Spatial Assembly of Functional Microbiomes via 3D Bioprinting for Modular Remediation of Mixed Organic Pollutants

Although bioremediation provides sustainable strategies for environmental cleanup, achieving effective microbial removal of cytotoxic organic contaminants, such as sulfonamide antibiotics and polycyclic aromatic hydrocarbons (PAHs), remains challenging. Three-dimensional (3D) bioprinting has emerged as a novel immobilization approach with the potential to engineer functional synthetic microbiomes for improved remediation. In this study, we developed a 3D-bioprinting platform using an optimized alginate-gelatin-cellulose (AGC) bio-ink and newly designed synthetic microbial consortia to fabricate biomaterials with defined microstructures for the biodegradation of sulfamethoxazole (SMX) and PAHs. A double-layered biomaterial, 3D-printed construct incorporating the synthetic microbiome SynBiom1 (comprising strains N39 and SD-1), which sequentially degrade SMX and its metabolite 3-amino-5-methylisoxazole (3A5MI), significantly enhanced bacterial stress tolerance and maintained high degradation activity. The platform was further extended to fabricate strain PD-1, which efficiently degraded multiple PAHs under heavy metal stress. Finally, an integrated synthetic microbiome (SynBiom2) was assembled through a modular combination of 3D-printed SynBiom1 and PD-1, enabling simultaneous removal of SMX and PAHs in co-contaminated scenarios. This work highlights the potential of 3D bioprinting as a versatile tool for constructing robust microbial systems for the remediation of complex pollutant mixtures. • A customizable 3D-bioprinting platform with optimized AGC bio-ink was developed. • 3D-printed biomaterials significantly enhanced stress tolerance and degradation efficiency. • The 3D-printed SynBiom1 achieved sequential degradation of SMX and 3A5MI. • The spatial assembly of degradation modules enables the simultaneous removal of pollutant mixtures.