Metagenomic Insights into Lignocellulose-Degrading Microbiota in High-Temperature (75°C) Chicken Manure and Straw Compost via Nanopore Sequencing

The discovery of robust biocatalysts from extreme environments is essential for advancing sustainable second-generation biofuel production. This study investigates the lignocellulose-degrading potential of a thermophilic microbial consortium derived from industrial chicken manure and straw compost maintained at 75°C. Using Oxford Nanopore Technologies (ONT) long-read sequencing, we performed a de novo metagenome assembly, resulting in a 25.1 Mb functional metagenome containing 40,763 coding sequences. Taxonomic analysis revealed a heavy dominance of the superkingdom Bacteria (99.41%), primarily represented by the phyla Deinococcota (73.18%), Pseudomonadota (19.17%), and Bacillota (4.92%), indicating a specialized community shaped by intense thermal selective pressure. Metagenomic mining identified five candidate genes encoding lignocellulose-degrading enzymes, including two xylanases and two peroxidases with relatively low amino acid sequence homology (75–82%) to known functional sequences. These genes were successfully expressed heterologously in Bacillus subtilis RIK1285. Recombinant strains demonstrated significant functional enhancement; the volumetric xylanase activity increased more than three-fold compared to the host strain, while the recombinant peroxidases exhibited a 20-fold increase in lignin-decomposition rates at pH 7.2. These findings validate the efficacy of long-read metagenomic pipelines in uncovering novel, highly active biocatalysts. The discovered enzymes, characterized by their exceptional stability and activity under thermophilic and acidic-to-neutral conditions, hold substantial promise for the industrial valorization of recalcitrant agricultural biomass.