When yeast spent media from vat fermentation is supplemented and reused, it can outperform fresh culture media, according to researchers at University College Dublin (UCD). This suggests a commercial-scale, circular green economy is feasible for biopharmaceutical fermentation operations.

In a recent paper, UCD PhD student Laura Murphy, postdoctoral researcher Ciara Lynch, PhD, and associate professor David O’Connell, PhD, compared spent yeast culture media to the original rich biological media used to produce recombinant proteins in E. coli fermentation.

When growing E. coli in either medium, the team identified substantial depletions in levels of six elements and 18 of 20 amino acids. They noted that 98% of the starting levels of magnesium were consumed and found that two to five mM of magnesium were considered ideal for E. coli growth.

Magnesium ups growth and titer

Concentrations of sodium and magnesium were lower in the spent media, while levels of phosphorus, sulfur, potassium, and calcium were comparable to those of rich biological media, the scientists report.

Therefore, they added magnesium and sodium to replenish those levels. In that solution, doubling time for E. coli was 1.23 hours, compared to 2 hours for the depleted medium alone. Simply adding magnesium to the spent solution resulted in a 1.15 hour doubling time, while only adding sodium resulted in a 2.15 hour doubling time.

These results suggest “that magnesium supplementation is the most important elemental addition for successful E. coli growth in spent yeast media,” O’Connell’s team noted.

Additionally, the work showed that all amino acid levels were “significantly depleted,” save that of cysteine, which increased, and isoleucine, which declined only slightly. Supplementing amino acids, therefore, helps support the culture’s growth rate and productivity but has little impact alone.

The highest recombinant protein titer occurred when 2 mM of magnesium and 1% tryptone solution (to restore amino acid levels) were added to the depleted culture. The resultant cell dry weight was 2.7-fold higher than adding magnesium alone, and 1.6-fold greater than cells grown in in the original rich biological media. This, they reported, was “the most striking observation.”

This approach not only increased protein titer, but also decreased energy use.

Other researchers determined that the water-related impact of energy, for example, ranged between 18 and 80 kg per kilogram of antibody produced. Approximately 95% of the water’s process mass intensity is related to media. “Calculations indicate that this media recycling could save upwards of 800 kg of waste per kg of product,” O’Connell told GEN.

The team concluded, “Yeast spent media are viable media for reuse in feeding secondary E. coli cultures with minimal supplementation steps. These cultures can be highly productive.”

“The next steps,” O’Connell said, “will be for collaboration with industrial teams to assess this approach to valorizing yeast spent media post-clarification, and retrieval of recombinant biopharmaceuticals.”

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