Semi-continuous propagation and synchronous differentiation of hyper-solventogenic cells of Clostridium thermosaccharolyticum in a xylan medium at 56 °C

Synchronized sporangia of Clostridium thermosaccharolyticum were prepared by diluting a Basal Xylan (BX) culture 10 times at each generation for two generations followed by 100 times dilution for unrestricted growth in a BX medium enriched with Ca-gluconate or CaCO3. This was a technique that enhanced the starter in which division synchrony and mass synchrony once established for synchrony in differentiation continued for almost 10 cycles, as long as the O2 purging was provided. Following the multi-step growth that correlated with the repetitive spikes of calcium incorporation up to 3.8 × 10−4 nmol/cell, cell density approached the stationary phase. These synchronized sporangia were used as a starter in the semi-continuous operation by repeating 50% draw-offs at each generation in the presence of 50 ppm O2 for nine generations, during which the percentage of sporangia increased from 60% to 88%. Traditional binary fission (TBF) of elongated sporangia (longer than 4×) must then be stopped by the addition of chemical growth inhibitors or temperature shift-down to 35 °C for steep solventogenesis that also promoted an increase of sporangia to 99%. It was evident that the stressful conditions were essential in the propagation process, and for retaining the highest percentage of sporangia, which had to be arrested at the solventogenic phase (without acidogenesis) to reach 7.05% final concentration in 27 h at 56 °C, or 10.57% in 105 h at 35 °C, giving a recovery yield of 97.9%. Differentiation and modified division without TBF required O2 purging to continue.