Production of Lactic Acid from Lignocellulosic Hydrolysates by Thermotolerant Bacteria

Abstract

Lactic acid (LA), is an industrially important organic acid with extensive applications in pharmaceuticals and food industries as well as biodegradable plastics. It has garnered significant attention for sustainable production from renewable lignocellulosic biomass resources. This study was aimed at lactic acid production by isolating thermotolerant and inhibitor-tolerant bacterial strains, optimizing lignocellulosic biomass utilization and employing co-cultivation strategies for enhancing lactic acid yields. Among 45 bacterial isolates obtained from compost, soil, and fermented food two strains Bacillus licheniformis DGB and Bacillus sonorensis DGS15 were identified as high lactic acid producers which were thermotolerant and inhibitor-tolerant. They demonstrated robust growth in Bushnell Haas medium and efficiently utilized glucose and xylose at elevated temperatures (45°C–50°C), while tolerating inhibitory compounds (furfural and hydroxymethyl furfural) commonly derived from lignocellulosic biomass pretreatment. Initial fermentation trials yielded 13.2 g/L and 12.8 g/L of lactic acid for DGB and DGS15, respectively. Morphological, biochemical and molecular analyses confirmed their identity, and their thermotolerance and inhibitor resistance ensured suitability for industrial applications. Furthermore, both strains exhibited efficient carbon catabolite repression (CCR) bypass features, enabling simultaneous glucose and xylose metabolism. Rice straw and wheat straw were selected as lignocellulosic feedstocks due to their abundance and high cellulose content. Pretreatment processes involving acid hydrolysis and enzymatic saccharification were optimized using Response Surface Methodology (RSM) to maximize sugar release. Pretreated rice straw yielded 50.8 g/L of total reducing sugars (TRS) from 100 g of biomass, while wheat straw yielded 48.6 g/L. Structural analysis using FTIR confirmed effective delignification, with the disappearance of lignin peaks at 1670 cm⁻¹, and SEM analysis revealed significant disruption of biomass structure. The crystallinity index (CrI) of pretreated rice straw increased by 15.5% compared to untreated material, highlighting enhanced accessibility for enzymatic digestion. In fermentation trials, DGB and DGS15 achieved lactic acid yields of 46.5 g/L and 44.7 g/L from rice straw and wheat straw hydrolysates, with yield efficiencies of 96.8% and 94.1%, respectively. Among the two biomass ix sources, rice straw emerged as the most suitable substrate due to its higher sugar release and conversion efficiency. To enhance productivity, a co-cultivation strategy was employed, leveraging the complementary metabolic pathways of DGB and DGS15. The co-culture system effectively utilized glucose and xylose, achieving 70% sugar consumption within 15 hours and complete substrate utilization within 48 hours. Optimized fermentation conditions (10% (w/v) mixed hydrolysate substrate (rice straw and wheat straw), 1:1 inoculum ratio, 50°C, pH 6.0) resulted in a maximum concentration of 64.3 g/L lactic acid, with a yield of 0.98 g/g and productivity of 1.036 g/L/h. Compared to monoculture fermentation, co-cultivation increased yields by 28% and reduced fermentation time by 12%. The Separate Hydrolysis and Co-Fermentation (SHCF) process addressed key challenges in lignocellulosic biorefining, demonstrating scalability and process efficiency. Co-cultivation proved to be the most suitable approach for lactic acid production, offering higher yields and better process efficiency compared to monoculture systems. Present study successfully demonstrated a comprehensive sustainable approach to lactic acid production by integrating thermotolerant and inhibitor-tolerant microbial strains, optimized lignocellulosic biomass utilization, and co-cultivation strategies. Among the strains, Bacillus licheniformis DGB was identified as the best performer due to its higher lactic acid yield and adaptability. Rice straw was identified as the most suitable biomass source for lactic acid production and co-cultivation emerged as the most efficient production strategy, offering enhanced yields, reduced fermentation times and robust process stability. Future studies could focus on refining biomass pretreatment, exploring genetic engineering of strains and incorporating integrated biorefinery models to enhance sustainability and economic feasibility.