Universidade do Minho. Departamento de Engenharia Biológica (DEB)
Abstract
Pichia pastoris is a methylotrophic yeast that can be genetically engineered to express proteins
for industrial use. One of the most important advantages of protein expression in P. pastoris is its
capability of growing on minimal medium and efficiently secreting heterologous proteins with
low secretion levels of endogenous proteins. Operational variables such as pH, temperature,
stirring rate, among others, usually affect the microorganism’s growth during the fermentation
processes. Therefore, the present work aimed to evaluate the influence of pH on cellular growth
of P. pastoris KM71H by fed‐batch process. The fermentation run was carried out in a 1.6 L (total
volume) bioreactor, being performed in two phases: In the first stage (24 h), the yeast was batchcultured
in BMGH medium; while in the second stage (72 h), it was cultivated by feed‐batch
operation with a feeding medium containing 50% glycerol and 12ml/l of trace metal solution.
During the overall process, which lasted after 96 h, the aeration and temperature conditions
were fixed at 10 ml\L.h, 1.5 vvm and 30°C, respectively. Different pH values were evaluated: 5.0,
5.5 and 6.0. Cellular growth was determined by measuring the fermentation broth UVspectrophotometric
absorbance at 600 nm, which was correlated to a calibration curve (dry
weight ´ optical density). Glycerol consumption was detected by HPLC analysis. P. pastoris KM71H
successfully grew in all the evaluated pH values; but the highest biomass production was
observed at pH 5.0 (98.79 g/L). Although P. pastoris is reported as being a microorganism able to
grow over a wide pH range (from 3 to 7); it was not observed high cell density of P. pastoris
KM71H strain when cultivated at pHs 5.5 and 6.0. High cellular growth is especially important for
proteins secretion, as the concentration of product in the medium is roughly proportional to the
concentration of cells in culture. Finally, these results reveal the possibility of obtaining high cell
density of P. pastoris KM71H by fed‐bach cultivation at pH 5.0, which can be a suitable condition
for the yeast application in heterologous proteins production.Conselho Nacional de Desenvolvimento Científico e Tecnológico Brazil (CNPq)Improving Skills Across Continents (ISAC ) - Erasmus Mundus External Cooperation Window (ERASMUS
