Anaerobes Wachstum von Corynebacterium glutamicum durch gemischte Säurefermentation

Corynebacterium glutamicum is able to grow under anaerobic conditions with nitrate as electron acceptor, but only very low cell densities are attainable because of the toxic nitrite that cannot be efficiently metabolized by C. glutamicum. Furthermore it is known that C. glutamicum is metabolically active under oxygen-free conditions and produces lactate, succinate, and acetate from glucose. However, fermentative anaerobic growth of C. glutamicum has not been described, yet. In this work, the ability of C. glutamicum for fermentative growth and amino acid production under anaerobic conditions was explored. The following results were obtained: 1. C. glutamicum wild type was able to grow anaerobically in minimal medium in serum bottles with glucose, fructose, sucrose, and ribose as sole carbon and energy source and forms lactate, succinate, and acetate as products. The optical density at 600 nm (OD600) increased from 1.0 to 1.9 (sucrose), 2.2 (glucose), and 2.7 (fructose and ribose). The cells could not grow with gluconate, pyruvate, lactate, acetate, and propionate. Growth experiments in glucose-minimal-medium supplemented with differently composed amino acid mixes indicated that energy limitation is one of the reasons for poor anaerobic growth. The anaerobic production of amino acids (lysine, leucine, and valine) with strains developed for aerobic production was not possible under the tested conditions. 2. Anaerobic fermentative growth in glucose minimal medium could be improved from OD600 1.0 to 11.8 by the addition of peptides. The peptides were presumably taken up by the cells and, after hydrolysis to amino acids, used predominantly for protein synthesis. Consequently, less amino acids had to be synthesized de novo and the cells saved energy in form of ATP, which could be used e. g. for building up proton motive force or for pH homeostasis. The C. glutamicum mutant ? actA? pqo? pta-ackA, which lacks all genes currently known to be involved in acetate formation, could grow to higher cell densities (OD600 = 16.8) in glucose minimal medium supplemented with peptides in the bioreactor than the wild type (OD600 = 12.7). This mutant produced 64% less acetate per CDW (cell dry weight) than the wild type. It is known that acetate can act as an uncoupler and therefore inhibits growth. 3. Growth experiments in the bioreactor in glucose minimal medium at constant extracellular pH values showed that under anaerobic conditions the pH tolerance range of C. glutamicum was drastically reduced (pH 6.5 to 8.0) in comparison to aerobic conditions (pH 4.0 to 10.0). The intracellular pH analysis of C. glutamicum pEKEx2_pHluorin with the pH-sensitive fluorescence protein pHluorin revealed that pH homeostasis occurred much slower and the intracellular pH did not reach the same value after a shift to acidic conditions under anaerobic conditions. Under aerobic conditions the protons which enter the cell under acidic conditions can be pumped out effectively and quickly by the respiratory chain.