Design of Solid-State Fermentation Systems for Polymer Hydrolytic Extracellular Enzyme Production by Filamentous Fungi

Solid-state fermentation (SSF) is a bioconversion process that utilizes a solid substrate that does not dissolve in an aqueous medium. Microorganisms grow on the surface of the substrate and penetrate its solid matrix to extract essential nutrients for their development. SSF is characterized by minimal free water, with a substrate moisture content maintained above 70%, and involves three interconnected phases -- gaseous, liquid, and solid. This protocol describes the use of wheat bran, an agro-industrial byproduct, as the base substrate for enzyme production in a rotary system. The substrate is supplemented with an inducer, such as chitin, chitosan, starch, or cellulose, to promote the synthesis of hydrolytic proteins. The system is highly adaptable, allowing the use of different fungal forms, including mycelium, spores, or pellets. In the methodology described, the inducer and substrate are mixed at a ratio of 1:100 (w/w), sterilized via autoclaving, and adjusted to the desired moisture level with sterile water. The fungal inoculum is then added, and the rotary system operates at 10 rpm to ensure adequate mixing and oxygenation. The system is incubated for 6-8 days under optimal growth conditions for mesophilic or thermophilic/thermotolerant fungi, enhancing its versatility. Following incubation, the enzyme is easily extracted using an appropriate cold buffer (e.g., acetate, citrate, or phosphate), depending on the type of enzyme. The extract is clarified through centrifugation and filtration to obtain a cell-free supernatant. The enzyme can then be further concentrated or purified as needed. The results demonstrated a 4-6-fold increase in enzyme activity compared to submerged fermentation (SmF), highlighting the effectiveness of the system. Its adaptability to different substrates, inducers, and fungal species makes it a valuable tool for various biotechnological applications.