| Summary: | 博士 === 國立成功大學 === 化學工程學系碩博士班 === 101 === In this study, an effective cellulase-producing strain was isolated from forest soil in southern Taiwan using the approaches associated with microbiology, enzyme engineering and fermentation engineering. The enzymes produced were used for the development of sustainable and commercially feasible bioenergy producing processes. Twelve strains were first isolated from the environmental samples. Among them, Pseudomonas sp. CL3 was found to secrete a number of highly active hydrolytic enzymes the best activity, so was selected as the target strain for further studies.
The isolated indigenous bacterium Pseudomonas sp. CL3 was able to produce novel cellulases consisting of endo-β-1,4-D-glucanase (80 and 100 KDa), exo-β-1,4-D-glucanase (55 KDa), β-1,4-D-glucosidase (65 KDa) and xylanase (hemicellulase) with a molecular weight of 20 KDa characterized by enzyme assay and zymography analysis. In addition, the CL3 strain also produced amylase and pectinase. The optimal temperature for enzyme activity was 50, 45, 45 and 55oC for endo-β-1,4-D-glucanase, exo-β-1,4-D-glucanase, β-1,4-D-glucosidase and xylanase, respectively. All the enzymes displayed optimal activity at pH 6.0.
In addition, the effect of reaction temperature and environmental factors (metal ions and surfactant) on the activity of the cellulolytic enzymes originating from Pseudomonas sp. CL3 was intensively studied. It was found that the cellulases produced by Pseudomonas sp. CL3 belong to mesophilc enzymes. The metal ions (Mn2+ and Fe2+) were also significant factors affecting the cellulase activity. Addition of biosurfactant (e.g., surfactin) can enhance the cellulase activity. However, for glycolipid-type biosurfactant (e.g., rhamnolipid), since the cellulases will bind to the sugar structure of the biosurfactant, the addition of rhamnolipid led to a decrease in the hydrolytic activity. The cellulases produced by strain CL3 were also immobilized on the magnetic particles. These results indicate that the β-glucosidase activity of the immobilized enzyme had extremely high stability, and could maintain 90% of its original activity even when the immobilized enzyme particles were reused for 5 cycles.
To further enhance the production of cellulases from the CL3 strain, statistical experimental design methodology was applied to optimize the culture medium composition favoring enzyme synthesis. Four key parameters (CMC, rice straw, NH4NO3, yeast extract) were selected by two-level factorial design. Response surface methodology was then used to identify the optimal composition of the selected parameters, giving an optimal concentration of 1.32% and 0.32% for rice straw and yeast extract, respectively. With this optimal medium, the cellulolytic enzymes production could be markedly elevated to a maximum concentration of 3.8 FPU/ml, which is 3.7 times compared with original medium.
In order to develop efficient and cost-effective process for producing cellulases from CL3 strain, different fermentation strategies were examined. In the CSTR fermentation process, the cellulase titer in the fermentor could be maintained at around 3.3 FPU/ml at a HRT of 17 h with a cellulase productivity of 194.12 FPU/L/h, which is 3.4 times higher than that obtained from the batch system.
Nest, the cellulases produced from CL3 strain was used to perform hydrolysis process for the saccharification of lignocellulosic materials (such as sugarcane bagasse and rice straw). The effects of pretreatment process (chemical and physical method) on the enzymatic hydrolysis were investigated. The alkaline pretreatment seems to be the most effective method able to achieve the highest monosaccharide yield from bagasse through enzymatic saccharification, which is 3.2 times compared with non- pretreated bagasse. Kinetic analysis shows that the dependence of cellulase activity on cellulose substrate can be described by Michaelis-Menten model with good agreement. The estimated kinetic constants for cellulases obtained from CL3 strain were Vmax= 0.133 g/L/h and Km=7.4 g/L, respectively (with an enzyme loading of 4.5 FPU/ml). That is, the maximum glucose productivity from the enzymatic hydrolysis was 0.133 g/L/h at a bagasse loading of 14.8 g/L. The cellulose hydrolysis experiments were also conducted under different concentrations of alkaline-pretreated bagasse. Under different bagasse loadings (50 to 70 g/L), a cellulase dosage of higher than 9 FPU/ml can obtain a great glucose productivity of around 0.55 g/L/h. Moreover, our study also shows that the enzyme from Pseudomonas sp. CL3 can also hydrolyze microalgal biomass (e.g., Chlorella vulgaris), giving a glucose productivity of 0.13 g/L/h, and the yield of around 90%.
The cellulases were also applied in producing biohydrogen from cellulosic feedstock. In the cellulosic hydrogen production process, the experiments were divided into two groups, namely separate hydrolysis and fermentation (SHF) process and simultaneous saccharification and fermentation (SSF) process using alkaline pretreated sugarcane bagasse. It was found that lower H2 content and productivity were obtained when the glucose concentration in the fermentation medium was at a lower level (under 4 g/L). Therefore, the SHF process seems to be more suitable for the biohydrogen production from the pretreated bagasse due to the requirement of high glucose concentration in the medium. Therefore, we used the bagasse hydrolysate to produce hydrogen with Clostridium pasteurianum CH4 using CSTR operations at a HRT of 12 h. Under this condition, the H2 content in the biogas was around 56.5% and the maximum H2 production rate was 326.7 ml/L/h. These results show that the cellulases produced form a newly isolated indigenous bacterium Pseudomonas sp. CL3 were able to combine with dark H2 fermentation system to produce cellulosic hydrogen. Finally, we combine the cellulases production strategy, optimal hydrolysis process, dark H2 fermentation, and microalgae-CO2 capture system to develop a sustainable and low-carbon-emission biohydrogen producing system.
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