Comparison of Different Bioremediation Methods in Reducing Petroleum Hydrocarbons of a Contaminated Soil (Case Study: Evaporation Ponds of Tabriz Refinery)

Document Type : Research Article

Authors

1 Department of Soil Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.

2 Department of Applied Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran.

Abstract

Background and Objectives
Petroleum and its derivatives serve as the primary energy source for industrial and domestic use today. However, soil contamination by crude oil and its refined products represents one of the most hazardous forms of environmental pollution. Bioremediation has emerged as an effective strategy for eliminating petroleum hydrocarbons from contaminated soils. Among bioremediation approaches, the application of beneficial free-living bacteria and plant growth-promoting rhizobacteria (PGPR) associated with the rhizosphere has gained significant attention for their bioremediation potential. Substantial experimental evidence supports the efficacy of these microbial agents in reducing both contaminant concentrations and ecotoxicity in polluted soils through bioremediation processes. Three principal bioremediation strategies have demonstrated effectiveness in petroleum hydrocarbon removal: 1- Intrinsic bioremediation - relying on natural biodegradation by indigenous microorganisms without human intervention; 2- Biostimulation - enhancing microbial activity through fertilizer addition, organic amendments, surfactants, and optimization of aeration and moisture conditions; 3- Bioaugmentation - introducing specialized hydrocarbon-degrading microorganisms into the soil microbiome while providing optimal growth conditions and nutrient supplementation.

Methodology
In the present study, a petroleum-contaminated soil was collected from evaporation ponds at Tabriz refinery, East Azerbaijan province, Iran to evaluate different bioremediation methods for hydrocarbon degradation. Following collection, hydrocarbon-degrading bacteria were isolated from the contaminated soils. Three bioremediation approaches were tested: (1) intrinsic bioremediation (control, with no amendments), (2) biostimulation (including aeration, NPK fertilization, organic amendments [cow manure, chicken manure, compost], and Tween 80 surfactant addition), and (3) bioaugmentation (using either individual inoculations of five crude oil-degrading bacterial strains or their consortium). The experiment was conducted in 2 kg pots over a four-month period. Upon completion, we measured: (1) total petroleum hydrocarbons (TPH), (2) soil enzyme activities (dehydrogenase, urease, phosphatase, catalase), and (3) biological indicators (BR: basal respiration and SIR: substrate-induced respiration).

Results
Among all treatments, the use of Tween 80, cow manure, Nutrient Broth (NB) without bacteria, and NPK fertilizers resulted in hydrocarbon degradation rates of 89.0%, 86.3%, 57.6%, and 67.4%, respectively, representing the highest degradation efficiencies in the soil. Among the bioaugmentation treatments, Arthrobacter sp. COD 2-3 and the bacterial consortium showed the highest TPH degradation rates, at 69.7% and 63.3%, respectively. The activity of dehydrogenase and urease enzymes increased in all treatments at the beginning of the experiment but declined after 2 and 4 months, respectively. Initially, dehydrogenase activity was highest in the bioremediation treatments involving Pseudochrobactrum sp. COD 1-4, Stenotrophomonas sp. COD 1-1, the bacterial consortium, and chicken manure, while Tween 80 exhibited the lowest activity. Urease activity was highest in NB, watering, chicken manure, and the consortium treatments, but lowest in the compost treatment and the Arthrobacter sp. COD 2-3 inoculation. Catalase enzyme activity increased significantly 2–3 days after treatment in the NB, Tween 80, and consortium treatments compared to other treatments. However, over time, activity in these three treatments declined, whereas in other bioremediation treatments and the contaminated control soil, it increased gradually, indicating sustained microbial viability and petroleum-degrading activity. The activity of acidic and alkaline phosphatase enzymes remained relatively unchanged throughout the experiment. Basal respiration (BR) and substrate-induced respiration (SIR) increased initially in all treatments but decreased significantly after four months. At baseline, the bacterial consortium, compost, and cow manure treatments exhibited the highest basal respiration rates, followed by microbial inoculations and chicken manure. Tween 80, aerated soil, and the contaminated control had the lowest basal respiration. Finally, the percentage of TPH degradation showed positive correlations with soil basal respiration, substrate-induced respiration, and dehydrogenase activity.

Conclusion
Finally, the treatments for remediation of oil-contaminated soil were introduced, including the addition of cow dung, NPK fertilizer, and a bacterial consortium as the most effective bioremediation method. Adding animal manure improved the soil's physical and chemical properties such as enhancing soil structure and facilitated oxygen transport, providing energy for microorganisms and thereby boosting microbial activity. Dehydrogenase, an intracellular enzyme, is directly linked to the activity of the soil's microbial population. An increase in this enzyme's activity at the beginning of the experiment in oil-contaminated soil indicated heightened microbial degradation activity. However, the subsequent decline in enzyme activity by the end of the experiment was likely due to the prolonged test duration and the microbes entering the death phase. In general, it is recommended to use shorter sampling intervals and testing periods for all enzyme activities to better monitor changes over time. This approach would help clarify the trends in enzyme activity throughout the experiment.

Data Availability Statement
Data is available on reasonable request from the authors.

Acknowledgements
This paper is published as a part of a Ph.D. thesis supported by the Vice Chancellor for Research and Technology of the University of Tabriz, Tabriz, Iran. The authors are thankful to the University of Tabriz for financial supports.

Conflict of interest
The authors declare no conflict of interest.

Ethical considerations
The authors avoided data fabrication, falsification, plagiarism, and misconduct.

Keywords

Main Subjects

References

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Journal of Soil and Plant Science
Volume 35, Issue 2
September 2025
Pages 23-41

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