pasteurianum C8
To evaluate the PDO production capacity of C. pasteurianum C8, fed-batch fermentation using pure glycerol as substrate was first carried out with pulse feeding strategy. As shown in Fig.2A and Table1 , no lag phase of cell growth was observed after inoculation for the fermentation. A maximum specific cell growth rate (umax) of 0.52 ± 0.04 h-1was reached, and glycerol consumption and PDO production increased rapidly along with the fast accumulation of biomass. The concentration of glycerol decreased from initially 80 g/L to less than 20 g/L within 7 h of fermentation, and then the feeding of 50% glycerol was initiated. The maximum glycerol consumption rate of 19.18 g/L/h and PDO production rate of 10.44 g/L/h both occurred in this feeding period. The final PDO concentration and overall productivity reached 74.65 g/L and 5.33 g/L/h with a high PDO yield of 0.54 gPDO/gGly. Furthermore, no butanol was produced during the whole process of pure glycerol fed-batch fermentation, which indicates a high metabolic selectivity and a high production efficiency of C. pasteurianumC8 for PDO.
Compared to pure glycerol, crude glycerol from biodiesel production has a better potential to serve as a cheap and abundant carbon source for a cost-effective bio-production of PDO. However, a lot of studies have showed that the impurities such as free fatty acids in crude glycerol may inhibit the growth of Clostridium species (Venkataramanan et al., 2012 ;Samul et al., 2014 ;Tan et al., 2018 ). In order to evaluate the possibility of using crude glycerol in the fed-batch fermentation for PDO production, an initial tolerance test for C. pasteurianumC8 was conducted. It can be seen that the growth of the strain decreased by more than 50% as the concentration of crude glycerol exceeded 60 g/L (Fig. 3 ). After evaluating the tolerance, a crude glycerol concentration of 40 g/L, where the strain can still maintain 50% growth in the tolerance test, was set as the initial glycerol concentration in the fed-batch fermentation using the same three-pulse feeding strategy. As shown in Fig.2B and Table1 , although no lag phase of growth was observed after inoculating the strain into the crude glycerol containing medium, the umax (0.41 ± 0.01 h-1), maximum glycerol consumption (12.13 g/L/h) and PDO production rate (6.68 g/L/h) were much lower than those achieved with pure glycerol. Finally, PDO production stopped at 58.36 g/L with significantly lower overall productivity and yield of 3.43 g/L/h and 0.46 gPDO/gGly, respectively. To our surprise, 1.75 g/L butanol was produced at the end of the fed-batch fermentation. It is possible that high PDO concentration and the accumulation of crude glycerol impurities together evoked a stress environment, forcing the strain to produce butanol as a stress release for cell maintenance. Moreover, the butyrate production was also much higher (13.66 g/L with crude glycerol vs. 4.58 g/L with pure glycerol) in the crude glycerol fermentation. It is well known that acetic acid production can provide more NADH and leads to the theoretical maximum PDO yield of 0.72 mol/mol glycerol, while the butyric acid production can provide more ATP for cell growth and maintenance (Zeng, 1996 ). When the strain was in a highly stressful condition, higher butyrate production was more preferable for the strain to produce more energy for supporting its survival, which also led to more carbon loss in the oxidation pathway and thus reduced PDO yield from glycerol. Therefore, apart from the specific growth rate and the butanol production, the acetate / butyrate ratio can be also considered as an indicator for evaluating the tolerance of C. pasteurianum to crude glycerol.
In order to further avoid toxicity from crude glycerol, a lower initial glycerol concentration of 30 g/L, and a continuous feeding of 50% crude glycerol with a feed rate of 15 g/L/h were used in the fed-bath fermentation. The most significant improvement observed was a reduced production of butyrate (7.34 g/L) and butanol (0.56 g/L), which led to a sharp increase of PDO yield from 0.46 to 0.54 gPDO/gGly by 17%. At the same time, the final PDO titer increased from 58.36 to 62.78 g/L (Fig.2C and Table1 ). In addition, the acetate / butyrate ratio also increased from 0.82 to 1.69, indicating a reduced stress ofC. pasteurianum C8 in the continuous fed-batch fermentation. Although the strategy of low initial glycerol concentration with low feeding rate can effectively avoid instant exposure of the cells to a large amount of crude glycerol, which reduced the stress response of the strain, and as a result improved the PDO yield significantly, the PDO productivity was greatly reduced owing to the longer time of feeding. The maximum and overall PDO production rate were only 7.54 and 2.59 g/L/h in the continuous fed-batch fermentation of crude glycerol, which were 28% and 51% lower than those in the pulse fed-batch fermentation of pure glycerol, respectively. The results from pure and crude glycerol fed-batch fermentations indicate that C. pasteurianum C8 has potential as an efficient PDO producer, but its tolerance to crude glycerol should be improved in order to fully exploit the production capacity of this strain.