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Background   Caloramator celer  is a strict anaerobic, alkalitolerant, thermophilic bacterium capable of converting glucose to hydrogen (H 2 ), carbon dioxide, acetate, ethanol and formate by a mixed acid fermentation. Depending on the growth conditions  C. celer  can produce H 2  at high yields. For a biotechnological exploitation of this bacterium for H 2  production it is crucial to understand the factors that regulate carbon and electron fluxes and therefore the final distribution of metabolites to channel the metabolic flux towards the desired product.    Results  Combining experimental results from batch fermentations with genome analysis, reconstruction of central carbon metabolism and metabolic flux analysis (MFA), this study shed light on glucose catabolism of the thermophilic alkalitolerant bacterium  C. celer . Two innate factors pertaining to culture conditions have been identified to significantly affect the metabolic flux distribution: culture pH and partial pressures of H 2  ( P  H2 ). Overall, at alkaline to neutral pH the rate of biomass synthesis was maximized, whereas at acidic pH the lower growth rate and the less efficient biomass formation are accompanied with more efficient energy recovery from the substrate indicating high cell maintenance possibly to sustain intracellular pH homeostasis. Higher H 2  yields were associated with fermentation at acidic pH as a consequence of the lower synthesis of other reduced by-products such as formate and ethanol. In contrast,  P  H2  did not affect the growth of  C. celer  on glucose. At high  P  H2  the cellular redox state was balanced by rerouting the flow of carbon and electrons to ethanol and formate production allowing unaltered glycolytic flux and growth rate, but resulting in a decreased H 2  synthesis.    Conclusion   C. celer  possesses a flexible fermentative metabolism that allows redistribution of fluxes at key metabolic nodes to simultaneously control redox state and efficiently harvest energy from substrate even under unfavorable conditions (i.e. low pH and high  P  H2 ). With the H 2  production in mind, acidic pH and low  P  H2  should be preferred for a high yield-oriented process, while a high productivity-oriented process can be achieved at alkaline pH and high  P  H2 .

microbial cell factories

Assessment of metabolic flux distribution in the thermophilic hydrogen producer Caloramator celer as affected by external pH and hydrogen partial pressure