Chair of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, Ackerstr. 76, ACK24, 13355, Berlin, Germany.
Biotechnol Lett. 2023 Dec;45(11-12):1487-1493. doi: 10.1007/s10529-023-03436-1. Epub 2023 Oct 13.
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.
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).
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.
对荚膜红假单胞菌的氢化酶的研究已经进行了二十多年，至今仍在使用常见的接种培养方法。这些方法从未适应过改良细菌菌株的要求，导致前培养物中细菌的生理状态不同，从而导致延长和不同的延迟期。
为了获得用于接种的均匀且始终适应良好的前培养物，我们在这项研究中为荚膜红假单胞菌 HF210（荚膜红假单胞菌 HP80）的衍生物建立了一种优化的前培养物方案，该方案用于同源过表达 NAD 还原可溶性氢化酶（SH）的基因。我们比较了用于前培养物生长的不同培养基，并确定了收获的最佳时间点。本研究中获得的方案基于两个后续的前培养物，第一个在前复杂营养肉汤培养基（NB）中进行，第二个在前果糖-氮矿物质盐培养基（FN）中进行。
尽管有两个后续的前培养物，但我们的方案将前培养时间缩短到 30 小时以下，并为荚膜红假单胞菌 HP80 的培养提供了可重复的前培养物。
