High cell density cultivation of Methylorubrum extorquens using a modified pH-stat supplemented with intermittent formate feeding

Abstract

Microbial utilization of formic acid as a carbon source represents a promising route for CO<inf>2</inf> mitigation and carbon resource valorization, which requires high cell density cultivation of formatotrophs. In this study, we developed a modified pH-stat fed-batch system for polyhydroxybutyrate (PHB) production by Methylorubrum extorquens ATCC 55366 to automatically trigger intermittent feeding based on the formic acid addition signal. In a simple pH-stat operation, exponential growth was maintained during the early phase; however, as cultivation proceeded, the rate of acid addition declined, leading to growth deceleration and a final cell concentration of only 6.2g/L. When formic acid was externally supplemented during growth arrest, cell growth immediately resumed, indicating that pH feedback alone did not reflect the actual substrate demand of the cells. To overcome this limitation, a control logic was implemented to automatically initiate intermittent feeding when the hourly acid addition rate dropped below a specified threshold. Among the tested C/N ratios (1:1, 2:1, and 3:1) for the feeding solution, the highest cell concentration (18.3g/L) was achieved at 2:1. However, overfeeding occurred during the late high cell density phase, and the number of intermittent feedings was therefore limited to seven. As a result, the final biomass and PHB concentrations reached 23.3g/L and 6.77g/L, respectively, with biomass and PHB productivities of 0.258g/L/h and 0.100g/L/h. The proposed modified pH-stat fed-batch process provides a simple yet effective strategy for automated control and can be readily extended to high cell density cultures of diverse formatotrophic microorganisms. © The Author(s), under exclusive licence to The Korean Society for Biotechnology and Bioengineering 2025.