Experimentation of various co-culture fermentation strategies for better efficiency bioethanol production using wild cassava flour (Manihot glaziovii Muell. Arg) as substrate

• Wild cassava presents a promising raw material option for bioethanol production. • Co-culture fermentation is able to increase the efficiency of bioethanol production. • Local isolates were simultaneously proven to be able to convert cassava flour into bioethanol. • Bioethanol production from wild cassava flour is a viable alternative energy source. The continuous increase in global demand for fossil-based fuels has driven the need for sustainable and renewable energy alternatives such as bioethanol. Wild cassava ( Manihot glaziovii Muell. Arg), a non-edible starch-rich crop with high cyanogenic content, represents a promising feedstock for bioethanol production without competing with food resources. This study aimed to enhance bioethanol production efficiency from wild cassava flour (WCF) through various co-culture fermentation strategies combining Aspergillus niger FNCC 6018, isolate R5I4, and isolate R5I3 under Simultaneous Saccharification and Fermentation (SSF) and Simultaneous Saccharification and Co-Fermentation (SSCF) conditions. The fermentation was conducted at 35°C and pH 5 for 7–8 days in a 250 mL bioreactor containing 33.33 g of WCF and 166.67 mL of distilled water, with agitation at 100 rpm. Among 17 treatment combinations, the co-culture of R5I4, A. niger FNCC 6018, and R5I3, added at the 96th hour, yielded the highest ethanol concentration (28.277 ± 0.228 g/L), efficiency (46.20 ± 0.37%), productivity (0.168 g/L/h), and yield coefficient (0.028 g/g). SEM and HPLC analyses confirmed efficient starch hydrolysis and glucose-to-ethanol conversion. Phylogenetic analysis identified R5I3 as closely related to Candida orthopsilosis (91 %) and R5I4 to Klebsiella variicola (83 %). These findings demonstrate that wild cassava flour is a viable substrate for efficient bioethanol production using co-culture SSF/SSCF systems. Future work should focus on scaling up fermentation, optimizing environmental parameters, and exploring the metabolic engineering of the microbial consortium to improve ethanol yield and process economics further.