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SummaryIn large‐scale production processes, metabolic control is typically achieved by limited supply of essential nutrients such as glucose or ammonia. With increasing bioreactor dimensions, microbial producers such as Escherichia coli are exposed to changing substrate availabilities due to limited mixing. In turn, cells sense and respond to these dynamic conditions leading to frequent activation of their regulatory programmes. Previously, we characterized short‐ and long‐term strategies of cells to adapt to glucose fluctuations. Here, we focused on fluctuating ammonia supply while studying a continuously running two‐compartment bioreactor system comprising a stirred tank reactor (STR) and a plug‐flow reactor (PFR). The alarmone ppGpp rapidly accumulated in PFR, initiating considerable transcriptional responses after 70 s. About 400 genes were repeatedly switched on/off when E. coli returned to the STR. E. coli revealed highly diverging long‐term transcriptional responses in ammonia compared to glucose fluctuations. In contrast, the induction of stringent regulation was a common feature of both short‐term responses. Cellular ATP demands for coping with fluctuating ammonia supply were found to increase maintenance by 15%. The identification of genes contributing to the increased ATP demand together with the elucidation of regulatory mechanisms may help to create robust cells and processes for large‐scale application.

Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO 2 footprint by converting industrial emissions containing CO 2 , CO and H 2 into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO 2 + H 2 , CO and CO + CO 2 + H 2 (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood-Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD + provider for CO 2 + H 2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fd red) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fd red formation when CO is used as substrate, whereas Fd red is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO 2 + H 2. Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.

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