Improved preculture management for Cupriavidus necator cultivations

OBJECTIVES: Research on hydrogenases from Cupriavidus necator has been ongoing for more than two decades and still today the common methods for culture inoculation are used. These methods were never adapted to the requirements of modified bacterial strains, resulting in different physiological states of the bacteria in the precultures, which in turn lead prolonged and different lag-phases. RESULTS: In order to obtain uniform and always equally fit precultures for inoculation, we have established in this study an optimized protocol for precultures of the derivative of C. necator HF210 (C. necator HP80) which is used for homologous overexpression of the genes for the NAD(+)-reducing soluble hydrogenase (SH). We compared different media for preculture growth and determined the optimal time point for harvest. The protocol obtained in this study is based on two subsequent precultures, the first one in complex nutrient broth medium (NB) and a second one in fructose –nitrogen mineral salt medium (FN). CONCLUSION: Despite having two subsequent precultures our protocol reduces the preculture time to less than 30 h and provides reproducible precultures for cultivation of C. necator HP80.