Salt accumulation resulting from base added for pH control, and not ethanol, limits growth of Thermoanaerobacterium thermosaccharolyticum HG-8 at elevated feed xylose concentrations in continuous culture

Thermoanaerobacter thermosaccharolyticum HG-8 was grown in continuous culture to characterize growth limitation at high feed substrate and product concentrations. Continuous fermentation of 50 and 73 g/L xylose at a dilution rate based on the feed flow, D-f, of 0.053 h(-1) and with the pH controlled at 7.0 by addition of KOH resulted in steady state utilization of >99% of the xylose fed and production of ethanol and acetic acid at a mass ratio of about 2:1. Continuous cultures of T. thermosaccharolyticum growing at D-f = 0.053 h(-1) achieved complete utilization of 75 g/L xylose in the presence of 19.1 g/L K+ (0.49 M) and an ethanol concentration of 22.4 g/L ethanol. When the feed to a culture initially at steady state with a 75 g/L xylose feed and D-f = 0.053 h(-1) was increased to 87.5 g/L xylose, limitation of growth and xylose utilization was observed. This limitation was not relieved by repeating this feed upshift experiment in the presence of increased nutrient levels and was not reproduced by addition of ethanol to a steady-state culture fed with 75 g/L xylose. By contrast, addition of KCl to a steady-state culture fed with 75 g/L xylose reproduced the K+ concentration, limitation of growth and xylose utilization, and product concentration profiles observed in the feed upshift experiment. The maximum concentration at which growth of batch cultures was observed was 0.43 M for KCl, NaCl, and equimolar mixtures of these salts, suggesting that the observed limitation is not ion-specific. These data support the interpretation that inhibition salt accumulation resulting from addition of KOH for pH control is the limiting factor manifested in the feed upshift experiment and that both nutrient limitation and ethanol inhibition played little or no role as limiting factors. More generally, salt inhibition would appear to be a possible explanation for the discrepancy between the tolerance to added ethanol and the maximum concentration of produced ethanol reported in the literature for fermentation studies involving thermophilic bacteria.