A buffered media system for yeast batch culture growth

Abstract

Background Fungi are premier hosts for the high-yield secretion of proteins for biomedical and industrial applications. The stability and activity of these secreted proteins is often dependent on the culture pH. As yeast acidifies the commonly used synthetic complete drop-out (SD) media that contains ammonium sulfate, the pH of the media needs to be buffered in order to maintain a desired extracellular pH during biomass production. At the same time, many buffering agents affect growth at the concentrations needed to support a stable pH. Although the standard for biotechnological research and development is shaken batch cultures or microtiter plate cultures that cannot be easily automatically pH-adjusted during growth, there is no comparative study that evaluates the buffering capacity and growth effects of different media types across pH-values in order to develop a pH-stable batch culture system. Results We systematically test the buffering capacity and growth effects of a citrate-phosphate buffer (CPB) from acidic to neutral pH across different media types. These media types differ in their nitrogen source (ammonium sulfate, urea or both). We find that the widely used synthetic drop-out media that uses ammonium sulfate as nitrogen source can only be effectively buffered at buffer concentrations that also affect growth. At lower concentrations, yeast biomass production still acidifies the media. When replacing the ammonium sulfate with urea, the media alkalizes. We then develop a medium combining ammonium sulfate and urea which can be buffered at low CPB concentrations that do not affect growth. In addition, we show that a buffer based on Tris/HCl is not effective in maintaining any of our media types at neutral pH even at relatively high concentrations. Conclusion Here we show that the buffering of yeast batch cultures is not straight-forward and addition of a buffering agent to set a desired starting pH does not guarantee pH-maintenance during growth. In response, we present a buffered media system based on an ammonium sulfate/urea medium that enables relatively stable pH-maintenance across a wide pH-range without affecting growth. This buffering system is useful for protein-secretion-screenings, antifungal activity assays, as well as for other pH-dependent basic biology or biotechnology projects.

Optimizing medium composition with wastewater from Coffea arabica processing to produce single‐cell protein using Candida sorboxylosa

“…Figure 3a and b show the contours of DCW and SCP yields, respectively, revealing that substantial DCW production is not accompanied by high SCP production. This is likely because the increase in (NH 4 ) 2 SO 4 concentration caused a decrease in biomass production due to the capacity of ammonium cations to repress the expression of catabolic pathways used for other nitrogenous sources 55 or due to the acidification produced by sulfate anions in culture media 56 …”

“…This is likely because the increase in (NH 4 ) 2 SO 4 concentration caused a decrease in biomass production due to the capacity of ammonium cations to repress the expression of catabolic pathways used for other nitrogenous sources 55 or due to the acidification produced by sulfate anions in culture media. 56 The adjusted-R 2 values for DCW and SCP were 44.72% and 90.96%, respectively, indicating that a better relationship between the experimental and predicted values is expected for SCP yield than for DCW (Table 6). Additionally, a higher concentration of DCW (4.35 g/L) was obtained in the absence of (NH 4 ) 2 SO 4 (run #2), while higher SCP yield (30.6%) and lower DCW concentration (3.6 g/L) were obtained by partially replacing the main nitrogen source (YE) with (NH 4 ) 2 SO 4 (run #3).…”

Inducible Synthetic Growth Regulation Using the ClpXP Proteasome Enhances cis,cis-Muconic Acid and Glycolic Acid Yields in Saccharomyces cerevisiae

“…cerevisiae strains were grown in biological duplicate or triplicate at 30 °C with 200–220 rpm shaking in SDM: 6.7 g/L YNB w/o amino acids (BD Diagnostic Systems) and 20 or 40 g/L d -glucose (VWR Chemicals). For the GA strains, 0.5x pH 5.6 synthetic defined medium with ammonium sulfate (SD-AS) w/o amino acids with 40 g/L d -glucose was used . Cultures were induced with 1, 2, 5, 10, or 20 μg/mL doxycycline (MERCK).…”

“…For the GA strains, 0.5x pH 5.6 synthetic defined medium with ammonium sulfate (SD-AS) w/o amino acids with 40 g/L d -glucose was used. 67 Cultures were induced with 1, 2, 5, 10, or 20 μg/mL doxycycline (MERCK).…”

“…with 40 g/L D-glucose was used 67. Cultures were induced with 1, 2, 5, 10, or 20 μg/mL doxycycline (MERCK).Plasmid Construction.…”