Uncovering the potential of Bacillus licheniformis in the utilisation of lignin-derived aromatics: Insights from transcriptomic analysis

Lignin is the second major component of lignocellulosic biomass (LCB) and the largest renewable source of aromatics, being recalcitrant due to its complex structure. In nature, gram-positive Bacillus species, contribute to the utilisation of low molecular weight lignin-derived aromatics (LMW - LDAs) and produce bioactive enzymes of biotechnological relevance. However, the ability of Bacillus licheniformis to metabolise LDAs from different technical lignins remains poorly characterized. Here, B. licheniformis was cultivated in batch cultures on glucose-based M9 medium supplemented with hydrolysis (HL), kraft (KL), and lignosulfonate (LS) as co-substrates. Metabolite profiling by gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), combined with transcriptomic analysis, was used to identify modified LDAs and the enzymes involved in their utilisation. B. licheniformis exhibited robust growth on LDAs derived from HL, KL, and LS. Notably, KL-grown cultures maintained active growth, whereas HL- and LS-grown cultures entered a decline phase after reaching stationary phase. Metabolite profiling revealed an active upper aromatic catabolic pathway, with efficient conversion of aromatic aldehydes and acids to protocatechuic acid (PCA), which accumulated under all lignin conditions. KL-grown cultures exhibited a distinct growth profile, including acetate re-assimilation, odd-chain fatty acids production, and resin acid removal. Transcriptomic analysis showed the greatest diversity of catabolic gene expression during growth on HL, followed by KL and LS, with differential expression of lignin-specific accessory genes, including peroxiredoxins, alongside core oxidoreductases and dehydrogenases. Overall, these results indicate that B. licheniformis modifies LDAs under all lignin conditions, and channels side-chain-derived carbon into central carbon metabolism under KL conditions.