Bioethanol Production from Lignocellulosic Biomass Using <i>Aspergillus niger</i> and <i>Aspergillus flavus</i> Hydrolysis Enzymes through Immobilized <i>S. cerevisiae</i>

Lignocellulose, the main component of a plant cell wall, is a potential renewable bioenergy source. It is composed of cellulose, hemicellulose, and lignin structures. Cellulose is a linear polysaccharide that is hydrolyzed chemically or enzymatically by cellulase. The addition of lignocellulosic biomass, such as wheat bran and coffee pulp, into the fermentation culture, induces the production of cellulases. Cellulose accounts for 20% of the enzyme market worldwide, demonstrating benefits in diverse applications, especially bioethanol and biogas generation. The aim is to evaluate the optimal condition for bioethanol production by previously isolated fungal species from different soil types in the eastern region of the Kingdom of Saudi Arabia. This study attempts to evaluate and optimize the culture conditions of lignocellulosic biomass under SSF using the highest cellulases-producer strains in the region: Aspergillus niger and Aspergillus flavus (GenBank Accession No. MT328516 and MT328429, respectively) to produce raw sugar that consequently is used in the next step of bioethanol production. This process has two parts: (1) hydrolyze lignocellulosic biomass to obtain raw sugar using A. niger and A. flavus that produce cellulase, and (2) produce bioethanol through the conversion of the raw sugar produced from the cellulolysis into ethanol using Saccharomyces cerevisiae. The optimal conditions under SSF were seven days of incubation, 5% glucose as a carbon source, 1% ammonium sulfate as a nitrogen source, and 80% moisture for both isolates. Biochemical characterization showed stability for the immobilized enzyme in all temperature ranges (from 20 °C to 70 °C), while the free enzyme exhibited its maximum at 20 °C of 1.14 IU/mL. CMCase production was the highest at pH 4.0 (1.26 IU/mL) for free enzyme and at pH 5.0 (2.09 IU/mL) for the immobilized form. The CMCase activity increased steadily with an increase in water level and attained a maximum of 80% moisture content. The maximum enzyme activity was with coffee pulp as a substrate of 7.37 IU/mL and 6.38 IU/mL for A. niger and A. flavus after seven days of incubation, respectively. The Carboxymethyl Cellulase (CMCase) activity in immobilized enzymes showed good storage stability under SSF for six weeks, maintaining 90% of its initial activity, while the free enzyme retained only 59% of its original activity. As a carbon source, glucose was the best inducer of CMCase activity with coffee pulp substrate (7.41 IU/mL and 6.33 IU/mL for A. niger and A. flavus, respectively). In both fungal strains, ammonium sulfate caused maximum CMCase activities with coffee pulp as substrate (7.62 IU/mL and 6.47 IU/mL for A. niger and A. flavus, respectively). Immobilized S. cerevisiae showed an increase in ethanol production compared to free cells. In the case of immobilized S. cerevisiae cells, the concentration of ethanol was increased steadily with increasing fermentation time and attained a maximum of 71.39 mg/mL (A. niger) and 11.73 mg/mL (A. flavus) after 72 h of fermentation