Synthetic microalgal-bacterial symbiotic system integrating oxidase-protease fusion enzyme for simultaneous removal of recalcitrant nitrogen-containing organic compounds and PET microplastics.
Abstract
Industrial wastewaters-particularly those from the printing and dyeing sector-contain complex mixtures of recalcitrant nitrogen-containing organic compounds and microplastics that resist conventional treatment. Here we report a previously undescribed oxidase-protease fusion enzyme (A20674), discovered through transcriptomic analysis of wastewater-acclimated Chlorella vulgaris. Domain dissection reveals that the oxidase-like region drives broad-spectrum removal of N-heterocyclic and aromatic compounds, while the protease-like domain removes organic nitrogen. Engineered overexpression of this enzyme boosted organic nitrogen removal up to fifty-five-fold (final concentration 13-100 mg/L) across different industrial wastewaters. Capitalizing on this metabolic specialization, we constructed a synthetic microalgal-bacterial consortium in which Pseudomonas putida uses microalgal extracellular polysaccharides (EPS) as a carbon source to sustain growth, while supplying indole-3-acetic acid that stimulates microalgal biomass and EPS production. Reciprocal engineering of EPS overproduction in C. vulgaris and enhanced polysaccharide-catabolism in P. putida amplified this mutualistic loop. An evolved, PETase/MHETase-expressing P. putida strain simultaneously acquired elevated IAA output, further strengthening the symbiosis. The optimized consortium reduced organic nitrogen concentrations by sixteen-fold to discharge-compliant levels (≤5 mg/L), enhanced removal of recalcitrant organic nitrogen compounds by five-fold (final concentration 34 mg/L), and improved PET microplastic removal by nine-fold (initial concentration 1 g/L and final concentration 684 mg/L) in printing and dyeing wastewater. These findings uncover a bifunctional enzyme architecture for degrading structurally diverse industrial pollutants, and establish a synthetic-ecology framework for integrated removal of dissolved nitrogen-containing organic compounds and particulate microplastics-a combination unattainable by any single organism or conventional treatment process.