Preliminary Screening and Identification of Biosurfactant-Producing Bacteria Derived from Olive Waste and Petroleum Hydrocarbon–Contaminated Soils | ||
| زیست شناسی خاک | ||
| Articles in Press, Accepted Manuscript, Available Online from 21 February 2026 PDF (1.23 M) | ||
| Document Type: Research Paper | ||
| DOI: 10.22092/sbj.2026.371894.292 | ||
| Authors | ||
| Ali Ebadi* ; Sepideh Al-sadat Jamali; Mehran Kamrani | ||
| Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NSTRI), Karaj, Iran. | ||
| Abstract | ||
| Background and Objectives: The global shift toward sustainable agriculture has intensified interest in environmentally friendly alternatives to conventional chemical inputs. Biofertilizers, which exploit beneficial microorganisms and their secondary metabolites, play a critical role in improving nutrient availability, solubility, and uptake efficiency in plants. Among these metabolites, biosurfactants—surface-active compounds of microbial origin—have attracted increasing attention due to their high biodegradability, low toxicity, and production from renewable resources. These characteristics make biosurfactants promising substitutes for synthetic surfactants in both agricultural and environmental applications. In agricultural systems, biosurfactants contribute to soil quality improvement by enhancing the solubilization of hydrophobic compounds, increasing the bioavailability of micronutrients such as iron and phosphorus, and facilitating the mobilization of heavy metals. These properties are particularly relevant in contaminated or nutrient-deficient soils, where biosurfactants can support phytoremediation processes and improve plant growth under stress conditions. Anionic biosurfactants, especially rhamnolipids, are of particular interest because of their strong surface activity, metal-chelating capacity, and compatibility with soil–plant systems. Despite their considerable potential, the practical application of biosurfactants is constrained by the limited availability of efficient, high-yield microbial producers, particularly indigenous strains adapted to harsh or contaminated environments. Systematic screening and comparative evaluation of biosurfactant-producing bacteria are therefore essential to identify promising candidates for future development of biofertilizers and nano-biofertilizer formulations. The present study aimed to isolate, screen, and characterize indigenous bacterial strains from olive oil mill waste and petroleum-contaminated soils, with a specific focus on identifying efficient producers of anionic biosurfactants using a combination of qualitative, semi-quantitative, chromatographic, and molecular approaches. Materials and Methods: Soil and waste samples were collected under sterile conditions from olive oil mill residues in Tarom region and from petroleum-contaminated soil at Pond No. 4 of the Shahid Tondguyan Oil Refinery, Tehran. Samples were transported to the laboratory under cooled conditions and subjected to an enrichment process in mineral salt medium supplemented with 3% (v/v) vegetable oil as the sole carbon source. Enrichment was conducted at 30 °C with shaking at 120 rpm over three successive cycles, totaling 21 days. Following enrichment, serial dilutions were prepared and plated on blood agar medium. Isolates exhibiting alpha or beta hemolysis were selected and purified on tryptic soy agar. A total of 30 bacterial isolates were retained for further analysis. Hemolytic activity was evaluated by measuring colony diameter and hemolytic zone, and the halo-to-colony ratio was calculated. To specifically screen for anionic biosurfactant production, isolates were tested on CTAB–methylene blue agar, and complex formation around colonies was recorded. Biosurfactant production was further assessed in liquid biosurfactant-forming broth, after which cell-free supernatants were obtained by centrifugation. Oil spreading and drop collapse assays were performed as indicators of surface tension reduction. Thin-layer chromatography (TLC) was used for preliminary chemical characterization of biosurfactants. Extracted compounds were separated using chloroform–methanol–water solvent systems and visualized with phenol–sulfuric acid, ninhydrin, and iodine reagents to detect glycolipid, lipopeptide, and lipid components, respectively. Molecular identification of the four most promising isolates was carried out by amplification and sequencing of the 16S rRNA gene. Phylogenetic analysis was performed using the Maximum Likelihood method with the Tamura–Nei model, and statistical analyses were conducted using one-way ANOVA and Duncan’s multiple range test at a 5% significance level. Results: Analysis of variance revealed significant differences among the isolates for all evaluated traits at the 1% probability level. All 30 isolates exhibited hemolytic activity, with halo-to-colony ratios ranging from 1.28 to 2.90. Isolate 23 showed the highest hemolytic index, while isolate 21 displayed the lowest value. However, only approximately 23% of the isolates formed visible complexes on CTAB agar, indicating the production of anionic biosurfactants. The halo-to-colony ratio in this assay ranged from 1.42 to 2.23, with isolate 28 exhibiting the highest anionic biosurfactant activity. Oil spreading diameters varied widely, from 3.46 to 50.6 mm. Isolates 19 and 17 demonstrated significantly greater oil displacement compared to other isolates, reflecting superior surface tension–reducing capability. Drop collapse assay results showed droplet diameters ranging from 3.93 to 14.8 mm, with more than 73% of isolates outperforming the distilled water control. A significant positive correlation was observed between CTAB complex formation and both oil spreading and drop collapse assays, whereas hemolytic activity did not correlate significantly with other traits. TLC analysis revealed distinct biosurfactant profiles among the superior isolates. Isolates 17 and 23 reacted positively with all three detection reagents, suggesting the production of polymeric lipo-glyco-protein biosurfactants with strong emulsifying properties. Isolate 19 showed positive reactions with phenol–sulfuric acid and iodine, indicating glycolipid biosurfactants, likely rhamnolipids. Isolate 28 reacted with phenol–sulfuric acid and ninhydrin, suggesting mixed carbohydrate–peptide biosurfactants. Phylogenetic analysis identified isolate 17 as closely related to Klebsiella sp., isolates 19 and 28 as members of the genus Pseudomonas, and isolate 23 related to Microbacterium paraoxydans. Conclusion: This study demonstrated substantial diversity among indigenous bacterial isolates in terms of biosurfactant production, chemical composition, and functional performance. While hemolytic activity served as a useful preliminary screening tool, it was insufficient for identifying anionic biosurfactant producers without complementary assays. Indigenous Pseudomonas isolates exhibited the highest surface activity and glycolipid production potential, whereas Klebsiella and Microbacterium isolates primarily produced polymeric biosurfactants with strong emulsifying properties. The strong agreement between chromatographic profiles, functional assays, and phylogenetic identification highlights the importance of an integrated screening strategy. Overall, isolates 17, 19, 23, and 28 represent promising candidates for further optimization and development of biosurfactant-based biofertilizers and bioremediation agents. These findings provide a solid foundation for future studies focusing on production optimization, molecular characterization, and application of indigenous biosurfactants in sustainable agriculture and environmental management. | ||
| Keywords | ||
| Biosurfactant; Molecular identification; Native strains; Pseudomonas; Screening of biosurfactant production | ||
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