A Method for Adapting Euglena gracilis to Cassava-Based Substrates for Heterotrophic Cultivation

Cassava processing generates nutrient-rich by-products that remain underutilized due to variable composition, cyanogenic compounds, and active microbial communities. This protocol presents a multistage adaptation strategy for establishing a cassava-tolerant lineage of Euglena gracilis suitable for heterotrophic cultivation in cassava-derived substrates. The method begins with simultaneous exposure of Euglena cultures to autoclaved and non-autoclaved cassava media and to a garri concentration gradient of 10-30 g/L to assess tolerance to microbial complexity and substrate load. Cultures are then subjected to a higher concentration gradient of 30-50 g/L to evaluate performance with denser substrates. The resulting lineage is compared with a non-adapted wild-type strain across five cassava products; garri, dried pulp, pellets, peels, and animal feed to assess growth consistency in field-relevant materials. The protocol includes measurement of pH changes, cyanide levels using colorimetric strips, and preparation of dried biomass for protein analysis based on total nitrogen quantification. Together, these procedures provide a reproducible framework for adapting Euglena gracilis to cassava-derived substrates and for evaluating strain performance across diverse cassava products. This method enables the development of robust algal lineages for use in cassava-focused bioprocessing and waste-valorization applications.