Abstract
Microbial consortia are essential for the degradation of complex hydrocarbon mixtures in coastal environments, yet the functional basis of consortium performance rarely resolved at strain level. In this study, we employed a Plackett-Burman experimental design to rationally assemble and optimize a synthetic consortium for the degradation of medium-grade crude oil in a sand-seawater microcosm. Six strains were selected: Stutzerimonas stutzeri CBC1 and CBC2, S. balearica GOM11, Pseudomonas sp. CBC3, Halomonas litopenaei GOM23 and Enterobacter kobei GOM24. After 45 days, the consortium removed 15.72% of total hydrocarbons relative to the abiotic control, with markedly higher fraction-specific removal determined by GC-MS: 94.7% of phenanthrene, 63.1% and 41.0% of the C8-C27 and C18-C27 aliphatic fractions respectively. Comparative genomic analysis revealed a non-uniform distribution of catabolic functions consistent with metabolic complementary. Stutzerimonas and Pseudomonas strains carried ring-hydroxylating dioxygenases (nah/nag-like clusters) together alkane-degrading alcohol/aldehyde dehydrogenases (alkJ, alkH), whereas H. litopenaei GOM23 and E. kobei GOM24 encoded alternative long-chain alkane-hydroxylating systems (ladA). GOM24 further carried an expanded repertoire of ABC transporters and oxidative-stress genes (ahpC/ahpF), consistent with a role in substrate uptake and stress tolerance rather than in primary ring cleavage. Enzymatic assays supported this partitioning, catechol 1,2-dioxygenase activity was confined to the same four strain that encoded the gene and was highest in CBC1 and GOM11 (0.0472 and 0.0443 U·mg⁻1 protein, respectively). No genome carried the classical alkB alkane-monooxygenase, pointing to alternative and partially redundant alkane-oxidation routes. By combining statistically guided assembly with comparative genomics and targeted enzyme assays, this work identifies a six-strain combination that removes more crude oil than the full eleven-isolate community and resolves how its catabolic functions are partitioned among strains.