Valorisation of perennial rye grass pressed cake to Γ – polyglutamic acid

Poly-γ-glutamic acid (γ-PGA) is a valuable biopolymer whose production costs limit widespread application. Utilising lignocellulosic biomasses such as rye grass pressed cake (RGPC) as alternative feedstock offers economic and sustainability advantages but requires addressing key technical challenges including identifying microbial strains capable of fermenting lignocellulosic hydrolysates, reducing biomass recalcitrance through effective pretreatment, and optimising fermentation conditions for high γ-PGA yields. This thesis develops an integrated approach to γ-PGA production from RGPC by systematically addressing each of these challenges. Seven Bacillus strains from B. licheniformis and B. subtilis species were screened for γ-PGA production capacity. B. licheniformis strains demonstrated superior performance, with B. licheniformis DPC6338 achieving the highest titre (59.5 g/L) and was selected as the candidate strain. Genomic analysis confirmed the presence of γ-PGA metabolic pathways, including genes for precursor biosynthesis, polymerization, racemization, and degradation in B. licheniformis DPC6338, while in-silico safety assessment validated its suitability for industrial application. Systematic optimization of nutritional and culture parameters including carbon and nitrogen sources, mineral composition, temperature, initial pH and inoculum concentration, improved γ-PGA titre and peak productivity in shake flask fermentation to 75.35 ± 0.38 g/L and 1.3 g/L/h, representing a 27 % and 4 % improvement respectively over screening conditions. Scale-up to bioreactor conditions further enhanced final titre and early-phase volumetric productivity by approximately 30 % and 80 %, respectively. Natural deep eutectic solvents (NADES) are eutectic mixtures of naturally derived compounds that associate through hydrogen bonding. NADES are finding increasing applicability in biomass pretreatment due to the ease of preparation, low-cost, greenness, sustainability, chemical and thermal stability, high solubilisation power, high stabilizing ability and tunability. In this study, NADES prepared from different molar ratios of citric acid (CA) and glycerol (Gly) were characterised, examined as a medium for the pretreatment of RGPC and optimized using Taguchi orthogonal array design. Optimal conditions were identified through signal-to-noise ratio analysis and ANOVA. NADES pretreatment achieved 72 - 84 % hemicellulose solubilization, 59 - 82 % lignin removal, and 50 - 71 % cellulose recovery, with structural characterization confirming significant biomass modification. The optimized pretreatment condition (citric acid:glycerol 1:2, 2 hours) yielded 95 % enzymatic saccharification efficiency when validated experimentally. Fermentation studies using RGPC hydrolysates demonstrated the practical application of this integrated approach. Batch fermentation of combined NADES and water extract produced 10.2 g/L γ-PGA at 72 h, which improved to 14.77 g/L under fed-batch conditions. Enzymatic hydrolysate alone yielded a maximum titre of 25.88 g/L after 72 h. Collectively, these fractions represent a total γ-PGA production of 40.65 g/L from the original RGPC biomass, demonstrating the technical feasibility of converting agricultural residues to high-value biopolymers. This work establishes B. licheniformis DPC6338 as a robust platform for γ-PGA production and demonstrates that RGPC can support commercially relevant γ-PGA titres in an integrated fermentation process. These findings provide a foundation for scaling γ-PGA lignocellulosic production from renewable feedstocks, contributing to the development of circular bioeconomy approaches in biopolymer manufacturing.

Funder

Department of Agriculture, Food and the Marine

Publisher

University of Galway

Rights

CC BY-NC-ND

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Collections

University of Galway Theses (PhD Theses)