Bioremediation of Diesel-Contaminated Soil: Hydrocarbon Degradation and Soil Toxicity Reduction by Constructed Bacterial Consortia
Abstract
1. Introduction
2. Materials and Methods
2.1. Preparation of Petroleum-Degrading Bacterial Consortia
2.2. Preparation of Experimental Soil
2.3. Experimental Design for the Out-Door Soil Remediation Trial
- 18% Nitrogen (11.7% ammoniacal nitrogen, 6.3% nitric nitrogen)
- 60% Phosphorus (6% soluble in neutral ammonium citrate and water; 54% water-soluble)
- 12% Potassium (water-soluble)
- Treatment 1 (CSC): untreated clean soil control;
- Treatment 2 (DCS): 2% (v/w) diesel-contaminated soil;
- Treatment 3 (BT1): 2% (v/w) diesel-contaminated soil + 10% bacterial consortia;
- Treatment 4 (BT2): 2% (v/w) diesel-contaminated soil + 10% bacterial consortia;
- Treatment 5 (BT3): 2% (v/w) diesel-contaminated soil + 10% bacterial consortia.
2.4. Extraction and Quantification of Petroleum Hydrocarbons in Soil
2.5. Total Viable Bacterial Cell Counts in Soil
2.6. Estimation of TPHs Degraders Counts in Soil Using the Most Probable Number (MPN) Method
2.7. Soil Sampling, DNA Extraction, and Sequencing Analysis
2.8. Earthworm Toxicity Test
2.9. Higher Plant Toxicity Test
2.10. Acute Toxicity Test of Daphnia magna
2.11. Acute Toxicity Test of Luminescent Bacteria
2.12. Statistical Analysis
3. Results and Discussion
3.1. Diesel Hydrocarbon Biodegradation and Fractional Analysis Across Treatments Diesel Degradation Study with GC-FID Analysis
3.2. Soil Microbial Population Dynamics During Bioremediation
3.3. Soil Bacterial Community Dynamics Analysis During Bioremediation
3.4. Toxicity Evaluation of Soil and Soil Water Extracts During Bioremediation
3.4.1. Effect of Diesel-Contaminated Soil (Before and After Remediation) on Earthworm (Eisenia foetida) Survival and Body Mass
3.4.2. Phytotoxicity of Diesel-Contaminated Soil (Before and After Remediation)
3.4.3. Daphnid (Daphnia magna) Toxicity Test
3.4.4. Luminescent Bacterial (Aliivibrio fischeri) Toxicity Test
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
TPH | Total Petroleum Hydrocarbons |
ONF | Octofluoronaphthalene |
PAH | Polycylic Aromatic Hydrocarbons |
LOI | Loss on Ignition |
CFU | Colony Forming Unit |
TVC | Total Viable Count |
MPN | Most Probable Number |
IG | Germination Index |
LMW | Low Molecular Weight |
ASV | Amplicon Sequence Variant |
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Species/Strain ID | Bacterial Treatment 1 (BT1) | Bacterial Treatment 2 (BT2) | Bacterial Treatment 3 (BT3) |
---|---|---|---|
Acinetobacter seifertii (BHA_1) | ✓ | ✓ | ✓ |
Bacillus subtilis (BHAD_1) | ✓ | ✓ | |
Rhodococcus qingshengii (BHAD_2) | ✓ | ||
Acinetobacter seifertii (NA_1) | ✓ | ||
Cellulosimicrobium cellulans (NA_2) | ✓ | ✓ | |
Acinetobacter seifertii (TRI_1) | ✓ | ||
Microbacterium algeriense (TRI_2) | ✓ | ||
Acinetobacter vivianii (TRI_3) | ✓ | ✓ |
S2 | S3 | |||||||
