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.