It was previously isolated from ruminal fluid and put in the collection of the Department of Biotechnology and mTOR inhibitor Food Microbiology, Poznan University of Life Sciences, Poland, as wall as deposited

at the Polish Collection of Microorganisms (PCM). Culture medium The strain was maintained in Reinforced Clostridial Medium (RCM, Oxoid, UK) in serum bottles at 4°C. Pre-cultures of pure culture inoculum were cultivated in Hungate test tubes in an appropriate cultivation medium (37°C, 18 h). Clostridium bacteria were cultured in a chamber for cultivation of anaerobic microorganisms (Whitley MG500, Don Whitley Scientific, Shipley, United Kingdom), without pH regulation or stirring. Fermentation medium The composition of the fermentation medium was (per liter of deionized water): 0.26 g K2HPO4; 0.02 g KH2PO4; 1.23 g (NH4)2SO4; 0.1 g MgSO4 × 7H2O; 0.01 g CaCl2 × 2H2O; 0.01 g FeCl2 × 7H2O and 2.0 g yeast extract. The fermentation medium was supplemented with crude glycerol (Wratislavia-Bio, Wroclaw, Poland) at a concentration of 70.0 ± 1.0 g/L in batch fermentation, and 50 g/L ± 1.0 g/L in fed-batch fermentation. The crude glycerol composition was (w/w) 85.6% glycerol, 6% NaCl, 11.2% moisture, and pH 6.5. The media were autoclaved (121°C, 20 min.).

Fermentation experiments The batch experiments were performed at three reactor scales, 6.6 L, 42 L (Sartorius Stedim, Germany) and 150 L (BIOFLO III, New HMPL-504 Brunswick Sci. Edison, N.J., USA). All bioreactors were equipped with controls for temperature, pH, agitation PLX3397 mw speed and aeration rate. The pH was controlled at 7.0 by automatic addition of 1 M NaOH and all fermentation experiments were carried out at 37°C. In the 6.6 L and 42 L bioreactors the anaerobic conditions were sustained by continuous nitrogen sparging at a flow rate of 0.1 vvm whereas in the 150 L bioreactor the medium was sparged with N2 for 3 h before and for 1 h after inoculation.

As the fermentation process progressed, the medium was sparged Molecular motor with N2 for 30 min. once every 24 h. All the bioreactors were inoculated with 10% (v/v) of the pre-inoculum cultures. The fed-batch experiments were performed at two reactor scales, in 6.6 L and 150 L fermenters. The fermentation was carried out at 5% of the initial glycerol concentration. The major dimensions of the bioreactors used in this study are presented in Table 1. The following equations were used to calculate the main fermentations parameters: Table 1 Stirred-tank reactor characteristics Dimension/operating condition Scale Nominal volume, V (L) 6.6 42 150 Working volume, VL (m3) 0.005 0.030 0.120 Impeller tip speed, TS (m/s) 0.20096 0.20096 0.20096 Agitation speed, N (rmp) 60.00 36.57 26.50 Number of impeller 2 3 3 Impeller type Rushton Rushton Rushton Liquid height, HL (m) 0.25 0.46 0.72 Impeller diameter, DI (m) 0.064 0.105 0.150 Reactor diameter, DT (m) 0.16 0.29 0.45 Reactor hight, HT (m) 0.34 0.63 0.98 HT/DT 2.12 2.17 2.18 DI/DT 0.40 0.36 0.