Studies On Kinetics Of Cell Growth And Polysaccharide Synthesis In Suspension Cultures Of Protocorm-like Bodies From Dendrobium Huoshanense

Dendrobium huoshanense, a precious wild medicinal plant in China, distributes in Huoshan County, Anhui Province. Polysaccharides isolated from Dendrobium can inhibit the growth of tumor cells and possess immunological activity. Because of a deficiency in the wild source of Dendrobium huoshanense and excessive collection, it has been on the verge of extinction. Tissue culture techniques have been used for the in vitro mass multiplication of Dendrobium huoshanense, but the culture system has not been shown to be more efficient. Protocorm-like bodies (PLBs)are actually somatic embryos that can be induced from the explants of Dendrobium and produce some active substances. A new approach has been considered as a feasible alternative for the production of active polysaccharides using protocorm-like body cultures of Dendrobium huoshanense. A structural kinetic model was developed to describe the cultivation process. Furthermore, the impact of phytic acid and polyamines on cell growth and polysaccharide synthesis, the regulation of cell growth and polysaccharide synthesis by two-stage cultivation and primary scale-up of PLB suspension culture in 10 L air-lift bioreactor were investigated.The kinetics of cell growth, polysaccharide synthesis and consumption of nutrients were analyzed in suspension cultures of PLBs. The suspension culture process of PLBs could be divided into three phase: lag phase, rapidly growth phase, and stationary phase under different conditions. Sucrose was a growth limiting nutrient on low concentration condition. Increase in sucrose concentration stimulated cell growth and increased final PLB harvest. Sucrose at 30 g/L gave the highest biomass of PLBs. Substrate inhibition was observed when the concentration was above 30 g/L. Nitrate at 30 mmol/L was beneficial for cell growth. Phosphate was a limiting factor for cell growth. Increase in phosphate concentration increased cell growth rate and shortened the culture period. A total of 2.5 mmol/L medium phosphate gave the highest biomass(28.7g DW/L).The trends of polysaccharide production variation were the same under different conditions. Increase in sucrose concentration could improve polysaccharide production to some extent, while higher sucrose concentration inhibited polysaccharide production. Nitrate at 30 mmol/L was beneficial to accumulation of polysaccharide. Initial phosphate concentration was the most effective on polysaccharide accumulation. The maximum polysaccharide production(2.46g/L) could be obtained at 0.312 mmol/L phosphate concentration. The accumulation of polysaccharides not only correlates with cell growth but also with intracellular reducing sugar level.PLBs rapidly hydrolyzed extracellular sucrose before absorbing it. The hydrolyzing process could be described using substrate saturation kinetic equation. Glucose and fructose were absorbed at the same time. Intracellular accumulation of reducing sugar was closely related to medium composition. Biomass yield coefficients of sucrose under different culture conditions were different. Intracellular phosphate accumulation level related to the initial phosphate concentration in the medium. Intracellular phosphate level affected the cell growth and accumulation of polysaccharides.A structural model was developed. The whole culture system was separated into biotic phase and abiotic phase. The former was divided into 4 compartments: reducing sugar, middle metabolites, product, and respiration loss. The later included sucrose and reducing sugar. Calculated result coincided well with experimental data. The model could be used for description of cultivation process.The medium supplemented with phytic acid 2.5g/L was beneficial for cell growth and polysaccharide synthesis. The cell dry weight and production of polysaccharides were 29.4g DW/L and 2.06g/L, respectively. The phytic acid could inhibit the activities of peroxidase and polyphenol. Putrescine and spermine at 0.6mmol/L improved intracellular polyamine contents and stimulated cell growth and polysaccharide production. The production of biomass and polysaccharides was 32.6g DW/L and 2.20g/L, respectively.Phosphate is an effective factor in the regulation of cell growth and the accumulation of polysaccharides. Two-stage cultivation can be used to produce polysaccharides. In the first step, the PLBs were grown in a medium with 2.5 mmol/l phosphate and in the second step, they were grown in a medium with 0.312 mmol/1 phosphate. By two-stage cultivation, polysaccharide production and content reached 5.22 g/l and 11.9 %, respectively, which were higher than that of other methods. The model could describe the two-stage cultivation process.Primary scale-up of PLB suspension cultures in 10 L air-lift bioreactor was investigated. Aeration rate at 0.5 L/min was beneficial for cell growth and polysaccharide synthesis. The maximum biomass and total polysaccharide production in bioreactor were 86.5 % and 145 %, respectively, compared to that in flask culture. The kinetics of cell growth and accumulation of polysaccharides in the bioreactor were the same as in flask culture. Consumption of nutrients was much more slowly and had lower utilization efficiency than that in flask culture. The biomass (44.7 g DW/L) and polysaccharide production (8.15 g/L) were obtained by feeding culture.