Sequential Production of Levan and Xanthan Gum via Integrated Fermentation for Enhanced Carbon Utilization

Microbial exopolysaccharides (EPS) have gained significant attention in recent years due to their diverse applications in food and healthcare sectors. These polymers can exist as either homoor heteropolysaccharides depending on their monomeric sugar composition. Among these, levan, a fructose-based homopolymer, holds considerable potential in pharmaceutical and food applications. Its biosynthesis by Bacillus species using sucrose as the sole carbon source has been extensively studied. Similarly, xanthan gum, a heteropolysaccharide produced by Xanthomonas campestris, consists of a β-(1→4)-D-glucose backbone with trisaccharide side chains comprising mannose and glucuronic acid. Xanthan is widely utilized in the food industry as a stabilizer and rheology modifier. Bacillus spp. produces levan using a two-step enzymatic process. In the initial step, sucrose is hydrolysed into glucose and fructose. In the second step levan is formed by the polymerization of fructose units. This process leaves a glucose-rich spent medium, which is typically underutilized. The present study proposes a sequential fermentation strategy to valorize the residual glucose and improve overall carbon efficiency. In the first stage, fermentation of 100 g/L of pure sucrose was conducted using B. subtilis MTCC 441. Levan was precipitated from the culture broth using isopropyl alcohol (IPA), resulting in a yield of 0.39 g levan/g sucrose. Post-precipitation, the IPA was recovered and the clarified supernatant was concentrated and subjected to sugar profiling via HPLC. The analysis revealed 92.16 g/L of glucose and 10.43 g/L of residual sucrose, indicating significant glucose availability for further processing. To utilize, this residual glucose, the recovered broth was diluted and supplemented with essential micronutrients to formulate a medium containing 20 g/L of glucose for xanthan production. The second-stage fermentation was carried out using X. campestris NCIM 2956, which yielded 2.17 g/L of xanthan gum. The structural integrity and identity of the produced EPSs—levan and xanthan—were confirmed using NMR and FT-IR spectroscopy. This study presents a cost-effective and sustainable bioprocessing strategy that integrates levan and xanthan production via sequential fermentation, thereby enhancing carbon conversion efficiency and reducing downstream waste.