The efficiency of oil-degrading and phosphate-solubilizing bacteria in phosphorus availability of a oil-contaminated calcareous soil

Abstract

Background and Objectives: The leakage of petroleum compounds into ecosystems poses a significant threat to the health of living organisms and humans. Various physical and chemical methods are employed to address oil contamination in soils; however, many of these methods are underutilized due to high costs and adverse environmental impacts. Physical methods are cost-effective but often fail to completely remove contaminants, leading to phase changes in pollutants. Chemical methods, such as precipitation, chemical oxidation, and the use of Fenton's reagent, are costly and environmentally unfriendly. Bioremediation, on the other hand, is widely used for the treatment of petroleum-contaminated soils due to its cost-effectiveness and environmental compatibility. This method relies on microorganisms to enzymatically degrade petroleum compounds into water and carbon dioxide. A key challenge in bioremediation is the limited availability of essential nutrients such as phosphorus in contaminated soils. Studies have shown that oil contamination significantly reduces the bioavailable phosphorus in soil, which acts as a critical limiting factor for plant growth and the effectiveness of bioremediation. To address this phosphorus deficiency, phosphate-solubilizing bacteria (PSBs) can be used to solubilize inorganic phosphate in the soil. This study aimed to isolate microorganisms capable of degrading crude oil and solubilizing phosphate from drilling mud and evaluate the changes in soil phosphorus after inoculation with these bacteria.

Materials and Methods: To enhance bioremediation and increase soil phosphorus availability, bacterial strains were initially isolated via enrichment techniques from drilling mud contaminated with high levels of total petroleum hydrocarbons (TPH). Their ability to utilize crude oil as the sole carbon and energy source was evaluated through three assays: growth on mineral salt medium (MSM) agar, growth in MSM, and crude oil degradation in MSM using a gravimetric method. Then, phosphate solubilization capability was determined using the spot-on-plate method in Sperber-agar, employing tricalcium phosphate [Ca3(PO4)2] as the insoluble phosphate source. Next, biosurfactant production was assessed using oil spreading, surface tension, and emulsification index (E24) assays, with crude oil as the carbon source. The isolates were phenotypically identified, and a soil microcosm experiment was designed using a completely randomized design with two treatments and six replicates in glass containers containing 300 grams of contaminated soil finally. The three top-performing isolates were mixed in equal proportions and inoculated into contaminated soil. After 30 days of incubation at 27°C, the available phosphorus content in the soil was measured using the sodium bicarbonate extraction method on days 0 and 30. Data variance analysis and graphing were performed using Minitab and Excel software, respectively.

Results: A total of seven distinct bacterial isolates were obtained from contaminated drilling mud at the end of enrichment stage. Crude oil degradation assays in MSM showed that isolates NW1, NW5, and NW7 achieved degradation rates of 20.6%, 19.66%, and 15.53%, respectively, and isolates NW2, NW7, and NW4 exhibited growth rates (OD₆₀₀) of 0.35, 0.35, and 0.31, respectively, over seven days. Phosphate solubilization tests revealed that isolates NW7 and NW5 demonstrated the highest solubilization indices of 3.76 and 2.83 on Sperber-agar, respectively (P<0.05). Based on these results, isolates NW1, NW2, and NW7 were selected for further experiments. In biosurfactant production tests, isolate NW2 exhibited the largest oil spread zone (1.26 cm) and the highest emulsification index (46.7%), significantly outperforming other isolates. The cumulative results indicated that NW2 was the most effective biosurfactant producer. Phenotypic identification classified NW1, NW2, and NW7 as belonging to the genera Pseudomonas, Bacillus, and Rhodococcus, respectively. The bacterial consortium significantly increased soil-available phosphorus from 3.4 mg/kg to 8.46 mg/kg compared to the control (P<0.05) in the soil microcosm experiment.

Conclusion: Oil compound spills have detrimental effects on ecosystems and human health, and conventional remediation methods face cost and environmental limitations. Bioremediation provides a sustainable and economical solution by employing microorganisms to degrade petroleum hydrocarbons. However, nutrient deficiencies, especially phosphorus, in contaminated soils restrict its efficiency. This study aims to isolate and evaluate oil-degrading and phosphate-solubilizing microorganisms from contaminated drilling mud, with the aim of increasing bioremediation efficiency and soil phosphorus levels. Among the seven isolated bacteria, NW1, NW2, and NW7 showed superior hydrocarbon degradation and phosphate solubilization capabilities and were identified as Pseudomonas, Bacillus, and Rhodococcus, respectively. The inoculation of these strains significantly enhanced soil available phosphorus, highlighting their potential for improving bioremediation efficiency and soil fertility. It is recommended that future studies investigate the phosphorus solubilization capability of these isolates in soils with varying levels of salinity, pH, and total phosphorus. Additionally, the impact of bacterial immobilization on different carriers for phosphorus solubilization in soil represents an interesting topic that has not yet been explored.