The growth of S. cerevisiae free cells occurred slightly faster than for immobilized cells, however, there was little difference between numbers of free cells and immobilized cells with amino acid supplementation at 60 h. Previously, a decrease in viability and proliferation of immobilized cells was reported [359], and our data here agrees (Figure 6.1), but also shows this problem can be mitigated by amino acid supplementation, where ca.
90% of these immobilized cells were still viable in VHG conditions.
The glucose consumption, the formation of ethanol and glycerol are shown in Figure 6.2 and 6.3, with a number of kinetic parameters summarised in Table 6.1. During the early
stage of immobilized cell fermentation (both with or without amino acid supplementation), glucose fluctuation occurred in the broths, while this phenomenon was not observed for the free suspension process (see inset in Figure 6.2 for detail). A decrease in glucose concentration in the broths appeared within first two hours. This phenomenon can be explained by the diffusion of external glucose from the broths into the gel matrix when the beads were first transferred to the media. Cells need to acclimatise to the new media, therefore, the glucose consumption by immobilized cells during this period was insignificant. After this period (after the 2nd hour), when cells began to increase their consumption of glucose, we hypothesise that the CO2 generated during fermentation began to accumulate in the capillary gel matrix, resulting in significant gas in the beads [360], forcing media (and glucose) back out. From Figure 6.2, we can see that this process occurred within the first six hours of fermentation, thus we can divide the curves in Figure 6.2 into two states, the first stage being unstable (about 6 hours), and the second state being the stable stage. The stable stage commenced when there was a balance between the diffusion of CO2, ethanol (from cells to gel beads, and then to the broths), and glucose (from the broths to the gel beads, and then cells).
Time (h)
0 12 24 36 48 60
Numbers of cells/fermentation (x10-9 cells/100 mL of broth)
0 2 4 6 8 10 12
14 Non-viable cells Free cells Immobilized cells
Immobilized cells in media with an amino acids supplement
Figure 6.1. Numbers of cells during fermentations. All experiments were performed in triplicate.
Figure 6.2. Residual glucose concentrations in the broths with free cells (U) and immobilized cells without (c) or with ( ) an amino acid supplementation. Experiments were performed in triplicate. Figure inset illustrates the fluctuation of the glucose concentration in the broths during the early hours of fermentation.
0 20 40 60 80 100 120
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 Time (h)
Ethanol concentration (g/L)
(A)
0 2 4 6 8 10 12 14
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 Time (h)
Glycerol concentration (g/L)
(B)
Figure 6.3. (A) Concentrations of ethanol (open symbols), and (B) glycerol (filled symbols) in the broths with free cells (U) and immobilized cells under conditions of without (c) or with ( ) an amino acid supplementation. Experiments were performed in triplicate.
0 50 100 150 200 250 300
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96
Time (h)
Glucose concentration (g/L)
260 280 300
0 2 4 6
Table 6.1. The fermentation kinetic parameters using free and immobilized cells with various media.
Free cells Immobilized cells Parameters
VHG VHG VHG + amino acids Initial glucose concentration, (g/L) 300 ± 5.6 300 ± 6.3 300 ± 6.2
Fermentation time, (h) 102 84 72
Residual glucose concentration, (g/L) 86.3 ± 9.45 63.7 ± 12.2 45.9 ± 10.1 Final biomass concentration, (g/L) 4.7 ± 0.3 3.9 ± 0.5 4.8 ± 0.6 Final ethanol concentration, (g/L) 90.2 ± 4.6 104.6 ± 5.1 118.3 ± 5.8 Ethanol yield, (g/g) 0.30 ± 0.01 0.35 ± 0.01 0.40 ± 0.01 Final glycerol concentration, (g/L) 11.3 ± 0.6 12.0 ± 0.8 12.5 ± 0.9 Glycerol yield, (g/g) 0.038 ± 0.003 0.040 ± 0.004 0.042 ± 0.005 Max specific glucose uptake rate (g/g
dry weight.h) 1.76 ± 0.13 2.04 ±0.16 2.21 ± 0.15 Max specific ethanol production rate
(g/g dry weight.h)
0.61 ± 0.03 0.73 ± 0.04 0.98 ± 0.06 Max specific glycerol production rate
(g/g dry weight.h) 0.14 ± 0.08 0.12 ± 0.07 0.15 ± 0.04
Table 6.1 shows an increased generation of ethanol from glucose for immobilized cells and then for immobilized cells with amino acid supplementation of 0.35 g ethanol/g glucose, and 0.40 g ethanol/g glucose compared to 0.30 g ethanol/g glucose obtained with free cells.
