Hydrolase enzyme production by filamentous fungi through solid-state fermentation of spent coffee grounds

Hydrolase enzyme production by filamentous fungi through solid-state fermentation of spent coffee grounds Georgi Pramatarov 1,2*, Denis Borisov 1,2, Vanina Lozanova 1, Orlin Gemishev 1, Dilyana Nikolova 1,2, Yana Evstatieva 1,2 1Department of Biotechnology, Faculty of Biology, Sofia University “St. Kliment Ohridski”, 8 Dragan Tsankov Blvd., 1164 Sofia, Bulgaria 2Centre of Competence “Sustainable Utilization of Bio-Resources and Waste of Medicinal and Aromatic Plants for Innovative Bioactive Products” (BIORESOURCES BG), 1000 Sofia, Bulgaria *e-mail: g[email protected] This work was supported by SU-project 80-10-79 / 27.05.2025. The support of the Centre of Competence “Sustainable Utilization of Bioresources and Waste of Medicinal and Aromatic Plants for Innovative Bioactive Products” (BIORESOURCES BG), project BG16RFPR002-1.0140001, funded by the Program “Research, Innovation and Digitization for Smart Transformation” 2021-2027, co-funded by the EU, is greatly acknowledged. Acknowledgements Both Aspergillus awamori K1 and Trichoderma reesei M7 showed good growth, dense mycelium formation, and sporulation on spent coffee grounds (SCG), confirming the substrate’s suitability for fungal cultivation. Moisture levels strongly influenced enzyme activity: intermediate moisture (87%) enhanced endoxylanase activity, while lower levels (65%) favored CMCase activity. Zeolite supplementation (10%) significantly increased CMCase activity in T. reesei M7 during solid-state fermentation, highlighting its potential as an enzyme production enhancer. Extraction solvent (water vs. buffer) and extraction time (30 vs. 60 min) had no significant effect on enzyme activities in either strain. SDS-PAGE analysis confirmed the presence of endoglucanase in T. reesei M7 and the absence of endo-xylanase in A. awamori K1. Conclusions Introduction Methodology Results 0 1 2 3 4 5 6 90 110 130 150 170 Endoxylanase activity, IU/gds Time (h) Influence of initial moisture concentration on endoxylanaseactivity by A. awamori K1 65% 76% 87% 98% 0 2 4 6 8 10 12 90 110 130 150 170 CMCase activity, IU/gds Time (h) Influence of zeolite supplementation on CMCase activity by T. reesei M7 0% 10% 20% 30% 0 2 4 6 8 10 12 30 minutes 60 minutes CMCase activity, IU/gds Influence of extraction time and extragent on CMCase activity by T. reesei M7 Water Buffer 0 2 4 6 8 10 12 30 minutes 60 minutes Endoxylanase activity, IU/gds Influence of extraction time and extragent on endoxylanaseactivity by A. awamori K1 Water Buffer * Aspergillus awamori K1: no clear band at ~32 kDa (endo-xylanase). Distinct protein bands observed at ~87, 66, and 53 kDa → unidentified proteins of future interest. Trichoderma reesei M7: distinct band at ~57 kDa (endoglucanase). Additional bands at ~68 and 32 kDa require further identification. Aspergillus awamori K1: vigorous growth with dense mycelium, dark brown pigmentation (sporulation), and deep substrate penetration. Trichoderma reesei M7: compact colonies with greenish hue, abundant sporulation, uniform mycelial coverage, strong colonization of SCG. Maximum activity at 65% moisture (~4.5 IU/gds). Decreased gradually at higher moisture (87% → ~4.0; 76% → ~3.8; 98% lowest ~3.6). Confirms 65% as optimal for cellulase production under SSF Maximum activity at 87% moisture (~4.9 U/gds). Moderate at 98% (~4.0).Much lower at 76% (~2.6) and 65% (~2.3). Highlights the role of optimal water activity for xylanase synthesis A. awamori K1: endoxylanase activity too low → no conclusive data on zeolite effect. T. reesei M7: CMCase activity strongly enhanced by zeolite. Peak activity at 10–20% zeolite supplementation. Activity declined at higher concentrations. Similar activities with water (10.67 IU/gds) and buffer (10.63 IU/gds) after 30 min. Slight increase after 60 min (11.63 IU/gds with water, 11.71 with buffer). Conclusion: No significant difference between solvents. Extraction plateau reached within 30 min → enzyme is stable and easily recoverable. At 30 min: 2.23 IU/gds (water) vs 2.58 (buffer). At 60 min: 2.76 (water) vs 2.88 (buffer). Conclusion: Activities remained low under all conditions, likely due to SCG’s complex polysaccharide matrix and inhibitory phenolic compounds. Coffee as a global commodity: Global coffee consumption exceeded 10.37 billion kg in 2023, and its popularity continues to grow due to both cultural significance and reported health benefits, including reduced risks of cardiovascular diseases and certain cancers. Spent coffee grounds (SCG): Coffee preparation generates large amounts of residues known as spent coffee grounds (SCG), which remain rich in carbohydrates, fats, antioxidants, and other bioactive compounds. SCG composition varies with coffee type, cultivation, and brewing method. Biotechnological potential: SCG is composed predominantly of polysaccharides such as cellulose and hemicellulose, with up to 45.3% carbohydrate content. This makes SCG a promising feedstock for solid-state fermentation (SSF) aimed at producing valuable bioactive compounds, including enzymes and peptides. Role of filamentous fungi: Filamentous fungi such as Trichoderma spp. and Aspergillus spp. are well known for producing cellulases, xylanases, and other industrially relevant enzymes used in biofuels, paper, and food industries. Study aim: This work focuses on optimizing SSF of Trichoderma reesei M7 and Aspergillus awamori K1 on SCG for the production of carboxymethyl cellulase (CMCase) and xylanase respectively, evaluating key parameters including initial moisture content, zeolite supplementation, extraction method and time. Substrate preparation Solid-state fermentation Morphology observation, Enzymes activities determination, SDS-PAGE analysis Autor Information: 0 1 2 3 4 5 90 110 130 150 170 CMCase activity, IU/gds Time (h) Influence of initial moisture concentration on CMCase activity by T. reesei M7 65% 76% 87% 98%