Abstract
Bacillus species are widely recognised for their metabolic versatility, particularly their ability to utilise diverse carbohydrate sources and produce a broad spectrum of bioactive metabolites. These attributes make them promising candidates for applications in biotechnology, fermentation, and probiotic development. This study investigated the genomic and metabolic potential of Bacillus paranthracis strain MHSD3, with a specific focus on its carbohydrate- active enzymes (CAZymes), carbohydrate utilisation capacity, and the metabolite profiles during fermentation on carbohydrate different substrates. The overarching objective was to integrate genome-based data with experimental metabolomics to provide a holistic understanding of the strain’s functional capabilities. Genomic analysis was performed using the dbCAN2 meta-server to identify CAZymes encoded by B. paranthracis MHSD3. The workflow incorporated HMMER, DIAMOND, and dbCAN_sub tools to ensure high confidence
functional annotations. Results revealed a diverse CAZyme repertoire distributed across glycoside hydrolases, glycosyltransferases, auxiliary enzymes, carbohydrate esterases, polysaccharide lyases, and carbohydrate-binding modules. These findings suggest that MHSD3 possesses the genetic capacity to degrade, modify, and synthesise a wide range of carbohydrate structures, supporting its adaptability in carbohydrate-rich environments. To experimentally validate functional predictions, the strain was fermented on selected α-linked (starch and glycogen) and β-linked (cellulose and chitin) substrates, followed by metabolite profiling using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). The molecular network generated through the Global Natural Products Social Molecular Networking (GNPS) platform revealed distinct clustering patterns corresponding to the different carbohydrate substrates. Fermentation of α-linked carbohydrates resulted in larger and chemically diverse metabolite pools, while β-linked substrates produced smaller but chemically distinct profiles. Principal Component Analysis (PCA) and heatmap analysis further confirmed substrate-specific metabolic signatures, reinforcing the relationship between carbohydrate structure and metabolite complexity. Chemical classification of detected metabolites showed a wide distribution across aromatic compounds, esters, fatty acyls, organic acids, heterocyclic compounds, alcohols, amino-acid derivatives, and nitrogen-containing metabolites. Several metabolites identified are known for biological relevance, including anti-inflammatory, antioxidant, antimicrobial, and quorum-modulating properties, highlighting the potential of strain MHSD3 to generate functionally beneficial biomolecules. Generally, the study provides an integrated genomic-metabolomic perspective on B. paranthracis strain
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MHSD3, demonstrating that it does not only encodes a functionally diverse CAZyme repertoire but also translates this capability into substrate-dependent metabolic outputs. These findings support the potential application of MHSD3 as a candidate for novel probiotic development, carbohydrate-rich fermentation processes, and biotechnological utilisation. The study further contributes to the broader understanding of how Bacillus species metabolise structurally distinct carbohydrates to produce metabolites relevant to health, industry, and microbial ecology.