9 ± 3.7 0 Substrate changes Transfer from cellobiose to cellulose 6.2 ± 3.7 0 Starvation Depletion of substrate during steady state growth 0 98.0 ± 0.017 Conditions predicted to be unfavorable for growth were tested to BAY 57-1293 cost determine which stressors cause C. thermocellum to form spores or L-forms. The percentage of resting cells to total cells is shown. Error Z-IETD-FMK mw represents one standard deviation, n = 3.

Conditions that resulted in sporulation included oxygen exposure and changes between growth on soluble and insoluble substrates. As C. thermocellum is an obligate anaerobe, oxygen was chosen as a stressor. Varying amounts of oxygen were tested and as is shown in Figure 1, the addition of 20% v/v sterile air to the headspace of a sealed serum vial grown culture was optimal for inducing spore formation. Oxygen selleck compound induced spore formation in approximately 7% of the cells. Additionally, approximately 7% of the cells sporulated when transferred from cellobiose to Avicel or from Avicel to cellobiose (Table 1). C. thermocellum can grow equally well on both substrates, and when cultures are transferred or subcultured in media with the same substrate, sporulation was not observed. L-forms were not observed in any of the conditions mentioned above. Figure 1 Sporulation

induced by aerobic cultivation. The effects of oxygen on spore formation were determined by exposing C. thermocellum cultures to increasing volumes of sterile air. Error bars represent one standard deviation, n = 3. Evaluation of conditions under which L-forms were observed Abrupt termination of the feed to a steady-state continuous culture at several dilution rates (0.03 h-1, 0.1 h-1, and 0.15 h-1) and with several cellobiose concentrations (2.5, 3.0 and 5.0 g/L) was used to evaluate the impact of sudden substrate exhaustion in C. thermocellum. This treatment,

independent of dilution rate or cellobiose concentration, was found to cause nearly all of the cells to shift to the L-form morphology (Table 1, Figure 2) with no spores observed. L-forms were tuclazepam readily distinguished from spores by light microscopy, appearing phase dark and nearly translucent whereas spores are phase bright and opaque. Further analysis by TEM clearly showed structural differences between L-forms and spores (Figure 3). We, as well as others [11], have observed C. thermocellum spores to exhibit a thick spore coat (Figure 3C and 3D), whereas the L-form cells appeared to lack a cell wall (Figure 3B) and often exhibited dark protrusions (Figure 3A and 3B). Essentially all cells following substrate exhaustion in continuous culture exhibited transition to the L-form cell type. This is in contrast to the sporulation responses observed, in which complete spore formation was never above 10% of the total cells under any of the conditions tested. Figure 2 L-form induction occurs after cellobiose depletion.