Formulation of Freeze-Dried Microbial Preparations from Selected Bacterial Strains for the Treatment of Oil Pollution in Aquatic Environments 

Abstract

The effective implementation of bioremediation is often constrained by the stability and delivery of microbial inocula. This study investigated the development and optimization of a hydrocarbon-degrading consortium comprising three indigenous bacterial strains: Rhodococcus ruber JN5.2, Stenotrophomonas acidaminiphila ZB2.1, and Bacillus amyloliquefaciens MD3.3. Phylogenetic analysis confirmed the taxonomic diversity of the members, while cross-streak assays established total biological compatibility, enabling stable coexistence. In synthetic wastewater containing 5% (v/v) oil, the bacterial consortium achieved a total petroleum hydrocarbon (TPH) removal efficiency of 78 ± 6% within 14 days. This performance significantly exceeded that of individual strains (51-54%), supported by a Synergy Index (SI) of 1.48 and a peak biomass density of 8.6 ± 0.9 × 10⁸ CFU/mL. To enhance practical applicability, freeze-drying conditions were optimized. Harvesting biomass at the 24-hour growth phase and utilizing a protective formulation of 10% skim milk, 5% sodium glutamate, and 20% bentonite preserved bacterial viability at ≥ 9.3 log₁₀ CFU/mL after one month of storage. The consortium was evaluated in three formulations: liquid culture, freeze-dried powder (FDO-VN25), and alginate-immobilized beads. Validation using real car-washing wastewater (TPH ≤ 100 mg/L) revealed that while all the formulations improved degradation, the alginate-immobilized system consistently exhibited the highest stability and efficacy, achieving over 60% TPH removal after 14 days. These results demonstrate that integrating a native multi-species consortium with optimized lyophilization and alginate encapsulation provides a robust microbial solution for treating low-strength petroleum-contaminated effluents in complex environments.