Abstract
The Bacterial Ghost (BG) platform technology evolved from a microbiological expression system incorporating the ϕX174 lysis gene E. E-lysis generates empty but structurally intact cell envelopes (BGs) from Gram-negative bacteria which have been suggested as candidate vaccines, immunotherapeutic agents or drug delivery vehicles. E-lysis is a highly dynamic and complex biological process that puts exceptional demands towards process understanding and control. The development of a both economic and robust fed-batch production process for BGs required a toolset capable of dealing with rapidly changing concentrations of viable biomass during the E-lysis phase. This challenge was addressed using a transfer function combining dielectric spectroscopy and soft-sensor based biomass estimation for monitoring the rapid decline of viable biomass during the E-lysis phase. The transfer function was implemented to a feed-controller, which followed the permittivity signal closely and was capable of maintaining a constant specific substrate uptake rate during lysis phase. With the described toolset, we were able to increase the yield of BG production processes by a factor of 8–10 when compared to currently used batch procedures reaching lysis efficiencies >98%. This provides elevated potentials for commercial application of the Bacterial Ghost platform technology.
Highlights
Bacterial Ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria
It was shown that E-mediated lysis (E-lysis) is dependent on a certain level of physiological activity as well as an intact membrane potential [53], whereas growth stagnation before lysis induction resulted in lysis-incompetent populations [1]
One of the major constraints for successful E-lysis is that, until the time-point of lysis induction, the bacteria are maintained in lysis-competent conditions, i.e., that the population is in a physiological state that allows every bacterium to go through the cycle of cell division [1]
Summary
Bacterial Ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria. Controlled expression of the single cloned bacteriophage φX174 lysis gene E [1] leads to the fusion of the cytoplasmic (inner) and outer cell membrane (IM/OM, respectively) followed by expulsion of the cytoplasmic content due to the osmotic pressure difference. Monitoring of E-lysis by dielectric spectroscopy has recently been reported for technical applications of BGs [26] Another tool for on-line estimation of viable biomass is a so-called soft-sensor. In contrast to dielectric spectroscopy, which measures the total volume of viable cells, soft-sensors compute the actual biomass from the metabolic activity of the culture [32] and provide capability for dynamic process control [35]. A real-time signal describing the cells’ physiological state under dynamic conditions would allow a tailored feeding strategy circumventing excess substrate feeding or critical substrate limitation Within this contribution, we want to demonstrate that one can establish a transfer function correlating the permittivity signal with the viable biomass concentration estimated by a soft-sensor during non-induced fed-batch fermentation. The subsequent control strategy is discussed as a PAT approach for high-density production of Bacterial Ghosts
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