纖維素水解菌Cellulomonas sp.和酵母菌於SSF程序以藻粉產生生質酒精之研究

Abstract

Microalgae biomass is a potential substrate for bioethanol production because (1) microalgae can exhibit a higher productivity than lignocellulosic crops; (2) cultivation of microalgae can avoid the competition between energy production and food supply when using energy crops; (3) microalgae, compared with terrestrial plants, have a low lignin content to facilitate the lignin removal. In order to generate bioethanol from microalgae biomass, cellulose in the microalgae biomass should be hydrolyzed by a variety of cellulases and the hydrolysates (reducing sugars) are subsequently fermented by yeast to produce ethanol. However, this traditional process, named separate hydrolysis and fermentation (SHF), revealed a great inhibition in the cellulase activity that is reduced by the accumulated reducing sugars. Nevertheless, it has been indicated that a simultaneous saccharification and fermentation (SSF) process can achieve a high ethanol yield than SHF process due to its maintenance of cellulase activity. Therefore, the aim of this study was to investigate the feasibility of bioethanol production from microalgae biomass by Cellulomonas sp. and Saccharomyces cerevisiae in a batch-mode SSF system. In this study, a commercial powder of Chlorella sp. biomass and a self-cultivated Graesiella sp. biomass, were used to estimate their bioethanol production. The effect of HCl or NaOH concentrations for hydrolysis pretreatment, composition of culture medium, microalgae biomass concentration, and the temperature for cellulose hydrolysis on the production of reducing sugars were all carried out, using the commercial powder of Chlorella sp. biomass as experimental substrate. On the other hand, the optimal conditions of temperature, pH value, and the initial amount of Saccharomyces cerevisiae inoculating biomass in the fermentation of glucose were examined. Considering the cellulase-producing bacterium, Cellulomonas sp. would consume the reducing sugars after the hydrolysis, the spent medium that contained the extracellular cellulase (crude cellulase) was used to understand the effect of above experimental parameters on the reducing sugar production and the inhibition of enzyme activity by hydrolysates. Finally, the efficiency of producing reducing sugar by the crude cellulase would be compared with that by a commercial cellulase. The results showed that the cellulose content of 55.18% in Graesiella sp. was observed in a 5-liter-photobioreactor cultivation, which can improve the cellulose content in 9.6 fold than a 200-milliliter-flask cultivation. For the hydrolysis of Chlorella sp. biomass by the crude cellulase, the maximum amount of reducing sugars was achieved in the Mandels Reese culture medium with 20 g/L inoculum at 30oC. With respect to the pretreatment procedure, treating the Chlorella sp. biomass with 2% NaOH and further mixing the treated biomass with neutralized supernatant from earlier base pretreatment can shorten the hydrolysis period and increase the reducing sugars production in 88%. In addition, the highest ethanol content of 43.0 g/L was obtained when the fermentation process was performed under 10 g/L of glucose with a inoculum of 0.07 g/L at 40oC and pH 6.5. Integrating those optimal parameters and utilizing the crude and commercial cellulase to hydrolyze the Graesiella sp. or Chlorella sp. biomass in both SHF and SSF processes, the results indicated that the commercial cellulase can yield reducing sugars of 0.6 g/L and ethanol of 4.09 g/L in the SHF process with unpretreated Chlorella sp. biomass. However, an obvious inhibition to endo-β-1, 4-gulcanase was observed and its enzyme activity decreased in 57% within this SHF experiment. On the contrary, using the crude cellulase in the SSF process showed a high ethanol production (6.59 g/L). Using the pretreated Chlorella sp. biomass as the substrate, the SSF process can improve the ethanol production in 3.03 fold than SHF process. Furthermore, hydrolysis of Graesiella sp. biomass produced more ethanol and exhibited a higher ethanol-producing rate than those of Chlorella sp. biomass no matter what kind of cellulase and process were performed.