n-Butanol was produced continuously in a two-stage fermentor system with integrated product removal from a co-feed of n-butyric acid and glucose. Glucose was always required as a source of ATP and electrons for the conversion of n-butyrate to n-butanol and for biomass growth; for the latter it also served as a carbon source. The first stage generated metabolically active planktonic cells of Clostridium saccharoperbutylacetonicum strain N1-4 that were continuously fed into the second (production) stage; the volumetric ratio of the two fermentors was 1:10. n-Butanol was removed continuously from the second stage via gas stripping. Implementing a two-stage process was observed to dramatically dampen metabolic oscillations (i.e., periodical changes of solventogenic activity). Culture degeneration (i.e., an irreversible loss of solventogenic activity) was avoided by periodical heat shocking and re-inoculating stage 1 and by maintaining the concentration of undissociated n-butyric acid in stage 2 at 3.4 mM with a pH-auxostat. The system was successfully operated for 42 days during which 93% of the fed n-butyrate was converted to n-butanol at a production rate of 0.39 g/(L × h). The molar yields Y(n-butanol/n-butyrate) and Y(n-butanol/glucose) were 2.0, and 0.718, respectively. For the same run, the molar ratio of n-butyrate to glucose consumed was 0.358. The molar yield of carbon in n-butanol produced from carbon in n-butyrate and glucose consumed (Y(n-butanol/carbon) ) was 0.386. These data illustrate that conversion of n-butyrate into n-butanol by solventogenic Clostridium species is feasible and that this can be performed in a continuous system operating for longer than a month. However, our data also demonstrate that a relatively large amount of glucose is required to supply electrons and ATP for this conversion and for cell growth in a continuous culture.
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