Growth of Candida Valida on Ethanol as Sole Carbon Source in Batch Culture: Preliminary Kinetics and Qualitative Detection of Glycogen and Residual Ethanol

Abstract

Candida valida (anamorph of Pichia membranifaciens) is an ascomycetous yeast capable of using ethanol as a sole carbon and energy source via the glyoxylate cycle and gluconeogenesis. Ethanol is an attractive feedstock for single-cell protein (SCP) production because it can be derived from low-emission sources. This study aimed to determine the apparent growth kinetics of C. valida on ethanol in batch culture, to detect intracellular glycogen qualitatively, and to assess residual ethanol qualitatively after cultivation. Batch cultivation was performed in mineral medium containing 1% (v/v) ethanol at 30 °C-over 168 h. Cell concentration was determined using a Goryaev–Thom counting chamber, intracellular glycogen was visualized with Lugol’s iodine stain, and residual ethanol was detected qualitatively by acidified dichromate oxidation. The initial cell density rose from 1.10 × 10⁶ to 5.50 × 10⁶ cells mL⁻¹, a 4.6-fold increase (approximately 2.2 doublings). The apparent net specific growth rate over the entire 168-h period, calculated as a single interval, was 0.0096 ± 0.0008 h⁻¹ (mean ± SD, n = 2 chamber loadings from one flask), with a 95% confidence interval of 0.0023–0.0170 h⁻¹. This value is approximately 8 to 16-fold lower than the 0.08–0.15 h⁻¹ reported for well-adapted C. valida cultures on ethanol. The estimated biomass-to-supplied-ethanol ratio was 0.028 g g⁻¹, indicating incomplete substrate utilization and suboptimal growth conditions. Brown intracellular granules were observed after Lugol staining in both the zero-time sample and the 168-h sample; however, because glycogen was present in the inoculum, this result does not demonstrate net accumulation during cultivation. Acidified dichromate produced a lighter green color in the post-cultivation filtrate compared with the zero-time sample, suggesting partial ethanol consumption. The data confirm that C. valida can grow on ethanol under the conditions tested, but the kinetics were severely suboptimal and the findings are limited by the absence of a growth curve, biological replication, and quantitative substrate and product measurements. Future work should incorporate frequent sampling, pre-adapted inocula, quantitative ethanol and biomass analysis, and vitamin supplementation to establish reliable kinetic parameters for SCP process development.