A significant change in maximum specific ethanol production rate was observed in immobilized cells (0.73 g/g dry weight.h) compared to free cells (0.61 g/g dry weight.h).
This increase is even more dramatic for immobilized cells with amino acid supplementation, where a maximum specific ethanol production rate of 0.98 g/g dry weight.h was observed. Moreover, in the case of immobilized cells with amino acid supplementation, a significantly enhanced maximum glucose turnover of 2.21 g/g.h was seen in comparison to free cells with 1.76 g/g.h. Concomitant with these kinetic data, a stimulation of glycolysis proteins was recorded. Compared with free cells, immobilized cells showed significant changes in proteome relative abundances (see Appendix F), as described below. This phenomenon was similar for immobilized cells grown in different conditions. As seen in Figure 6.3.A, the detection of ethanol starts after 12 hours and
finishes at the 72nd hour for immobilized cells with amino acid supplementation, at the 84th hour for un-supplemented immobilized cells, and at the 96th hour for free cells.
To estimate the affect of substrates on the reaction rates of immobilized cells and free cells, the data in the 2nd stage (Figure 6.2) were used and plotted in Figure 6.4. When the residual glucose concentration in the media remained high, the bioconversion rate was independent of glucose concentration, and the reaction rates fluctuated slightly. In this period, the activity of immobilized yeast depended not only on the activity of enzymes in the cell, but also on the gel matrix, since the diffusion of substrates and products relied on the size of gel matrix.
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
0 20 40 60 80 100 120 140 160 180 200
Glucose concentration (g/L)
Reaction rate (g/L.h)
Figure 6.4. Dependency of reaction rates on glucose concentration. (U) free cells, (c) and ( ) are immobilized cells without, or with an amino acids supplement respectively.
Even though fermentation is performed by a multi-enzyme system, the kinetics of the whole fermentation process are limited by the activity of particular key enzymes in the reaction chain [360]. Therefore, relatively simple enzyme kinetics can be used to illustrate the whole process [360]. To describe the kinetics quantitatively, the slope of log [v/(V-v)]
against log[glucose] (known as a Hill plot) [361] was used. Subsequently, the slope, known
as the Hill coefficient (h), was calculated using the linear regression function in Microsoft Excel (Figure 6.5). From Figure 6.5, the Hill coefficients were 1.87, 1.96, and 2.03 for free cells, immobilized cells without, and with amino acid supplementation. The effect of immobilization as well as amino acid supplementation on the kinetic behaviour is clear.
The consumption rate of glucose was enhanced by immobilization, and a further increase was obtained by the addition of an amino acid supplementation, where h rose from 1.96 to 2.03. These results show that ethanol fermentation was enhanced by using immobilized cells with amino acid supplementation.
y = 1.8714x - 3.422 R2 = 0.9887 y = 2.0278x - 2.7807
R2 = 0.9698
y = 1.9615x - 2.8865 R2 = 0.9722
-0.2 0.2 0.6 1.0 1.4 1.8
1.6 1.7 1.8 1.9 2.0 2.1
Log[Glucose]
Log(v/(V-v))
Figure 6.5. A Hill plot representing the activity of a particular key enzyme in free cells and immobilized cells. (U) represents free cells, while (c) and ( ) represent immobilized cells without or with an amino acid supplementation respectively.
For immobilized cells with amino acid supplementation, the fermentation time was reduced by 12 hours to 72, with a corresponding increase in specific glucose uptake rate (see Table 6.1). Amino acid supplementation appears to aid cell viability, thus enhancing ethanol production productivity. The metabolism of immobilized cells was enhanced, since an up- regulation of most glycolysis proteins compared to free cells was observed at the proteomic level (see Appendix F, and sections below).