Key Parameters Of Photobioreactor Cultivation Of Macroalgal Cells

Macroalgae, the primitive oceanic plants, contain numerous primary and secondary metabolites with important or potential pharmaceutical values. The extremely low content in the algae,and the complicated molecular structure of those rare secondary metabolic compounds, however, discourage the direct extraction and chemical synthesis. Photolithotrophic cultivation of macroalgal cells is an effective way to optimize culture conditions, especially those that are specifically important, in the stimulation of secondary metabolic biosynthesis, and to achieve future commercial production of relevant biochemicals. Till now, cultivation of macroalgal cells in photobioreactors is a great challenge. And because of the weak biological techniques, there are no macroalgal cells cultivated in vitro. Only gametophyte cells and microplantlets are chosen for macroalgal tissue or cell culture. And the former resemble much the real macroalgal cells. In this study, gametophyte cells of brown macroalgae Laminaria japonica were employed to stimulate macroalgal cell growth. Effects of all conditions involved in photobioreactor cultivation, such as temperature, light intensity, photoperiod, and aeration modes, on algal cell growth rates and cell physiological status, were comprehensively studied. Further research included the effects of different initial nutrient concentration and different pulse fed-batch modes on biomass production under the above optimal cultivation conditions. In addition, the influence of agitation intensity and agitation time on cell growth and cell injury was studied. Corresponding engineering and biology explanation was specifically elucidated.The innovation of this experiment is the comprehensive and systemic study on the key conditions of photobioreactor cultivation of the gametophyte cells. It is the first systemic study on cell growth rates and cell physiological status under different subcultural systems of macroalgal cells. Bubble-less silicone tubular membrane aeration mode for macroalgal cell culture is applied for the first time in this paper. Because algae will accumulate much more of nutrient than is required for normal metabolism, pulse fed-batch modes are felicitously applied in this research for macroalgal cell proliferation. Moreover, the systemic study on macroalgal clones'responses to different agitation intensity and the exposure time is another innovation of this experiment. This work has provided rational methods and important data for large-scale production of biomass and important secondary metabolites of macroalgal cells in the future.Photosynthetic rate, increment of chlorophyll a, cell injury ratio, cell growth curve, cell morphology, gas-liquid mass transfer co-efficient (kLa) indicated that the optimal temperature and photoperiod of Laminaria japonica gametophytic cells were 13℃and 16:8 LD, with the light saturation point of 28μE m-2 s-1. kLa of spinning, shaking and bubbling aeration modes versus input rates accorded well with the equation of Y=aXb. High biomass production with fine macroalgal cell physiological status was obtained under the environment of low suspension (low turbulence) and moderate gas-liquid transfer rates. In this experiment, compared with static, bubbling, and spinning mode, increase of biomass accumulation under membrane aeration mode was 103%, 29%, and 69%. However, larger cell aggregates formed because of quick cell growth under lower turbulence condition. The internal cells of the large aggregates were in lack of light, nutrient and gas delivery. Low shearing, on the other hand, could contribute to moderate aggregate size, and further the assurance of continuous cell proliferation. The self-designed modified stirred tank photobioreactors (with bubbling) were proved feasible for the cultivation of Laminaria japonica gametophyte cells.With the increase of initial nutrient concentration in the artificial Pacific seawater (APSW) medium, specific growth rate of gametophyt cells cultivated in the above photobioreactors increased gradually, its lag phase shortened step by step. When initial nutrient concentration exceeded 1.189 mmol N L-1 and 0.0742 mmol P L-1, biomass increasing times decreased, with the potential toxicity of high nutrient concentration emerged. The optimal nutrient concentration in this work was 0.594 mmol N L-1 and 0.0371 mmol P L-1,under which biomass increasing 7.2 times. As to the pulse fed-batch modes, the results showed that feeding the culture frequently with small nutrient quantity was beneficial for the synchronization between nitrate and phosphate absorption. Feeding when ambient nutrient was abundant or depleted would result in the divergence absorption between nitrante and phosphate, nutrient storage, or the decrease of nutrient absorpsion. Feeding nutrient frequently with small quantity from mid-exponential growth of macroalgal cells, that is maintaining medium nutrient concentration between 1/3 and 1/2 of its initial concentration in this study, was the most effective way for biomass production, with 76% increase of biomass accumulation compared with the batch mode.Results of short-term (60 h) continuous agitation on Laminaria japonica gametophytic cells under stirred tank photobioreactors (with surface aeration) indicated that chlorophyll content, medium nitrate and phosphate concentration, and cell injury ratio are suitable indicators for the evaluation of cell injuries under hydrodynamic agitation shear stress. Moderate shearing was beneficial for biomass accumulation and nutrient absorption. Greater shear stress or longer shearing time would lead to negative cell growth, the release of cellular N pool and P pool, the rise of cell injury ratio, and tremendous alteration of cell micro-morphology. In addition, hydrodynamic characteristics, such as Kolmogoroff eddy sizes, Kolmogoroff eddy velocity, and maximal turbulent stress were preferable engineering explanation for those differences under different agitation speeds. Results of long-term (21 d) continuous agitation on gametophytic cells showed that larger biomass accumulation and better cell physiological status were obtained under the agitation speed of 90 rpm (the stirrer tip speed of 0.254 m s-1). A three-phase of ascending, decending, and re-ascengding trend of cell growth was presented under the agitation speeds of 120-270 rpm. Corresponding cell injury ratio increased firstly, and then decreased. These results were due to the variation of cell wall fraction (CWF) during the agitation period. CWF under the agitation speeds of 120-270 rpm decreased, and then increased, even exceeded CWF under the control. These explained well cell injury and cell resistance to hydrodynamic shear stress.