Vol.:(0123456789) Folia Microbiologica https://doi.org/10.1007/s12223-024-01238-0 ORIGINAL ARTICLE Mixed‑species Pseudomonas bioﬁlms: a novel and sustainable strategy for malachite green dye decolorization and detoxiﬁcation Nabanita Ghosh 1 · Arup Ratan Biswas 2 · Arindam Chakraborty 1 · Arnab Ganguli 1 Received: 29 May 2024 / Accepted: 21 December 2024 © Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2025 Abstract This study investigated the application of mixed bioﬁlms formed by two Pseudomonas strains (NAA22 and NAA23) for bio-decolorization of malachite green (MG) dye. The isolated strains displayed bioﬁlm formation and MG decolorization capabilities. Mixed bioﬁlms exhibited signiﬁcantly greater bioﬁlm formation and MG decolorization (94.3%) compared to individual strains, suggesting synergistic interactions. This decolorization eﬃciency surpassed previously reported values for single strain decolorization. The mixed bioﬁlms tolerated a broad range of temperatures (20–40 °C) and pH (5–9), with optimal decolorization at neutral or slightly acidic conditions (pH7.0). Enzyme analysis revealed laccase, NADH-DCIP reductase, and azoreductase as key contributors to MG decolorization, with signiﬁcantly higher activity in mixed bioﬁlms. Importantly, the bio-decolorization process transformed MG into non-phytotoxic compounds, demonstrated by seed germi- nation and growth assays. These ﬁndings propose a promising and environmentally safe approach for MG bioremediation using mixed Pseudomonas bioﬁlms. Keywords Malachite green · Bioﬁlms · Decolorization · Detoxiﬁcation · Pseudomonas spp Introduction Environmental pollution from synthetic dyes is a global issue due to their widespread use and persistence in ecosystems (Tkaczyk et al. 2020). Their complex aromatic structures resist natural degradation, and dye-laden wastewater disrupts aquatic ecosystems by blocking sunlight and altering water chemistry, harming aquatic life (Sharma et al. 2021). Addi- tionally, synthetic dyes and their byproducts pose serious health risks due to their toxicity, carcinogenicity, and muta- genicity (Gürses et al. 2016; Manzoor and Sharma 2020). Traditional dye removal methods, such as adsorption and chemical oxidation, are limited by high costs, secondary pol- lutants, and incomplete removal (Lee et al. 2006; Issa Ham- oud et al. 2017). This has prompted interest in eco-friendly solutions like microbial bio-decolorization, which uses bac- teria to degrade dyes eﬀectively and sustainably (Manjarrez Paba et al. 2021). While research has often focused on planktonic bacte- ria, these cultures face challenges such as limited metabolic capacity and sensitivity to environmental stresses. In con- trast, bioﬁlm-forming bacteria present a more robust alterna- tive (Haque et al. 2022). Bioﬁlms, structured microbial com- munities encased in an extracellular polymeric substance (EPS) matrix, enhance dye degradation through increased enzyme production, resource-sharing, and protection from harsh conditions (Kravvas et al. 2018). Bioﬁlms are also eas- ily immobilized on surfaces, improving treatment eﬃciency and system stability (Sfaelou et al. 2015). These advantages highlight the potential of bioﬁlm-based approaches for eﬀec- tive dye remediation. The genus Pseudomonas comprises metabolically versa- tile bacteria known for their ability to produce a wide array of enzymes (Kuddus et al. 2013) and their propensity for bioﬁlm formation (Vetrivel et al. 2021), making them attrac- tive candidates for bioremediation applications. The degra- dation of MG by Pseudomonas species has been extensively studied. Several Pseudomonas strains, such as Pseudomonas veronii (Song et al. 2020), Pseudomonas sp. DY1 (Du et al. * Arnab Ganguli ganguli.arnab009@gmail.com 1 Department of Microbiology, Techno India University, West Bengal EM-4 Sector-V, Salt Lake City, Kolkata, West Bengal 700091, India 2 Department of Chemistry, Techno India University, West Bengal EM-4 Sector-V, Salt Lake City, Kolkata, West Bengal 700091, India