A closely related study used 500 ml batch cultures controlled at
pH between 4.5 and 7.5 using 50 g/L pure glycerol as substrate and
a similar media formulation otherwise as the current study
(6.54 mM phosphate) (Biebl, 2001). The study reported finding
great variation in product formation, however no correlation to
pH was found accept for ethanol. Cell growth was found to be more
or less equal over the pH range applied, in contrast to the data
reported in this study. In general there was great variability in product
formation under equal or slightly different conditions, a cause
that was hypothesized to be due to weak pathway regulation and/
or multiplicity effects. However, it is likely that the use of 50 g/L
glycerol could have led to multifactorial effects involving substrate
and/or product inhibition, masking the single variable effect of pH.
The pH has been shown elsewhere to effect cell growth in continuous
1 L cultures of C. pasteurianum using glucose as substrate
(40 g/L) over a pH range from 4.8 to 7.0 (Dabrock et al., 1992). It
was reported that for pH below 5.5, growth and substrate utilization
decreased and at pH below 4.8 steady state conditions could
not be obtained. Using a phosphate-limited (0.5 mM) minimal
medium it was concluded that there was almost no effect of pH
on solvent production (ethanol and butanol). Unfortunately, data
on PDO production was not provided. However, the different substrates
make a meaningful comparison difficult. Variation between
PDO and butanol production from crude glycerol have been
reported elsewhere (Gallardo et al., 2014), however the pH was
not controlled and the amount of crude glycerol, including the
associated impurities, was varied instead. Studies investigating
the initial pH (no control after inoculation) with pure glycerol
show increased butanol yields with lowering pH by C. pasteurianum
in 50 ml batch cultures (Ahn et al., 2011), in good agreement
with the results reported here. Similarly an optimization study on
Fig. 4. Butanol and PDO formation during glycerol fermentation by C. pasteurianum
at varying controlled pH values. The symbols (open circles) represent off-line
measurements via HPLC, while the solid lines (first order splines) are added for
visual clarity only.
Fig. 5. Butanol and PDO production rates during glycerol fermentation by
C. pasteurianum at varying controlled pH values. Rates were determined via
numerical differentiation of the data shown in Fig 4.