Exploration For Oscillation In Continuous VHG Ethanol Fermentation With Saccharomyces Cerevisiae

The widely using of fuel ethanol was limited by its high production cost.Energy cost accounted for 30%of total production,only after that of raw material consumption,was the second largest in ethanol production cost.Very high gravity(VHG) ethanol fermentation was the most promising technology for energy saving in distillation of fermented broth and waste water.Markedly oscillatory behaviors of parameters,which destroyed steady run of fermentation process,occurred usually in continuous ethanol fermentation under VHG condition,and no rational explanation was given yet.Parameter oscillations characterized by long oscillation periods and large oscillation amplitudes were observed in the continuous ethanol fermentation with Saccharomyces cerevisiae under VHG conditions,and the mechanism was speculated to be different from those oscillations observed in the continuous culture of the yeast,which were triggered by the synchronization of the cell cycles and characterized by short oscillation periods and small oscillation amplitudes.Metabolic flux analysis(MFA) was applied to the oscillatory VHG ethanol fermentation system,and the results indicated that the carbon fluxes at the keynotes within the metabolic network oscillated correspondingly,with over 80%of the total carbon flux to the ethanol production to generate energy to support the metabolism of the yeast cells. Ethanol was validated to be the main inhibitor of the yeast cells under oscillatory conditions, and the overall metabolic activity of the yeast cells was found not exactly out of phase but lag behind the ethanol concentration accumulated within the fermentation system and its inhibition on the yeast cells as well,which experimentally supported the mechanistic speculation for the process oscillation:ethanol inhibition in yeast cells and the lag response of the yeast cells to the ethanol inhibition.The synchronization of the intracellular trehalose,an effective protectant of yeast cells to environmental stresses,with ethanol production further supported this mechanistic speculation since time was needed for the stress protectant to provide protection again the ethanol inhibition.And in the meantime,analysis of the yeast cell cycle using the flow cytometry approach showed that no cell cycle-dependent synchronization of the daughter and mother cells occurred within the duration of the oscillation.A bioreactor system composed of a stirred tank and tubular bioreactors in series was established,and the impact of yeast cell immobilization with supporting materials on the oscillation of the fermentation generated within the tank ahead was investigated through packing the tubular bioreactors with wood chips and polyurethane particles,two packings with significant difference in their surface properties and microstructures.It was found that the immobilized yeast cells with the wood chips effectively attenuated the oscillation,and sugar consumption and ethanol tolerance were improved compared with the tubular bioreactor without packing,which could be the root reason for the oscillation attenuation.Either oscillatory or steady state could be generated at the dilution rates of 0.027 and 0.04 h^-1,which depended on the initial conditions applied to the fermentation system. However,the productivity increased by 12.3%compared to the steady state,when the diluation rate of 0.04 h^-1 was applied to the fermentation system under oscillation conditions. Further investigation revealed that besides the lag response of the yeast cells to ethanol inhibition under the oscillation conditions,the oscillatory dynamic kinetics of the yeast cells possessed advantages over that under steady state conditions,with 53.8%increase in the specific growth rate,indicating the fermentation system could be operated at higher dilution rates without washing out of the yeast cells.Introduction of such oscillations in a CSTR multistage tubular reactors system for ethanol continuous fermentation using VHG medium was experimentally proven to be practical.Fermentation time was shorten remarkably in oscillatory mode compared to steady states by greatly increased the fermentation efficiency. Oscillations introduced were attenuated successfully by wood chips packed tubues,and steady final ethanol content and low level of final residue sugar which were satisfied to the industrial standards were realized.Forced oscillations were created by applying the dilution rates of 0.02 and 0.04 h^-1 to the fermentation system periodically.Compared with the fermentation system operated under steady state at the dilution rate of 0.03 h^-1,improved fermentation performance was achieved when the period of 4 d was applied to the periodic change of the two dilution rates,with a increases of 3.4%for ethanol productivity of the fermentation system,while no improvement was observed for another two fermentations under the oscillation periods of 2 and 1 d, respectively,indicating the effectiveness of the forced oscillation in improving the fermentation performance could be achieved when the forced oscillation period is close to the oscillation period naturally developed by the yeast cells within the fermentation system.