Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently supports production of recombinant protein by Escherichia coli

Abstract

Deriving new value from waste streams through secondary processes is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be recycled and used as a feed in secondary microbial fermentation to produce new recombinant protein products. Our results show that CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich microbiological media (LB). The titre of recombinant protein produced following induction in a 4-hour expression screen was approximately equivalent in the CDSM fed cultures to that of baseline, and this was maintained in a 16-hr preparative fermentation. To understand the protein production achieved in CDSM fed culture we performed a quantitative analysis of proteome changes in the E. coli using mass spectrometry. This analysis revealed significant upregulation of protein synthesis machinery enzymes and significant downregulation of carbohydrate metabolism enzymes. We conclude that spent cell culture media, which represents 100s of millions of litres of waste generated by the bioprocessing industry annually, may be valorized as a feed resource for the production of recombinant proteins in secondary microbial fermentations. Data is available via ProteomeXchange with identifier PXD026884.

Feeding secondary fermentations with mammalian and fungal culture waste streams increases productivity and resource efficiency

“…In a previous study, it was shown that E. coli grown in CSM-fed cultures underwent a proteomic shift with significant upregulation of amino-acid biosynthetic enzymes (Lynch and O’Connell, 2022). This finding is reinforced by our findings here on the amino-acid content of the CSM composition pre and post-secondary culture, with the concentrations of three different amino acids (alanine, cysteine, and valine) increased after the secondary E. coli culture.…”

“…Glutamate, another precursor, was also consumed at a high level in this condition (1.3 mM) and is one of the most essential amino acids as it has many uses outside of protein synthesis including assimilation of nitrogen as ammonium (Walker and van der Donk, 2016). Of the enzymes involved in the amino acid biosynthetic pathway found previously in CSM-fed secondary E. coli cultures, most were involved in the conversion of these precursors into other amino acids, including the most significantly upregulated enzyme, asparagine synthase A, with a difference in LFQ intensity of 2.79 (Lynch and O'Connell, 2022). These findings suggest that stringent response due to amino acid starvation may influence the CSM-fed E. coli culture in order to upregulate these biosynthetic pathways.…”

“…A number of pilot studies have investigated the principle of recycling spent media waste and show utility when used as a supplement to fresh media in the same bioreactor across a number of cell types and processes (Kempken, Büntemeyer and Lehmann, 1991; Hsiao, Glatz and Glatz, 1994; Riese et al ., 1994; Wu, Ruan and Lam, 1998). We have previously shown spent media from CHO cell culture supports growth and recombinant protein production in a secondary fermentation as a standalone feed (Lynch and O’Connell, 2022). The nutritional value remaining in spent culture media could potentially be employed to feed multiple, distinct secondary fermentations.…”

Feeding secondary fermentations with mammalian and fungal culture waste streams increases productivity and resource efficiency

“…In a previous study, it was shown that E. coli grown in CSM-fed cultures underwent a proteomic shi with signicant upregulation of amino-acid biosynthetic enzymes. 18 This nding is…”

Precision fermentation with mass spectrometry‐based spent media analysis

“…Additionally, metabolomic analysis also identifies potential opportunities to reuse and valorize spent media from industrial mammalian cell cultures. Chemically defined spent media from CHO culture, supplemented with 2% glycerol, has been demonstrated to be nutrient rich medium for E. coli fermentation for protein production (Lynch & O'Connell, 2022).…”