Optimization of bacteriophage propagation in high-yield continuous culture (cellstat) meeting the constraints of industrial manufacturing processes

Abstract

The growing use of bacteriophages in the fields of agriculture, agri-food, veterinary treatments, and medicine involves the quantitative production of these bacteriophages. In this study, we propose a bacteriophage production protocol that can easily be transposed to industry. We used a cellstat production system because the latest studies have shown that it is the most suitable process for the production of phages due to volumetric productivity, safety (limitation of co-evolution), and flexibility (choice of growth rate criteria). Sizing of the assembly used makes it possible to extrapolate the results to industrial production. The production conditions are indicated precisely, which would allow manufacturers to adapt the protocol to their own equipment. We propose experimental conditions in order to obtain a stable Escherichia coli population, qualitatively and over time, and production of high-titer T7 bacteriophages. The optimized production conditions (yield, cost and simplicity of the process) are: a buffered peptone water medium concentration of 11 g L^-1 and a dilution rate of 1.6 h^-1. Under these conditions, we obtained a production of 7.35×10¹⁶ plaque-forming units (PFU) L^-1 day^-1 with a concentration of 9.8×10¹² PFU mL^-1. The strength of this work lies in its focus on industrial applicability.

Keywords: Bacteriophages; Cellstat; Continuous production; Escherichia coli; Phage T7; Phage therapy.