Laboratory soil bacterium degrades phenanthrene and produces stable biosurfactant

A bacterium isolated from uncontaminated soil in southern India removed 72% of phenanthrene from laboratory cultures maintained under static, non-agitated conditions, according to a laboratory study of petroleum-hydrocarbon remediation. The isolate, identified as Stenotrophomonas maltophilia VITSVK4 through 16S rRNA gene sequencing, also produced a glycolipid-type biosurfactant that retained activity across a range of temperature, pH and salinity conditions. The results support the strain as a candidate for further investigation, but they do not establish performance in polluted field soils or sediments.

Phenanthrene is a three-ring polycyclic aromatic hydrocarbon used in the study as a representative petroleum contaminant. Its hydrophobic structure limits its availability to microorganisms, while its persistence and toxicity make its removal environmentally important. Bacteria from several genera have been reported to degrade petroleum hydrocarbons, including Achromobacter, Acinetobacter, Bacillus, Marinobacter, Micrococcus, Pseudomonas and Rhodococcus. Many bioremediation experiments use agitated or well-aerated cultures, but the researchers selected static conditions to test whether degradation could occur without continuous mixing. Under those conditions, VITSVK4 degraded phenanthrene without an externally imposed aeration or agitation regime.

The researchers obtained soil from a depth of 5–10 cm in the Vallimalai hills near Katpadi, Vellore, India, after removing the exposed surface layer. They enriched the sample for seven days in Bushnell Haas broth containing 100 mg/L phenanthrene as the sole carbon source, keeping the cultures static throughout. Two isolates with distinct colony morphologies, designated VITSVK4 and VITSVK5, were selected. Both formed biofilms, and a 96-well assay classified both as strong producers. In the initial comparison, VITSVK4 degraded 45% ± 2.08% of 100 mg/L phenanthrene over seven days, compared with 29% ± 1.73% for VITSVK5. VITSVK4 also produced stronger biosurfactant activity, with an emulsification index of 73% ± 2.30%, compared with 62% ± 2.08% for VITSVK5.

Additional screening showed positive lipase and laccase activity for VITSVK4, whereas VITSVK5 was positive only in the lipase assay. Researchers therefore selected VITSVK4 for molecular characterization and the detailed degradation experiments. They amplified and sequenced a 1,418-base-pair segment of the isolate’s 16S rRNA gene. A BLASTN comparison with 100% query coverage showed 99.44% sequence similarity to a reference S. maltophilia strain, supporting the tentative identification. The gene sequence was deposited in GenBank under accession PX485663, and phylogenetic analysis was used to assess its relationship to reference sequences.

Growth measurements showed that dry biomass increased progressively during a 10-day experiment in medium containing phenanthrene and 0.3% yeast extract. It reached a maximum of 0.74 ± 0.05 g/L on day eight and declined over the following days. One-factor-at-a-time testing examined temperature, pH, inoculum volume and incubation time under static conditions. Among the levels tested, 32 °C, pH 7, a 2% inoculum and an eight-day incubation produced the highest phenanthrene degradation. Yeast extract was the most effective nitrogen-containing supplement among those tested. In a separate supplementation experiment, 0.3% yeast extract yielded 66% ± 2.3% degradation, higher than the values obtained with the other nitrogen sources evaluated.

Under the selected laboratory conditions, VITSVK4 achieved its highest measured removal of phenanthrene at an initial concentration of 100 mg/L: 72% ± 1.73% after eight days. Removal decreased as the starting concentration rose, although the strain still degraded 59.3% ± 2.08% at 500 mg/L under static conditions over the same eight-day period. A pronounced decline began at concentrations of 600 mg/L and above. These percentages describe losses of the parent compound from the test medium after extraction and comparison with abiotic controls; they do not represent a uniform degradation rate across soils, sediments or mixed petroleum contaminants.

