Effect of initial loading rate
Based on the results obtained in the first part, two particle size
ranges, one small and one large were selected for the loading rate
study. Here, the gelling effect observed in <0.125 particle
size range become even worse and rendered the media highly viscous for
higher PPW loading rates, so it was discarded as an option and the next
size range (0.125-0.250 mm) was chosen as the small size. For the large
size, 1-2 mm range was chosen considering the unusual DP4+ production
observed for the larger size.
A higher PPW loading rate means more PPW and consequently, more starch
and carbon sources are available for R. oryzae to produce lactic
acid and ethanol. Therefore, concentrations of lactic acid and ethanol
are expected to be dependent on the PPW loading rate. In order to test
this, in addition to 2% PPW loading rate used in the previous section,
two more rates (4 and 8%) were tested on small (0.125-0.250 mm) and
large (1-2mm) PPW sizes. Due to high viscosity, PPW suspensions with a
loading rate above 8% (w/v) could not be appropriately mixed and were
therefore not investigated.
Time-dependent concentration profiles of measured compounds (DP4+,
glucose, lactic acid, ethanol) obtained under different PPW loading
rates (2, 4, 8%) for both small and large particle sizes are given in
Figure 2. Calculated bioprocess performance data (sugar recovery,
substrate conversion efficiency, maximum amounts produced, average
production rates, yields) were tabulated in Table 2.
Figure 2 shows that DP4+ and free glucose concentrations were dependent
on the PPW loading rate applied (Fig. 2A and B), a higher loading rate
resulted in more DP4+ and glucose for both small and large particle
sizes. Lag time for production was not affected by the loading rate for
DP4+, however, it was considerably increased for the glucose as the
loading rate increased.
It is also obvious from Figure 2 (C and D) that the PPW loading rate
significantly affected ethanol and lactic acid production. Increasing
PPW loading rates clearly increased the produced amounts, average
production rates, and yields of both ethanol and lactic acids, for both
small and large particle sizes (Table 2). Table 2 also shows that sugar
recovery was also dependent on the loading rate; it decreased with the
increasing loading rate, for both particle sizes tested.
Moreover, regression analysis revealed that produced lactic acid and
ethanol amounts increased linearly with increasing the PPW loading rate
(R2=0.98 (lactic acid small size),
R2=0.97 (lactic acid large size),
R2=0.99 (ethanol small size),
R2=0.99 (ethanol large size)). This is expected, as
the high loading rate means more substrate available to the cells which
in turn should result in more product obtained. Similarly, increasing
the PPW loading rate increased ethanol and lactic acid production rates.
Again, the correlation can be described with a linear regression with an
offset (R2=0.99 (lactic acid small size),
R2=0.93 (lactic acid large size),
R2=0.99 (ethanol small size),
R2=0.98 (ethanol large size)). This is also expected,
as the high loading rate may alleviate any substrate limiting step on
the bioprocess rate. On the other hand, lactic acid and ethanol yields
do not seem to depend on the PPW loading rate, with regression analysis
giving mixed results and poor fits. By definition, the yield is based on
amount produced per substrate, so increasing substrate does not
necessarily increase product conversion efficiency.
Lastly, it can be deduced from Figures 2C and D that higher PPW loading
rates caused slightly longer lag times for both ethanol and lactic acid
production.