High-throughput screening and optimization of fungal isolates for enhanced lignin biodegradation.

Lignin is a highly recalcitrant aromatic polymer and a major contributor to industrial wastewater pollution, particularly from chemical pulping processes, posing persistent challenges to environmental sustainability. Fungal bioremediation offers an eco-friendly alternative by exploiting extracellular ligninolytic enzymes capable of lignin depolymerization. In this study, a rapid high-throughput microtiter-plate assay quantifying chromophoric lignin reduction (absorbance decrease at 465 nm in relation to time) was employed to screen fungal isolates from diverse ecological niches. A total of 102 fungal isolates were evaluated, among which two top performing isolates, designated AP1 and AP2, were selected for further investigation. Under optimized conditions (0.75% fructose, pH 5, 25 °C, 120 rpm), lignin degradation efficiency increased to 73.2% for AP1 and 86.6% for AP2 within 24 h, as indicated by optical lignin reduction. Enzymatic profiling demonstrated manganese peroxidase (MnP) as the dominant ligninolytic enzyme, followed by lignin peroxidase (LiP) and laccase. ITS-based phylogenetic analysis indicated that the isolates (AP1 and AP2) are affiliated with ligninolytically competent Penicillium lineages and were recovered as sister lineage to Penicillium wotroi with 83% bootstrap support. Overall, this study demonstrates the effectiveness of a rapid high-throughput screening and optimization framework for identifying fungi with strong ligninolytic potential based on degradation proxy assays. Moreover, it highlights the first report of Penicillium wotroi AP1 and Penicillium wotroi AP2 as promising fungal biocatalysts (MnP dominated) for lignin bioconversion (within 24 h) and other environmental applications.