Results
Figure 4
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In the first shake flask study, addition of the plant hydrolysates produced protein yields of around 100–125 μg/mL (Figure
1). Culture viability using any of the hydrolysates did not extend beyond 12 days, while viabilities remained high with EfficientFeed
A for the 14-day duration of the experiment. Interestingly, the soy and rice hydrolysates maintained viability for an extra
day. This result is consistent with published data where the ability of soy hydrolysates to outperform other plant hydrolysates
has been previously noted.5 The chemically defined feed attained protein yields of greater than 400 μg/mL, a four-fold yield improvement compared to
any of the plant hydrolysates tested.
In the second shake flask study, addition of the plant hydrolysates and the chemically defined feed produced protein yields
from around 175–375 μg/mL (Figure 2). Addition of chemically defined feed and plant hydrolysates together extended cell viability
to 14 days with all plant hydrolysate combinations and resulted in increased protein yields by as much as three-fold when
compared to the performance of plant hydrolysate alone. Soy and rice hydrolysate combinations with the chemically defined
supplement generated the highest yield, while pea and wheat hydrolysate combined with chemically defined feed generated the
lowest bioproductivity. However, when the chemically defined feed was added alone, it achieved higher protein yield than the
feed and plant hydrolysate combinations. The lower yields obtained by adding plant hydrolysates and chemically defined feed
together may be explained by osmolality increases, although this was not investigated, and therefore, cannot be proved in
this study.
In the bioreactor study, after the daily addition of 3% v/v soy hydrolysates, a total cell count of 7 x 106 cells/mL was achieved at day 9, compared to 9–11 x 106 cells/mL with various combinations of defined supplement and feeding strategies (Figures 3 and 4). The addition of soy hydrolysate
resulted in a lower cell growth rate up to day 9, when the soy bioreactor was terminated because of contamination. The growth
rates of cells grown in the presence of 10% and 3% v/v chemically defined feeds were similar and both achieved a maximum cell
count of ~11 x 106 cells at day 9, while those grown in 15% v/v chemically defined feeds were lower, with cells achieving a maximum cell density
of ~9 x 106 at day 9.
Cell density does not necessarily have a direct relationship to protein expression. Cells supplemented with 15% feed A on
day 0 and an addition of 15% feed B on day 6, which achieved the highest cell density, expressed comparable protein production
values to those grown with other feed strategies. Cells grown in the presence of 3% v/v soy hydrolysate compared to the 3%
v/v chemically defined feeds showed slightly less than half the cell density, yet protein production was only around 10% lower
(0.7 mg/mL at day 9 with soy hydrolysate and 0.8 mg/mL at day 9 with chemically defined medium). Lower osmolality for the
soy hydrolysate condition may have contributed to the lower productivity seen. Overall, the best bioproductivity resulted
when using a counter-intuitive feeding strategy—feeding with a relatively large bolus of supplement, both at initiation of
culture and at one more timepoint before the culture lag phase. Therefore, consideration of multiple defined nutrient supplement
formulations is suggested when maximum protein expression is required.
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