Systematic evaluation and repurposing analysis of spent bioprocessing waste

Millions of litres of spent cell culture media are disposed each year from the production of biomolecules from monoclonal antibodies to food additives. The immediate disposal of this waste also entails disposing of the many leftover nutrients in this feedstock and uses harsh chemicals for inactivation and subsequent incineration. This thesis was founded around this central idea: could we reuse this feedstock in a second process and therefore contribute to building a more circular bioeconomy? The investigation undertaken went on to explore spent media from three different primary cell culture bioprocesses. Firstly, three different chemically defined spent mammalian media were tested for the capability to support a secondary fermentation producing recombinant protein in Escherichia coli. It was found that spent mammalian media from an adherent CHO cell culture was the most capable of supporting further E. coli cultures. These CHO spent media (CSM)-fed E. coli cultures managed to produce at least equivalent amounts of recombinant product as the control rich media LB at bioreactor scale (LB = 1.29 (± 0.09) g/L and CSM = 1.35 (± 0.08) g/L). CSM was discovered to have many useful nutrients leftover from the primary cell culture including glucose, host cell proteins, and many free amino acids which were not used by the CHO cells. In the second chapter, a fungal spent media (FSM) from a Trametes versicolor bioreactor bioprocess producing beta-glucan food additives was tested for the same capability. This FSM also achieved high recombinant protein titres from E. coli in bioreactors (1.19 (± 0.06) g/L). Detailed analysis showed it to be nutritionally rich post-fungal culture. In particular, this complex spent media contained very high levels of iron and zinc. Both of these essential nutrients have protective qualities for E. coli when dealing with the high lactic acid that was also found in this media as a fungal metabolite. The third chapter then investigated the usage of a yeast spent media from a Pichia pastoris culture. The primary yeast culture was shown to have consumed many of the nutrients necessary for E. coli growth including magnesium. Therefore, this spent media was less nutritious for E. coli culture with a protein yield in shake flask culture of only 27% of that achieved by LB-fed E. coli. Supplementation to the media with magnesium recovered recombinant protein production in bioreactors to equivalent productivity levels as the LB (~18% product/gram of biomass), however the growth was still inhibited resulting in less total yield (YSM = 0.79 (± 0.04) g/L). All three spent media-fed E. coli cultures had lower final biomass than the LB cultures. However, the higher protein productivity levels in the CSM and FSM fed cultures (22.12% and 22.89% product/gram of biomass respectively) meant these cultures still produced approximately equivalent final protein yields as the LB-fed cultures. Although the recombinant protein productivity of the YSM was equivalent to the LB, the lower final biomass of E. coli gained from these cultures meant the final yield was less than the LB. The reasons behind such are discussed within this thesis and future experiments proposed in order to improve this spent media to the same extent as the other two. A methodology is thus proposed for general spent media design for reuse based on the knowledge gained over this four-year investigation. Spent media has proven to be a valuable resource for reuse which could decrease the waste produced from the world's bioprocesses and increase resource efficiency for a cleaner, more circular future.