Obligate Mutualistic Growth in 1L Bioreactor

Starter cultures were prepared with a methodology similar to that described previously. Cells were co-cultured in modified TAP medium (pH 8), which consisted of 500 ml TAP medium lacking ammonium (NH₄Cl), supplemented with 3.6% mannose, 20 mM KNO₂ and 1X Hom’s vitamins (Table 1). Cells were inoculated to a cell density of 1 × 10⁶ cells/ml. Co-culture experiments were conducted at 25°C, with 50 rpm agitation under continuous light (∼200 μmol m^-2 s^-1) in a BioFlo 110 Vessel bioreactor system (New Brunswick Scientific) and the pH, dissolved oxygen concentration, agitation and temperature were monitored throughout the course of the experiment. The batch bioreactor system was set-up as described in the Guide to Operations manual no. M1273-0054 (Figure 4, New Brunswick Scientific). At appropriate experimental time-points (12-h interval during the day), co-cultures were thoroughly mixed by increasing agitation to 300 rpm for 5 min, thereafter the agitation was set back to 50 rpm. Five ml were sampled from the bioreactor every 12 h for 7 days. Yeast and microalgae growth was monitored by means of haemocytometer cell counts (cell/ml) and combined dry weight (g/l). Mannose (D-Mannose/D-Fructose/D-Glucose Assay kit, Megazyme) and nitrite (Nitrite/Nitrate Assay Kit, colorimetric, Merck) consumption were monitored throughout the experiment. The production of organic acids (citric acid, tartaric acid, malic acid, succinic acid, and acetic acid), glycerol and ethanol were measured using high-performance liquid chromatography (HPLC) analysis (Eyéghé-Bickong et al., 2011). The HPLC method (SUG_PY5) was run using an isocratic gradient of 5 mM H₂SO₄ at a flow rate of 0.5 ml/min for 39 min, and the injection volume was 10 μl. After HPLC analysis, the integrated standard area and known concentrations were used to plot each standard accordingly, to determine unknown sugar and organic acid concentrations.

Mutualistic growth of C. sorokiniana (CS) and S. cerevisiae (SC) under single and cc conditions at different temperatures. Cultures were grown from an initial inoculum of 0.1 × 10⁶ cells/ml for each species at temperatures of (A) 25°C, (B) 30°C, and (C) 37°C. Data represents the mean ± standard error (n = 3).