Growth was monitored by optical density (OD) at 600 nm and by the rate of base addition. Once the culture reached mid-exponential phase (OD600 = 0.4), the culture Selleck SHP099 was continuously diluted at a rate of 0.1 h-1 with fresh media, while waste media was expelled

from the fermentor to maintain a total volume of 1 L. The culture was maintained at a steady growth rate for 4 residence times, after which the continuous feed was stopped. Cells were sampled and observed under a microscope at different times thereafter to determine changes in morphology. Media samples were also analyzed via HPLC to determine cellobiose, acetic acid, lactic acid, and ethanol concentrations throughout. Viability of cells was determined 24 h after the feed was stopped via plating and determination of CFUs. To ensure culture purity, single colonies obtained from dilution plating were sequenced using 16 S rRNA universal primers 27 F (5’ – AGAGTTTGATCATGGCTCAG – 3’) and 1492R (5’ – GGTTACCTTGTTACGACTT

– 3’). Spore/L-forms determination To determine the number of spores or L-forms present in a culture after exposure to stresses, PD0325901 supplier all cultures were observed microscopically. Spores, L-forms and cells were quantified by manual counts of 5 randomly selected fields. Numbers reported are indicative of the averages of these counts, and the specified error indicates the standard deviation of each biological replicate. Spore purification and storage Phosphatidylinositol diacylglycerol-lyase C. thermocellum 27405 was grown on MTC medium with 5 g/L Avicel for 24 h, and then a 10% transfer was made to MTC medium with 5 g/L cellobiose to generate a population of spores and cells. This culture was harvested after 24 h of growth. Spores were separated from vegetative cells by centrifugation and a modified HistoDenz (Sigma) gradient [41] prepared in a 15 ml conical tube (Fisher). Tubes were prepared with a 1 ml 100% v/v Histodenz gradient on the bottom followed sequentially by 1 ml gradients of 75, 50, and 25% Histodenz. After

1 ml of cell culture was added, each gradient column was centrifuged for 1 hour at 3000xg at room temperature in a Beckman Coulter Allegra 6R centrifuge. Microscopic examination revealed that phase bright spores and terminal endospores settled primarily in the 50% Histodenz fraction. This fraction was isolated and spores were then pelleted at 15,000 rpm for 30 minutes using a Beckman Coulter Avanti T-25 centrifuge. The spore pellet was then resuspended in 50 ml sterile water and allowed to settle overnight. The bottom few milliliters of this suspension were recovered and found to be highly buy TPX-0005 enriched in spores with essentially no vegetative cells observed. Spores were then stored in sterile water at −80°C for later use. L-form purification and storage L-forms were generated using the starvation procedure described above, and quantified microscopically by counting the number of L-forms and cells in 5 randomly selected frames and averaging these quantities.