Probing the redox metabolism in the strictly anaerobic,extremely thermophilic,hydrogen-producing Caldicellulosiruptorsaccharolyticus using amperometry

Changes in the redox metabolism in the anaerobic, extremely thermophilic, hydrogen-forming bacterium Caldicellulosiruptor saccharolyticus were probed for the first time in vivo using mediated amperometry with ferricyanide as a thermotolerant external mediator. Clear differences in the intracellular electron flow were observed when cells were supplied with different carbon sources. A higher electrochemical response was detected when cells were supplied with xylose than with sucrose or glucose. Moreover, using the mediated electrochemical method, it was possible to detect differences in the electron flow between cells harvested in the exponential and stationary growth phases. The electron flow of C. saccharolyticus was dependent on the NADH- and reduced ferredoxin generation flux and the competitive behavior of cytosolic and membrane-associated oxidoreductases. Sodium oxamate was used to inhibit the NADH-dependent lactate dehydrogenase, upon which more NADH was directed to membrane-associated enzymes for ferricyanide reduction, leading to a higher electrochemical signal. The method is noninvasive and the results presented here demonstrate that this method can be used to accurately detect changes in the intracellular electron flow and to probe redox enzyme properties of a strictly anaerobic thermophile in vivo.