Enzymatic One-Pot Hydrolysis of Extracted Sugar Beet Press Pulp after Solid-State Fermentation with an Engineered Aspergillus niger Strain

Extracted sugar beet press pulp (SBPP) is a promising agricultural residue for saccharification and further bioconversion. Combining solid-state fermentation of SBPP with engineered Aspergillus niger for enzyme production followed by hydrolysis of additionally added SBPP in the same bioreactor was studied to produce a sugar solution (hydrolysate) in a one-pot process. The initial aerobic solid-state fermentations were carried out in duplicate on non-milled, wet SBPP (moisture content of 72% (w/v)) with an A. niger strain engineered for constitutive pectinase production for 96 h, and this resulted in polygalacturonase activities of up to 256 U mL⁻¹ in the wet media. Afterwards, water was added to the bioreactor, and the remaining solids were suspended by stirring to dissolve the hydrolytic enzymes. Metabolic activities of A. niger were inactivated by a N₂-atmosphere and by increasing the temperature to 50 °C. High solid loads of milled SBPP were added to the stirred-tank reactor with a delay of 24 h to enable sugar yield calculations based on the compositional analysis of the SBPP used. The resulting final sugar concentrations of the hydrolysate after 166 h were 17 g L⁻¹ d-glucose, 18.8 g L⁻¹ l-arabinose, and 12.5 g L⁻¹ d-galacturonic acid, corresponding to sugar yields of 98% d-glucose, 86% l-arabinose, and 50% d-galacturonic acid, respectively. Including the other sugars released during enzymatic hydrolysis in the one-pot process (d-xylose, d-mannose, d-galactose), a total sugar concentration of 54.8 g L⁻¹ was achieved in the hydrolysate. The one-pot process combining hydrolytic enzyme production in solid-state fermentation with high solid loads during enzymatic hydrolysis of the milled SBPP reduces hydrolytic process costs by replacing chemical pre-treatments, enabling the in situ production of SBPP-adapted hydrolytic enzymes, as well as avoiding intermediate enzyme extraction and preparation steps.