Succinate and Lactate Production from Euglena gracilis during Dark, Anaerobic Conditions

Abstract

Euglena gracilis is a eukaryotic, unicellular phytoflagellate that has been widely studied in basic science and applied science. Under dark, anaerobic conditions, the cells of E. gracilis produce a wax ester that can be converted into biofuel. Here, we demonstrate that under dark, anaerobic conditions, E. gracilis excretes organic acids, such as succinate and lactate, which are bulk chemicals used in the production of bioplastics. The levels of succinate were altered by changes in the medium and temperature during dark, anaerobic incubation. Succinate production was enhanced when cells were incubated in CM medium in the presence of NaHCO3. Excretion of lactate was minimal in the absence of external carbon sources, but lactate was produced in the presence of glucose during dark, anaerobic incubation. E. gracilis predominantly produced L-lactate; however, the percentage of D-lactate increased to 28.4% in CM medium at 30°C. Finally, we used a commercial strain of E. gracilis for succinate production and found that nitrogen-starved cells, incubated under dark, anaerobic conditions, produced 869.6 mg/L succinate over a 3-day incubation period, which was 70-fold higher than the amount produced by nitrogen-replete cells. This is the first study to demonstrate organic acid excretion by E. gracilis cells and to reveal novel aspects of primary carbon metabolism in this organism.

Physiological functions of pyruvate:NADP+ oxidoreductase and 2-oxoglutarate decarboxylase in Euglena gracilis under aerobic and anaerobic conditions

“…20) Furthermore, nitrogen-starved Euglena cells, when incubated anaerobically in the presence of NaHCO 3 , excrete succinate that is used as a building block for various compounds including plastic polybutylene succinate. 42) Results obtained in the present study on 2-oxoacid metabolism would be useful not only to understand the central carbon and energy metabolisms but also to improve the productivity of wax esters and succinate by gene manipulation in E. gracilis. Notes: The closed circle shows the conserved His residue among acetolactate synthases close to the catalytic center, and the open circles denote the proposed binding residues for thiamin pyrophosphate in acetolactate synthase family proteins.…”

Effect of pH on metabolite excretion and cell morphology of Euglena gracilis under dark, anaerobic conditions

“…In addition to the metabolites accumulated in the cells, E. gracilis can excrete value-added metabolites into the extracellular environment. Under dark, anaerobic conditions, nitrogen-starved E. gracilis cells excrete succinate, which is a precursor of many valuable products such as nanofibers and bioplastic resources [9,10]. At least 38 metabolites including various amino acids are excreted under dark, anaerobic conditions and glutamate is produced in a (NH 3 ) 2 HPO 4 -dependent manner [11].…”

Effect of pH on metabolite excretion and cell morphology of Euglena gracilis under dark, anaerobic conditions

“…gracilis cells excrete succinate, which is a precursor of many valuable products such as nanofibers and bioplastic resources [9,10].…”

“…At least 38 metabolites including various amino acids are excreted under dark, anaerobic conditions and glutamate is produced in a (NH 3 ) 2 HPO 4 -dependent manner [11]. The highest succinate titre achieved is 870 mg/L, excreted from nitrogen-starved E. gracilis cells [10]. The highest titres of glutamate and glutamine are 265 mg/L and 56 mg/L, respectively, excreted from E. gracilis cells grown in a pH-adjusted media with buffer salts [11].…”

“…incubation was performed as described previously[10,28] with certain modifications. E. gracilis cells were concentrated in 10 mL of 20 mM buffer ranging from pH 3-8 at OD 730 = 20, 140 or 200 in a GC vial.…”