Metabolic Regulation Of Growth And Ubiquinone Biosynthesis Of Suspension Roselle Cell

In this dissertation, a Roselle cell line was chosen as experimental material, which was induced by Bioengineering Lab of South China University of Technology several years ago. Through changing the ingredients and ratio of Gamborg B5 basal medium, and adding various types of substance, the effects of these changes on the cell growth and ubiquinone accumulation of Roselle cell were studied systematically. Under the different beginning conditions, not only the assimilation power of suspension Roselle to the main nutrients, but also the physical-chemical characteristics, were studied during the whole culturing time.On the regulation of cell growth of suspension Roselle cell, the results showed that the soluble starch could not be used by Roselle cell, but the glucose and sucrose could, further more the effect of sucrose was a little better than that of glucose. The 4% sucrose was the best to support growth of Roselle cell, and got the highest specific growth ratio (0.2994d^-1). The sucrose concentration lower than 2% showed substrate limiting phenomenon. The sucrose concentration above 5% could inhibit the growth of Roselle cell. In B₅ medium, the nitrogen source was provided by NK₄⁺ and NO₃^-, the Roselle cell could endure NO₃^- far more than NH₄⁺, and the toxic effect of NH₄⁺ to culturing cell could be eased some extent by adding NO₃^- but the results also showed that high NO₃^- with low NH₄⁺ was better than pure high NO₃^- to the growth of Roselle cell. The phosphate and calcium were the essential inorganic nutrients. The 1mg/L 2,4-D was the best to the growth of Roselle cell, the inhibition of 2,4-D to the growth of Roselle cell was obviously seen with higher 2,4-D concentration;the growth of Roselle cell was insensitive to KT. The light quality also had some effect on the growth of Roselle cell, purple and white light were bad to the growth of Roselle cell, but the blue light was the best. The inoculum amount had very obvious effect on the growth of Roselle cell, the bigger the inoculum was, the faster the cell began to grow and the shorter the time of highest biomass was reached.In this dissertation, the assimilation power of suspension Roselle cell to the main nutrients in the medium was also studied under the different starting conditions. The carbon sources which could be used by Roselle cell, such as sucrose and glucose,could give a reasonable biomass, and the other nutrients in the medium could also be absorbed quickly;but the carbon source which could not be used by Roselle cell, such as soluble starch, the results just reversed to the above results. In the medium which used soluble starch as carbon source, the rate of respiration of Roselle cell maintained at low level during the whole culturing time. In the medium which used sucrose or glucose as carbon source, the rate of respiration increased markedly after inoculum, and then decreased slowly to a maintaining respiration level. The 3% or 4% sucrose concentration was enough to support the growth of Roselle cell. If the sucrose concentration was too low (<2%) or too high (>5%), the substrate limiting or inhibiting was showed to the growth of Roselle cell, and the other nutrients assimilation were also slowed. The relationship between biomass and medium peroxidase activity or biomass and medium protein concentration was studied, through comparison we found that biomass and medium protein concentration had a higher linear relationship than biomass and peroxidase activity, and could be used to estimate biomass through medium protein concentration during exponential phase. The reason of pH changing during the culturing process was discussed, and we verified again at the cell culturing level that nitrate reductase was a strict substrate-inducing enzyme. Combination the known theories and the analysis of experimental results, the author proposed a new hypothesis to explain why NH4+ was not a good nitrogen source to plant cell.The effects of different physical-chemical conditions, including plant growth regulator, organic and inorganic nutrients, environmental conditions, precursors and sterol inhibitors, on the regulation of ubiquinone of suspension Roselle cell were studied. The results showed that ubiquinone was a primary metabolites which was stringently controlled by endogenous factors. Exogenous factors were difficult to influence the accumulation of ubiquinone. We got this conclusion from the results that various organic and inorganic nutrients, environmental conditions all had no obvious promotion to the accumulation of ubiquinone. The adding of precursors also did not show obvious effects by the restriction of penetration or enough endogenous provision. We only found that two substance (2,4-D and miconazole) had a good effect on the promotion of Ubiquinone.Through the above analysis to cell growth, substrate assimilation and productsaccumulation, a non-structural kinetic model of suspension Roselle cell was constructed as follows:Growth model:C.+15.5 l + C/65.6Sugar usage: qs =-------u +0.008 +-------qpe B s 0.556H 0.602 PProduct formation: q p = 0.009|i + 0.0185The calculated results coincided well with experimental one. This showed that this model was right, and could be used to predict the culturing process.