Gas chromatography–mass spectrometry provided chemical evidence consistent with removal of phenanthrene. The uninoculated control produced a prominent chromatographic peak at a retention time of 15.20 minutes, corresponding to phenanthrene, whereas the degraded culture had no detectable peak at that retention time. The researchers also identified a limitation in the extraction procedure: hexane is poorly suited to recovering polar and acidic phenanthrene transformation products. The chromatogram therefore supports disappearance of the parent compound from the extractable fraction, but it does not identify the complete transformation pathway, establish the extent of mineralization or demonstrate that every resulting product is environmentally safe.

Seed-germination experiments indicated that treatment with VITSVK4 reduced the phytotoxicity associated with phenanthrene. In mung bean, Vigna radiata, 90% of seeds germinated in liquid treated with VITSVK4, compared with 10% in untreated phenanthrene and 100% in distilled water. Estimated phytotoxicity was 24.5% for treated phenanthrene and 94.7% for untreated phenanthrene. In wheat, Triticum aestivum, 70% of seeds germinated in the treated material, compared with no germination in untreated phenanthrene and 90% in distilled water. The treated wheat had estimated phytotoxicity of 13.1%. Treated mung-bean roots and wheat shoots and roots did not differ significantly from their distilled-water controls, whereas treated mung-bean shoots were significantly shorter than the water control. These measurements apply to the two tested plant species and the laboratory exposure conditions used in the assay.

In additional screening assays, VITSVK4 degraded benzene, toluene and xylene, as well as phenol, mineral oil, crude oil and used engine oil. The strain’s cell-free supernatant showed biosurfactant activity in oil-displacement and drop-collapse tests, and its emulsification index reached 79.2% ± 2.51% under the optimized conditions. Fourier-transform infrared spectroscopy identified a peak at 2,984 cm−1 associated with C–H stretching, a peak at 1,736 cm−1 associated with C=O stretching and peaks at 1,233, 1,097 and 1,043 cm−1 associated with C–O–C and C–O stretching. The combination of lipid- and sugar-related bands supported classification as a glycolipid-type biosurfactant, rather than the identification of a particular purified molecule.

The biosurfactant retained substantial emulsifying activity from approximately pH 5 to 9, at temperatures up to 50 °C and at salinities up to 5%, with declining activity beyond those ranges. Residual activity was 94.9% ± 2.73% at 40 °C and 84.3% ± 2.59% at 50 °C. At pH 5 and 9, the respective values were 83.9% ± 2.59% and 81.8% ± 21.6%; at 1% and 5% salinity, they were 83.5% ± 2.04% and 67.4% ± 2.59%. In an oil-removal assay, the biosurfactant recovered 60% ± 2.51% of used engine oil from sterilized soil, compared with 19% ± 3.05% for distilled water and 73% ± 2.08% for Triton X-100.

The principal limitation is that all experiments were conducted under laboratory conditions. The study did not test field performance, environmental persistence or ecological compatibility in contaminated matrices. S. maltophilia is also a recognized opportunistic human pathogen, meaning that any environmental application of the living organism would require rigorous biosafety evaluation. The authors instead point toward possible use of cell-free extracts, purified enzymes or biosurfactant, but note that fuller characterization of the enzymes and biosurfactant would be necessary. The evidence therefore establishes laboratory degradation, reduced seed phytotoxicity and biosurfactant activity, while leaving field effectiveness, product safety and deployment readiness for further study.

Subject of Research: Laboratory evaluation of bacterial phenanthrene degradation and biosurfactant production for petroleum-contamination remediation

Article Title: Phenanthrene degradation and biosurfactant production by Stenotrophomonas maltophilia under static conditions: potential for bioremediation

Article References: Yuvaraja, S., Chakraborty, T., Gomathysankar, J., Kumar, S., & Krishnan, K. (2026). Phenanthrene degradation and biosurfactant production by Stenotrophomonas maltophilia under static conditions: potential for bioremediation. Frontiers in Bioengineering and Biotechnology, 14, Article 1983589. https://doi.org/10.3389/fbioe.2026.1983589

Image Credits: AI Generated

DOI: 10.3389/fbioe.2026.1983589

Keywords: phenanthrene degradation, Stenotrophomonas maltophilia, biosurfactants, bioremediation, petroleum hydrocarbons, static culture conditions, phytotoxicity, glycolipids, oil recovery, laboratory study, Phenanthrene, degradation