2.2 Microorganism, media and fermentation conditions
A wild-type strain of Clostridium pasteurianum which was previously isolated from an active sludge sample and proven to have significantly low butanol production by our group (Kaeding et al., 2015 ) was further purified by isolating a single colony from a cyro stock solution maintained at -80°C for several years. We checked the identity of the purified strain using 16S rDNA analysis again, and the analysis using NCBI BLAST showed that it has a high similarity (> 99%) to the C. pasteurianum type strain DSM 525 (ATCC 6013). This purified strain termed as C. pasteurianum C8 was used for the fermentations and adaptive evolution in this study and restored at -80°C in 2-mL cryo vials containing 75% active culture and 25% glycerol (v/v).
Seed cultures for fermentation experiments were prepared in anaerobic bottles as previously described by Kaeding et al . (Kaeding et al., 2015 ). The medium for all pre-cultivations consisted of (per liter of water): 40 g pure / crude glycerol; 3 g (NH4)2SO4; 0.75 g KCl; 2.45 g NaH2PO4; 4.58 g Na2HPO4; 0.28 g Na2SO4; 0.42 g citric acid; 0.2 g L-cysteine; 0.024 mg biotin; 0.015 mg calcium pantothenate; 5 mg FeSO4·7H2O; 0.26 g MgCl2·6H2O; 3 mg CaCl2·2H2O; 2 g/L CaCO3; 2 mL trace element solution (70 mg/L ZnCl2; 100 mg/L MnCl2·4H2O; 200 mg/L CoCl2·6H2O; 20 mg/L CuCl2·2H2O; 35 mg/L Na2MoO4·2H2O; 25 mg/L NiCl2·6H2O; 60 mg/L H3BO3; 0.9 mL/L 37% HCl). After a two-step cultivation procedure (first overnight and second 12 h at 35°C), 100 mL of seed culture with an OD600 value over 4.0 were inoculated in a 2-L glass bioreactor (Eppendorf, Germany) filled with 1 L unsterilized fermentation medium to initiate the batch or fed-batch fermentations. The compositions of the fermentation medium was (per liter of water): 30-120 g pure / crude glycerol; 3 g (NH4)2SO4; 0.5 g KH2PO4; 0.5 g K2HPO4; 0.42g citric acid; 0.26 g MgSO4·7H2O; 0.024 mg biotin; 0.015 mg calcium pantothenate; 20 mg CaCl2·2H2O; 0.5 g cysteine hydrochloride·H2O; 1-100 mg FeSO4·7H2O; 2 g/L CaCO3; 2 mL trace element solution. It is worth mentioning that yeast extract was used neither in the seed culture medium nor in the fermentation medium.
Unless otherwise stated, fermentation experiments were conducted at 35°C with agitation at 200 rpm, and pH was controlled at 6.5 by automatic adjustment with 25% ammonia solution. To achieve anaerobic conditions, the medium was only degassed with 0.6 vvm N2 for 30 min before the inoculation, and no sparging of N2 was needed throughout the process after inoculation. The feeding to the fed-batch fermentations was done with a feeding solution containing 50% pure or crude glycerol depending on the experimental purpose. In fed-batch fermentations, continuous feeding was initiated when the residue glycerol concentration was lower than 20 g/L, and the feeding rate was adjusted to maintain the glycerol concentration within the range of 10-20 g/L. In pulsed fed-batch fermentation, 80 g of 50% glycerol solution was added into the bioreactor 3-4 times, when the residue glycerol concentration was lower than 20 g/L.
Continuously fed-batch fermentation of crude glycerol using the same fermentation conditions described above was also carried out in a 1 m3 stainless steel bioreactor (Frings, Germany). The initial volume of the fermentation medium containing 80 g/L crude glycerol was 500 L. The process consisted of three seed cultures in anaerobic bottles and two more in 10 L and 100 L fermenters. Finally, 50 L seed culture with an OD reached 6 was inoculated in the 1 m3 fermenter to initiate the fed-batch fermentation.