Influence of nitrogen variation and 15% CO ₂ injection on wax ester production in <i>Euglena gracilis</i> isolated from Dieng Peatland, Indonesia

Biodiesel production requires alternative feedstocks that are both renewable and environmentally friendly. Euglena gracilis is a microorganism capable of producing wax esters, which show strong potential as alternative fuels due to their high productivity. Wax esters from E. gracilis also hold significant promise as raw materials in the pharmaceutical, health, and cosmetic industries. This study aimed to increase wax ester production in E. gracilis through nitrogen variation (1000, 500, 200, 0 mg l−1) and 15% CO₂ injection. Results showed that a nitrogen concentration of 500 mg l−1 yielded the highest biomass (3400 ± 0.000 mg l−1), carbohydrate (180 ± 16.782 mg l−1), lipid (255 ± 21.213 mg l−1), paramylon (151 ± 51.055 mg l−1), chlorophyll a (5.29 ± 0.0002 × 10−3 mg l−1), chlorophyll b (0.70 ± 0.0002 × 10−3 mg l−1), carotenoid (0.71 ± 0.084 × 10−3 mg l−1), and wax ester (16.88 ± 0.01%). At 500 mg l−1 nitrogen, E. gracilis also produced myristic acid methyl esters and palmitic acid, which are key biodiesel precursors. Under the same condition, E. gracilis produced the highest levels of diatoxanthin, trans-diatoxanthin, dinoxanthin, lutein, phaeophytin-a, β-carotene, β-cryptoxanthin, all-trans-zeaxanthin, phaeophytin-b, 9’-cis-neoxanthin, and violaxanthin. Modifying nitrogen concentrations and injecting CO₂ can increase lipid and wax ester levels in E. gracilis without reducing biomass growth, supporting its potential for biodiesel production.