Bacterial strains and growth conditions
Thermoanaerobacterium thermosaccharolyticum GSU5 was isolated from animal dung collected in a pasture plain in Buenos Aires, Argentina, in 1987. The strain was originally designatedClostridium thermopapyrolyticum due to its phenotypic characteristics (Mendez, Pettinari, Ivanier, Ramos, & Siñeriz, 1991). Stock cultures were kept at 4°C in Hungate tubes containing growth medium with a strip of filter paper for several decades.Thermoanaerobacterium thermosaccharolyticum DSM 571 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany).
Both strains were grown at 60 ºC in 5 ml screw cap tubes using the Hungate method (Hungate, 1950) in TSC medium (Shaw et al., 2012) containing per liter: 2.0 g sodium citrate tribasic dihydrate, 1.85 g of (NH4)2SO4, 0.1 g FeSO4.7H2O, 2.0 g MgSO4.7H2O, 1.0 g KH2PO4, 0.1 g CaCl2.2H2O, resazurin 2 mg 8.5 g yeast extract, 10 g glucose. The pH was adjusted to 6.7 with NaOH 10 M.
For metabolite production analysis, the strains were grown in TSC medium supplemented with 10 g/L of different carbon sources: monosaccharides xylose, arabinose, glucose, galactose and fructose; and disaccharides sucrose and cellobiose. The pH was adjusted to 6.7 with NaOH 10 M. The strains were grown at 60ºC for 48 hours.
Fermentations were performed in a 2.5 L BiostatA bioreactor with 1.5 L of TSC supplemented with 10g/L of carbon sources (glucose or xylose). After 17 hs 5g/L of the corresponding sugar were added to avoid carbon source depletion. The agitation was kept at 100 rpm. The pH was adjusted initially at 7 and was regulated with NaOH 2M and H2SO4 1M to keep it over 5. TSC pre-cultures were grown in serum bottles containing 50 ml of medium at anaerobic conditions, 150 ml of inoculum was added to the bioreactor (DO= 0,3). The strains were grown at 60ºC for 35 hours.