Assessment of Rhizopus stolonifer tolerance to methotrexate: morphological responses and biodegradation limitations

This study evaluates the tolerance of Rhizopus stolonifer to methotrexate (MTX) and its capacity to remove and transform this antifolate compound, highlighting both morphological responses and biodegradation limitations. The xenobiotic affected fungal differentiation more strongly than vegetative growth: although radial expansion remained unchanged up to 500 ppm, sporulation, hyphal integrity, and cellular organization were progressively impaired. Spore germination decreased sharply at low concentrations, yielding an average half maximal inhibitory concentration (IC₅₀) of 138 ppm. In pellet systems exposed to 30 ppm of the antifolate compound for 9 days, both active and heat-inactivated biomass achieved similar final removal rates (approximately 15% on average), demonstrating that biosorption is the dominant elimination mechanism. However, only active pellets produced a new chromatographic signal at 2.127 min, confirming for the first time that R. stolonifer can partially biotransform MTX, although biotransformation remained limited. Scanning Electron Microscopy analysis revealed severe MTX-induced damage, including hyphal collapse, surface roughening, and granular deposits consistent with persistent cell wall interactions. Overall, R. stolonifer exhibits clear tolerance to MTX but only limited biodegradation capacity, indicating that optimized environmental or physiological conditions will be necessary to enhance degradation efficiency.