 week, 3 weeks, 8 weeks) Record and analyze OD values over time for different isolates and substrates

2. 2,6-Dichlorophenol Indophenol (DCPIP) Assay: This redox indicator changes color from blue to colorless when reduced by microbial metabolic activity, specifically related to electron transfer during hydrocarbon degradation. Isolates were incubated in MSM with crude oil/fractions and DCPIP.
   • Several bacterial isolates (highlighted as numbers 7, 8, 11, 12, 14, corresponding to some of the top candidates) showed a strong ability to reduce DCPIP, causing the color to change completely from deep blue to colorless within 4 days when cultured with crude oil. Other bacterial isolates also showed some color reduction. However, bacterial isolates did not cause a color change with any of the isolated crude oil fractions using this method, indicating a lack of degradation activity on these lighter fractions under the assay conditions.
   • Fungal isolates Rhizopus arrhizus and Aspergillus lentulus also demonstrated a strong ability to reduce DCPIP with crude oil, changing the color from blue to colorless. Notably, these two fungal isolates also reduced DCPIP when cultured with the F1 fraction, indicating degradation of this lighter hydrocarbon component. Other fungal isolates degraded crude oil but not the fractions according to this assay.

     # Pseudocode for DCPIP Assay Initialize Minimal Salt Medium (MSM) Sterilize MSM, Crude Oil/Fraction, and DCPIP stock separately Add 1% Crude Oil or Fraction to 10ml MSM Add 1% (0.6 mg/L) DCPIP solution Inoculate with standardized bacterial suspension (OD 600nm = 1) or fungal mycelia Incubate at 30°C, 150 rpm shaking Observe and record color change (deep blue to colorless) every 24 hours for up to 2 weeks Compare color change to uninoculated control

In addition, fungal isolates were tested for their tolerance and ability to use crude oil as a sole carbon source by measuring mycelial radial growth on solid MSM plates supplemented with different crude oil concentrations (0%, 5%, 10%, 15%, 20%). All five fungal strains showed high tolerance and growth across all concentrations, confirming their ability to utilize crude oil. Rhizopus arrhizus and Aspergillus lentulus exhibited the highest growth rates.

# Pseudocode for Fungal Radial Growth Assay Prepare solid Minimal Salt Medium (MSM) plates Add varying concentrations of sterilized crude oil (0%, 5%, 10%, 15%, 20%) to MSM agar before pouring plates Inoculate the center of each plate with a small plug of fungal mycelia Incubate plates at 30°C for 7 days Measure the diameter of the fungal colony (radial growth) daily or at the end of the incubation period Calculate radial growth rate (cm/day)

The study concludes that the isolated indigenous microorganisms, particularly Bacillus anthracis, Bacillus cereus, Achromobacter sp., Pseudomonas aeruginosa, Rhizopus arrhizus, and Aspergillus lentulus, possess significant potential for the bioremediation of crude oil in contaminated soil environments.

Practical Implementation and Application:

This research provides foundational knowledge for developing biological treatments for oil spills and contaminated sites. Practical implementation steps based on these findings could include:

1. Site Assessment: Identify the specific types and concentrations of hydrocarbons present at a contaminated site.
2. Microbial Sourcing: Isolate indigenous hydrocarbon-degrading bacteria and fungi from the specific contaminated site, as performed in the paper. This ensures the microbes are already adapted to the local conditions and contaminants. Alternatively, obtain known, effective strains like the ones identified in the study (Pseudomonas, Bacillus, Achromobacter, Aspergillus, Rhizopus species) from culture collections, ensuring they are non-pathogenic or appropriately handled if potential pathogens like Bacillus anthracis are involved (though the paper notes most Bacillus spp. are non-pathogenic and widely used in biotech).
3. Strain Selection & Optimization: Screen the isolated strains (or select known strains) for optimal growth and degradation efficiency on the specific types of hydrocarbons found at the site using laboratory-scale experiments similar to those described (MSM media, spectrophotometry, DCPIP assay). Mixed cultures could potentially offer synergistic degradation benefits, as suggested for future research.
4. Pilot-Scale Testing: Conduct pilot studies on smaller contaminated areas to evaluate the efficacy of selected microbial consortia under real-world environmental conditions (soil type, temperature, moisture, nutrient availability).
5. Nutrient Amendment: Often, bioremediation requires adding nutrients (biostimulation) like nitrogen and phosphorus to enhance microbial activity, as hydrocarbon degradation is a high-energy process. The MSM used in the study provides essential salts and a vitamin mix, which mimics nutrient supplementation strategies.
6. Oxygen Supply: Aerobic respiration is the primary mechanism for hydrocarbon degradation by many of these microbes. For in situ bioremediation, ensuring adequate oxygen supply (e.g., tilling, aeration) is crucial, especially in compacted or waterlogged soils.
7. Monitoring: Implement monitoring programs to track the reduction in hydrocarbon concentrations over time and assess the health and activity of the microbial population. Analytical techniques like gas chromatography-mass spectrometry (GC-MS) would be used to quantify specific hydrocarbons, going beyond the qualitative/semi-quantitative methods (OD, DCPIP) used in this initial screening study.

Implementation Considerations:

• Computational Requirements: Microbial identification via sequencing and bioinformatics (BLAST analysis) requires standard computing resources for processing sequence data. No intensive computational AI/ML models are directly applied for the core degradation process itself based on this paper's methodology.
• Potential Limitations:
  • Environmental Factors: Temperature, pH, salinity, moisture, and nutrient availability can significantly impact microbial activity in the field. Lab results under optimized conditions may not directly translate to variable field conditions.
  • Hydrocarbon Complexity: Crude oil is a complex mixture. While Rhizopus arrhizus and Aspergillus lentulus showed degradation of the lighter F1 fraction, bacterial isolates in the DCPIP assay did not degrade tested fractions, suggesting limitations for some microbes on specific compound classes. Heavier fractions (asphaltenes, resins) are generally more difficult to degrade.
  • Toxicity: High concentrations of hydrocarbons can be toxic to microorganisms.
  • Competition: Indigenous non-degrading microbes may compete with the introduced or stimulated degraders.
  • Scale-Up: Scaling laboratory findings to field applications requires careful planning and significant logistical effort.
  • Pathogenicity: While beneficial, potential pathogenicity of isolated strains (e.g., Bacillus anthracis requires careful handling and assessment for field release) needs to be considered.
• Deployment Strategies:
  • Biostimulation: Adding nutrients and optimizing environmental conditions (most common approach).
  • Bioaugmentation: Introducing specific, highly effective degrading strains (isolated or commercial) to the site. This paper supports the potential for using indigenous strains for bioaugmentation.
  • Bioreactor Treatment: For highly contaminated soil or water, ex situ treatment in controlled bioreactors could be considered, offering more control over conditions.

The findings provide a strong starting point for utilizing these specific species, particularly Pseudomonas aeruginosa, Bacromobacter sp., Bacillus anthracis, Bacillus cereus, Rhizopus arrhizus, and Aspergillus lentulus, in bioremediation strategies for crude oil spills. Further research on enzyme mechanisms and mixed culture performance, as suggested by the authors, would enhance the practical applicability and optimization of these biological approaches.