This study compares the crude oil removal efficiency of free and biosurfactant-producing bacterial consortia (Bacillus tequilensis NR_104919 and Bacillus axarquiensis NR_115929) entrapped in chitosan (CH), chitosan/glutaraldehyde (CH/GA), and chitosan/Na-bentonite (CH/BNT) matrices. Additionally, the mechanical and thermal stability, swelling capacity, surface morphology, and chemical bonding of these composites were evaluated. Initially, the biosurfactant produced by the consortium was identified using screening methods, and its lipopeptide structure was confirmed through TLC and ¹H NMR analyses, with a yield of 0.2 g/L. Following characterization, the biosurfactant-producing bacterial consortium was immobilized onto CH, CH/GA, and CH/BNT beads for further assessment. When comparing the oil removal capacities of the composites, the consortium immobilized in the CH/BNT matrix exhibited significantly higher crude oil removal efficiency than those entrapped in CH, CH/GA, and the free consortium. The maximum oil removal by the CH/BNT-entrapped consortium was achieved under optimized conditions: 0.5 mL crude oil, 0.1 g Na-bentonite, pH = 6.0, at 35 ℃, over a 7-day incubation period. However, a decline in oil remediation performance was observed from the fourth reuse cycle onward. These laboratory-scale findings were further validated through ex-situ simulation and long-term experiments, confirming the potential of the CH/BNT composite for effective and sustainable petroleum pollutant remediation. The swelling capacity of inactivated CH was comparable to that of the CH/BNT composite, with both exhibiting greater swelling than CH/GA beads. Additionally, CH/BNT beads demonstrated superior mechanical stability relative to CH and CH/GA, which showed similar stability profiles. Thermal stability, surface morphology, and chemical bonding of the CH, CH/GA, and CH/BNT composites were characterized using TGA, FT-IR, and SEM analyses. CH/BNT composite represents an effective, economical, and environmentally benign alternative to costly synthetic materials for the remediation of oil pollution in aquatic environments. This study highlights the potential of chitosan/Na-bentonite-based composites as sustainable solutions for petroleum pollutant removal, while recommending pilot-scale investigations to assess their feasibility and performance under real-world conditions.

The purpose of this study was to identify and compare the degradation efficiencies of free and entrapped bacterial consortia (Staphylococcus capitis CP053957.1 and Achromobacter marplatensis MT078618.1) to different polymers such as Sodium Alginate (SA), Sodium Alginate/Poly (Vinyl Alcohol) (SA/PVA), and Bushnell Haas Agar (BHA). In addition to SA and SA/PVA, which are cost-effective, non-toxic and have different functional groups, BHA, which is frequently encountered in laboratory-scale studies but has not been used as an entrapment material until now. Based on these, the polymers with different surface morphologies and chemical compositions were analyzed by SEM and FT-IR. While the petroleum removal efficiency was higher with the entrapped bacterial consortia than with the free one, BHA-entrapped bacterial consortium enhanced the petroleum removal more than SA and SA/PVA. Accordingly, the degradation rate of bacterial consortia entrapped with BHA was 2.039 day⁻¹, SA/PVA was 1.560, SA was 0.993, the half-life period of BHA-entrapped bacterial consortia is quite low (t_1/2 = 0.339) compared with SA (t_1/2 = 0.444) and SA/PVA (t_1/2 = 0.697). The effects of the four main factors such as: amount of BHA (0.5, 1, 1.5, 2, 2.5, 3 g), disc size (4, 5, 6, 7, 8 mm), inoculum concentration (1, 2.5, 5, 7.5, 10 mL), and incubation period on petroleum removal were also investigated. The maximum petroleum removal (94.5%) was obtained at ≥ 2.5 mL of bacterial consortium entrapped in 2 g BHA with a 7 mm disc size at 168 h and the results were also confirmed by statistical analysis. Although a decrease was observed during the reuse of bacterial consortium entrapped in BHA, the petroleum removal was still above 50% at 10th cycle. Based on GC-MS analysis, the removal capacity of BHA-entrapped consortium was over 90% for short-chain n-alkanes and 80% for medium-chain n-alkanes. Overall, the obtained data are expected to provide a potential guideline in cleaning up the large-scale oil pollution in the future. Since there has been no similar study investigating petroleum removal with the bacterial consortia entrapped with BHA, this novel entrapment material can potentially be used in the treatment of petroleum pollution in advanced remediation studies.