Effect of particle size
In order to investigate how the bioprocess was affected by PPW particle size in the nutrient media, grounded PPW was separated into six fractions in size (<0.125, 0.125- 0.250, 0.250-0.500, 0.500-1.000, 1.000-2.000, and 2.000-4.000, all in mm) were tested. Nutrient media were prepared by using these PPW as the main substrate at a loading rate of 2%. In media prepared with a PPW particle size smaller than 0.125, some gelling was observed which resulted in a more viscous media. No such effect was observed for other PPW size ranges. The fermentations were carried out and DP4+, glucose, lactic acid, and ethanol concentrations were determined throughout the runs (Figure 1). Based on these data, sugar recovery, substrate conversion efficiency, produced amounts, average production rates, and yields of both ethanol and lactic acid were calculated and tabulated in Table 1.
R. oryzae utilized PPW as a substrate in all cases studied to produce glucose, DP4+, ethanol, and lactic acid.
It can be clearly seen in Figure 1 that the fungi show a distinct pattern of substrate utilization and consume back the metabolites they produced; all concentrations peak at the early stages of the fermentation and then decrease steadily. The consumption rates can be used as an indicator of the substrate preference order of the microorganism. Figure 1B shows that R. oryzae consumed the initial glucose available in the nutrient media within the first 10 hours, and after this is depleted, it started to degrade the starch available in the PPW, as suggested by an increase in glucose and DP4+ concentrations (Fig. 1A and B), followed by lactic acid and ethanol fermentations (Fig. 1C and D). After the depletion of glucose, lactic acid, and ethanol consumption started and lasted until the end of the runs. Ethanol production was much higher compared to lactic acid production, for all cases.
Regression analysis did not show any meaningful correlation between PPW particle size and the produced amounts, average production rates, and yields of both ethanol and lactic acids (R2< 0.4 for all). However, some significant findings were obtained for the extreme cases. The lag time of substrate utilization for particle size <0.125 (Fig. 1) was the longest, which was probably caused by the gelling observed for this particle size range, which created mass transfer limitations. On the other end of the spectrum tested, for particle size 2-4mm, DP4+ production was significantly higher (Fig. 1A) compared to the other size ranges. The maximum glucose produced (Fig. 1B) was also the lowest for this particle size. This is expected since starch hydrolysis due to amylase activity should yield less glucose and more high molecular weight products (DP4+) as the particle size increases. Sugar recovery percentages given in Table 1 support this claim, they decrease from 72% to 42% as the particle size increases.