The results of this study revealed that the pH of the medium was influenced by concentrations of sucrose. Actually, medium containing the 3% sucrose showed the highest pH at the end of the growth period while the lowest pH was observed in medium containing 6% sucrose (Figure 3). On the other hand, in the treatment of gradual sucrose increment, the pH was stand in the middle as compared to the 3 and 6% sucrose.
As shown in Figure 4, the concentration of MES also had a significant effect on the medium pH. In the medium with the highest MES content (5 mM), the lowest pH was observed while, no significant differences in pH were recorded between the medium containing 2.5 mM of MES and the control treatment (no MES).pH variation in medium containing different levels of sucrose during the growth period (6 weeks) has been presented in figure 5. In all media, pH during the first week was sharply decreased from 5.8 to 4.37, 4.47 and 4.6 in the medium enriched with 6%, gradually 6% and 3 %sucrose respectively. During the second week, a slight increase in pH was observed in all three media, although the pH increment in medium with 3% sucrose was not significant, in others it raised up significantly. In the third week, the pH was constant in all three media and no change was observed. In the fourth week, it was experienced a decrease in pH in all three culture media. Although the pH decrement in medium with 3% and 6% (gradual) was significant, but in the medium containing 6% sucrose, the decrement was not significant. In the fifth week, pH in all three treatments was sharply increased. The highest and the lowest pH was observed in the medium with 3% and 6% sucrose. However, it should be noted that at the end of the period, although, no significant difference was observed between the pH in 6% and 6% (gradual) sucrose, the pH in the medium containing 3% sucrose was significantly higher than the others.
The trend of pH changes under the influence of different MES concentrations was similar to those observed when it was influenced by the different levels of sucrose (Figure 6). In fact, in the first week, although a sharp decrement was observed in the pH, there was no significant difference between the MES treated and non-treated one. During the second week, an arising trend was observed in both concentrations of MES, which are significantly different from the previous week, but in the control, it was non-significant. Indeed, at the end of this period, no significant difference was observed between MES treated and non-treated samples. Although, the trend of pH increment was continued during the third week, it was not significant compared to the previous week. In the fourth week, a decrement in pH was observed in all treatments, so that, it was significant for MES treated and non-treated samples. However, it should be noted that at the end of fourth week, no significant differences was seen among all treatments. Finally, in the fifth week, the pH in all three treatments increased significantly compared to the previous week. Although no significant difference was observed between control (0 mM) and 2.5 mM MES, the medium containing 5 mM MES significantly showed lower pH than others.
As shown in Figure 7, cell growth index had the highest growth during the first week, from 0 to 0.39, while during the second week a dramatically growth was observed, so that, the growth index increased only 0.07. Since that till the end of sixth week, an exponential cell growth was seen and it reached to 1.28.
The interaction of different concentrations of MES and sucrose was found to be statistically significant on saffron cell growth index which has been presented in Figure 8. Results revealed that the highest growth index was happened in the medium contained 2.5 mM MES and 6% sucrose (gradual). Although this treatment does not show significant difference to the control (3% sucrose and no MES), there is a big difference compared to the others treatments. Results showed that in the sucrose content of 6%, though by increment of MES from 0 to 2.5 the cell growth was non-significant, by doubling the concentration of MES (5 mM) the cell growth significantly decreased. Similar trend in cell growth was also observed for 6% sucrose (gradual) except the cell growth significantly raised up with changing MES from 0 to 2.5 mM and then dropped down when the levels of MES increased up to 5 mM. In general, except for 3% sucrose, in the other sucrose contents (6% and 6& (gradual)), MES at 2.5 mM caused the acceleration of cell growth index, but at 5 mM, it had as a limited growth role.
Cell count in the cell suspension cultures showed that the highest number of viable cells was observed in treatment containing 6% sucrose (gradual) supplemented with 2.5 mM MES (Figure 9). In the medium containing 6% sucrose, both with and without MES, the number of cells was significantly reduced. In the medium with 3% sucrose, there was no significant difference with 6% sucrose in terms of cell number, either in the presence (2.5 mM MES) or in the absence of MES. The number of live cells increased up in the medium with 3% sucrose, while it decreased in medium containing 6% sucrose (gradual), when the MES level rose from 2.5 to 5 mM.
The spectrophotometry recorded data at 440 nm, related to the interaction of sucrose concentration and sampling time on crocin percentage are presented in Figure 10. Crocin levels were assessed at the end of the fourth and sixth week after the culture time. As shown in this figure, the cell sampling time has a significant effect on the crocin content of the extract. After the five weeks, the lowest and the highest amount of crocin was observed in the medium containing 3% and 6% sucrose (gradual) with 0.47 and 0.8% respectively (p≤0.05).
Based on the data recorded at the end of fourth and sixth weeks, the interaction effect between MES and sucrose concentrations on crocin production showed that, the highest amount of crocin was observed in medium containing 6% sucrose (gradual) and 2.5 mM MES after five weeks (p≤0.05). It was also revealed that in this medium, no significant difference was occurred in crocin content when the MES level increased up to 5 mM (Figure 11).
Investigation on cell biomass and crocin production in a stirred bioreactor
The results of this part of the study showed that the constant pH (adjusting pH by adding HCl and NaOH via peristaltic pumps which were automatically under the control of the pH sensing electrode during the growth period, the pH of the medium was kept constant at 5.8) with or without aeration severely reduced the cell growth (Figure 12).
The highest cell growth was observed when the pH variation was happened naturally during the culture period with no effort to keep it constant. Results also revealed that the constant pH and aeration simultaneously led to a decrease the cell biomass in the bioreactor. Consequently, it was also caused a negative effect on crocin contents of saffron cells extract. As shown in figure 12, although the non-constant pH of medium resulted higher crocin content as compared to the constant pH, it was significantly lower to those observed in medium without aeration. On the other hand it was also found that only the extracted cells from cultures with natural pH (non-constant) contained crocin.
The HPLC profiles of the extraction of cell sample were presented in Fig. 13. It showed that the cell extraction sample contains 2 analogues of crocin. The total amount of crocin in cells based on the HPLC results at 440 nm, and crocin standard curve was found to be 2 mg per gram of cell dry weight.
As the growth period of plant cells in suspension cultures takes several weeks, generally a significant volume of medium is losing due to evaporation particularly by aeration in bioreactor system. Accordingly, it the second experiment of the bioreactor, the effect of aeration and volume of medium was addressed. The results of this part of the experiment showed that adjustment of medium volume by adding water led to increased cell growth. Based on this result, it can be concluded that aeration, itself, is not a reason for reduction cell growth and cell browning as happened in the previous experiment. Therefore, increasing the concentration of elements in the medium caused by evaporation can be the reason for cell browning and reducing cell mass growth. Hence, in the aeration system, if the volume of medium can be kept constant during the culture, aeration can have a positive effect on cell growth. The results of this section are presented in Figure 14.