---|---|---|---|---|---|---|---|---|
DCS | BT1 | BT2 | BT3 | DCS | BT1 | BT2 | BT3 | |
C8-10 | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% | 100.00% |
C10-12 | 73.14 ± 3.40% | 79.85 ± 0.44% | 75.02 ± 2.89% | 79.73 ± 1.28% | 84.83 ± 1.03% | 85.21 ± 0.85% | 87.68 ± 1.31% | 82.44 ± 5.20% |
C12-14 | 28.21 ± 3.98% | 24.06 ± 4.90% | 39.86 ± 3.25% | 41.47 ± 0.92% | 34.95 ± 3.67% | 30.32 ± 5.18% | 39.60 ± 5.22% | 42.64 ± 4.15% |
C14-16 | 23.17 ± 8.68% | 46.54 ± 6.32% | 48.74 ± 12.82% | 78.67 ± 11.78% * | 30.13 ± 7.20% | 54.88 ± 5.68% * | 55.26 ± 8.97% * | 86.0 ± 2.47% * |
C16-18 | 41.24 ± 7.22% | 40.29 ± 9.30% | 51.74 ± 12.31% | 57.94 ± 2.97% | 44.61 ± 3.41% | 43.22 ± 1.39% | 60.41 ± 1.99% * | 65.81 ± 2.06% * |
C18-20 | 42.09 ± 3.48% | 62.02 ± 11.44% | 68.53 ± 10.74% | 77.21 ± 9.62% * | 47.28 ± 3.08% | 68.36 ± 11.89% * | 75.92 ± 14.98% * | 89.47 ± 2.02% * |
C20-22 | 70.43 ± 7.85% | 80.60 ± 12.96% | 86.96 ± 3.51% | 91.84 ± 3.81% | 75.50 ± 0.15% | 81.26 ± 13.17% | 92.30 ± 3.11% * | 97.17 ± 1.00% * |
C22-24 | 40.16 ± 8.65% | 73.59 ± 15.65% * | 73.18 ± 7.10% * | 70.88 ± 5.27% * | 50.11 ± 8.01% | 75.59 ± 15.58% * | 78.56 ± 11.10% * | 91.95 ± 10.84% * |
C24-26 | 40.42 ± 18.44% | 58.09 ± 1.81% | 39.67 ± 11.59% | 41.87 ± 17.49% | 55.43 ± 6.71% | 66.03 ± 2.90% | 64.88 ± 10.95% | 63.86 ± 1.75% |
C26-28 | 48.65 ± 6.36% | 36.29 ± 6.03% | 41.50 ± 11.74% | 49.35 ± 2.43% | 52.62 ± 3.70% | 72.47 ± 3.06% * | 76.07 ± 11.59% * | 77.09 ± 7.87% * |
Lepidium sativum | Sinapis alba | Sorghum saccharatum | |||||
TPH reduction S3 (%) | S1 | S3 | S1 | S3 | S1 | S3 | |
CSC | 0% | 0% | 0% | 3.7% | 0% | 3.7% | 7.4% |
DCS | 48.19% | 36.7% | 7.1% | 22.2% | 11.1% | 18.5% | 14.8% |
BT 1 | 63.42% | 25.0% | 3.6% | 11.1% | 0% | 11.1% | 3.7% |
BT 2 | 73.08% | 21.4% | 0% | 18.5% | 0% | 11.1% | 7.4% |
BT 3 | 78.34% | 25% | 0% | 14.8% | 0% | 14.8% | 7.4% |
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Wang, M.; Dowling, D.N.; Germaine, K.J. Bioremediation of Diesel-Contaminated Soil: Hydrocarbon Degradation and Soil Toxicity Reduction by Constructed Bacterial Consortia. Appl. Sci. 2025, 15, 10143. https://doi.org/10.3390/app151810143
Wang M, Dowling DN, Germaine KJ. Bioremediation of Diesel-Contaminated Soil: Hydrocarbon Degradation and Soil Toxicity Reduction by Constructed Bacterial Consortia. Applied Sciences. 2025; 15(18):10143. https://doi.org/10.3390/app151810143
Chicago/Turabian StyleWang, Mutian, David N. Dowling, and Kieran J. Germaine. 2025. "Bioremediation of Diesel-Contaminated Soil: Hydrocarbon Degradation and Soil Toxicity Reduction by Constructed Bacterial Consortia" Applied Sciences 15, no. 18: 10143. https://doi.org/10.3390/app151810143
APA StyleWang, M., Dowling, D. N., & Germaine, K. J. (2025). Bioremediation of Diesel-Contaminated Soil: Hydrocarbon Degradation and Soil Toxicity Reduction by Constructed Bacterial Consortia. Applied Sciences, 15(18), 10143. https://doi.org/10.3390/app151810143