Presentation Information
[P02-300]Conversion of Kraft Pulp (KP) into a glucose-rich medium for yeast Lactic acid fermentation
○Arthur Oliveira1, Chiaki Ogino1, Prihardi Kahar1, Filemon Jalu Nusantara Putra1 (1. Kobe University (Japan))
Keywords:
Biomass,Kraft pulp,Flocculant yeast,D-lactic acid,Hydrolysis
[Purpose]
To investigate the relationship between the chemical composition of different kraft pulps and their enzymatic digestibility, and to develop an integrated process for lactic acid production from pulp-derived hydrolysates.
[Method]
Four Kraft pulps (ozone-bleached and chlorine-bleached, from both softwood and hardwood) were characterized through compositional analysis following the NREL method. Enzymatic hydrolysis was then performed using Acremonium cellulase at 50 °C for 72 hours in citrate buffer (pH 4.5), with solid loadings of 15–25% (w/v) and an enzyme dosage of 6 FPU/g cellulose. The resulting glucose-rich hydrolysates were subsequently used as fermentation media. An engineered S. cerevisiae strain (F118), containing the D-LDH transgene in the CYB2 locus to enable D-lactic acid production, was employed. Different media containing only 50 g/L of glucose or with the addition of yeast extract (10 g/L) and peptone (20 g/L) were prepared from the hydrolysates and used for lactic acid production. A single colony of the yeast strain was inoculated into 12 mL of medium and incubated at 30 °C with shaking at 90 rpm for 24 hours. Key fermentation-related compounds, including glucose, D-lactic acid, glycerol, acetic acid, and ethanol, were analyzed by high-performance liquid chromatography (HPLC). All experiments were conducted in triplicate.
[Results]
At a solids loading of 20% (w/v), all kraft pulps produced the highest glucose concentrations, with softwood reaching 44.52 g/L (ozone-bleached) and 57.6 g/L (chlorine-bleached), and hardwood reaching 38.1 g/L (ozone-bleached) and 55.4 g/L (chlorine-bleached). At 25% solids loading, the pulp absorbed all the buffer, leaving limited liquid availability, which decreased the uniform distribution of the enzyme and enzyme–substrate contact, resulting in slower pulp liquefaction. D-lactic acid production achieved above 20 g/L in all media containing yeast extract and peptone, with the highest at 22.82 g/L in YPD medium. In all media containing only hydrolysate, nutrient limitations prevented the strain from consuming all the glucose, limiting D-lactic acid production to 7 g/L.
[Consideration]
Variations in kraft pulp composition influenced enzymatic digestibility, particularly in relation to cellulose accessibility and residual lignin content. While higher solid loadings increased glucose concentrations, they also introduced mass transfer and mixing limitations. Fermentation results further indicated that, although hydrolysates can serve as substrates, nutrient limitations significantly reduced glucose utilization and D-lactic acid production, highlighting the need for supplementation or process optimization to improve overall yields.
[Conclusion]
Chlorine-bleached kraft pulps derived from both softwood and hardwood resulted in elevated glucose titers, measuring 57.6 and 55.4 g/L, respectively. The engineered strain F118 demonstrated robust growth across all media containing yeast extract and peptone, attaining a superior lactic acid titer of 22.82 g/L, a productivity of 0.95 g/L/h, and a yield of 0.4564 g of lactic acid per gram of glucose in YPD medium.
To investigate the relationship between the chemical composition of different kraft pulps and their enzymatic digestibility, and to develop an integrated process for lactic acid production from pulp-derived hydrolysates.
[Method]
Four Kraft pulps (ozone-bleached and chlorine-bleached, from both softwood and hardwood) were characterized through compositional analysis following the NREL method. Enzymatic hydrolysis was then performed using Acremonium cellulase at 50 °C for 72 hours in citrate buffer (pH 4.5), with solid loadings of 15–25% (w/v) and an enzyme dosage of 6 FPU/g cellulose. The resulting glucose-rich hydrolysates were subsequently used as fermentation media. An engineered S. cerevisiae strain (F118), containing the D-LDH transgene in the CYB2 locus to enable D-lactic acid production, was employed. Different media containing only 50 g/L of glucose or with the addition of yeast extract (10 g/L) and peptone (20 g/L) were prepared from the hydrolysates and used for lactic acid production. A single colony of the yeast strain was inoculated into 12 mL of medium and incubated at 30 °C with shaking at 90 rpm for 24 hours. Key fermentation-related compounds, including glucose, D-lactic acid, glycerol, acetic acid, and ethanol, were analyzed by high-performance liquid chromatography (HPLC). All experiments were conducted in triplicate.
[Results]
At a solids loading of 20% (w/v), all kraft pulps produced the highest glucose concentrations, with softwood reaching 44.52 g/L (ozone-bleached) and 57.6 g/L (chlorine-bleached), and hardwood reaching 38.1 g/L (ozone-bleached) and 55.4 g/L (chlorine-bleached). At 25% solids loading, the pulp absorbed all the buffer, leaving limited liquid availability, which decreased the uniform distribution of the enzyme and enzyme–substrate contact, resulting in slower pulp liquefaction. D-lactic acid production achieved above 20 g/L in all media containing yeast extract and peptone, with the highest at 22.82 g/L in YPD medium. In all media containing only hydrolysate, nutrient limitations prevented the strain from consuming all the glucose, limiting D-lactic acid production to 7 g/L.
[Consideration]
Variations in kraft pulp composition influenced enzymatic digestibility, particularly in relation to cellulose accessibility and residual lignin content. While higher solid loadings increased glucose concentrations, they also introduced mass transfer and mixing limitations. Fermentation results further indicated that, although hydrolysates can serve as substrates, nutrient limitations significantly reduced glucose utilization and D-lactic acid production, highlighting the need for supplementation or process optimization to improve overall yields.
[Conclusion]
Chlorine-bleached kraft pulps derived from both softwood and hardwood resulted in elevated glucose titers, measuring 57.6 and 55.4 g/L, respectively. The engineered strain F118 demonstrated robust growth across all media containing yeast extract and peptone, attaining a superior lactic acid titer of 22.82 g/L, a productivity of 0.95 g/L/h, and a yield of 0.4564 g of lactic acid per gram of glucose in YPD medium.
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