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.