proposalstudyabroad学习计划.doc

华南理工大学“国家建设高水平大学公派研究生项目”学习计划Study Plan for CSC Scholarship Program, PhD South China University of TechnologyName/SCUT Student IDGender女/femaleDate of birth 12/1985School轻工与食品学院/College of Light Industry and Food Science所学专业/Major制浆造纸/pulp and paper making留学国别/Hosting foreign country加拿大/Canada国外留学院校/Hosting foreign institution英属哥伦比亚大学/The University of British Columbia留学院系/Hosting faculty or department林学院 / Forestry国外导师/Hosting foreign supervisorXXX/ Jack (John) N Saddler研究领域/Area of research生物能源/Bioresources学习期限/Duration of study 48 months ( Jan.1st 2012 to Jan.1st 2016)题目名称/ Research Title: The Potential of Enzyme Re-adsorption in an Enzyme Recycling Strategy as a Means of Reducing the Cost of Hydrolysis学习计划 Study Plan(直接用英文书写,内容完整，篇幅1000字以上)：Study PlanResearch BackgroundAlthough intensive work has been done to improve the efficiency of the pretreatment technologies to increase substrate accessibility to enzymes, an efficient enzymatic hydrolysis still requires a high loading of enzymes, making the whole process too expensive for a commercial production of lignocellulosic ethanol 1-5. Three ways of reducing the cost of enzymatic hydrolysis have so far been identified: minimizing the costs of producing the enzymes 1, 5, increasing the specific activity of the enzymes 6, 7, or recycling the enzymes for multiple rounds of hydrolysis 8, 9.A recent review article estimated that the first two strategies have led to a 20 to 30-fold reduction in the cost of enzymes for ethanol production from acid-treated corn stover 3, and the current enzyme cost is estimated at about $1/ gallon ethanol 10. However, to achieve the goal of commercializing biomass conversion process for bioenergy and other bio-products, a further 5-10 fold cost reduction in the use of enzyme is still direly needed. The third strategy, which is to recycle the enzymes for multiple rounds of hydrolysis, has not received as much attention as the previous two strategies. Recycling the enzymes for multiple rounds of hydrolysis is just as important a strategy as the cost-reduction strategies mentioned before for two reasons: 1. high enzyme loadings are still required to achieve efficient hydrolysis 11, 12 and 2. The activity of the cellulases is remarkably stable over extended period of hydrolysis 13. Theoretical ReviewThe costs of enzymatic hydrolysis are still a major bottleneck for the commercial production of cellulosic ethanol 1, 2. Efforts to reduce costs of enzymatic hydrolysis are mainly focused on improving the economics of enzyme production and the efficiency of enzyme mixtures, and these efforts have led to a 20-30 fold reduction in the costs of enzymatic hydrolysis over the last decade 3. A further reduction in costs is, however, still needed. The stability of the enzymes and the high loadings of enzymes used for hydrolysis of lignocellulosic biomass make the possibility of recycling the enzymes an attractive additional strategy to reduce the enzyme costs. Recycling the enzymes requires a good understanding of how the enzymes interact with the substrates during the course of hydrolysis. Unfortunately, our knowledge on this fundamental interaction is limited, hampering our effort to devise an efficient enzyme recycling strategy. Therefore, the proposed work aims to bridge this knowledge gap by monitoring and understanding the distributions of different enzymes during hydrolysis of a lignocellulosic substrate.The Aim and Expectation of the ResearchThe high stability of the cellulase enzymes makes the possibility of recycling the enzymes an attractive strategy to reduce costs of enzymatic hydrolysis. Understanding adsorption/ desorption patterns of specific enzyme components in a cellulase mixture will give insights into designing an efficient enzyme recycling strategy which, in turn, will improve the economics of enzymatic hydrolysis for cellulosic ethanol production. The Current Research Condition and Level of the Research Project at Home and AbroadThere are mainly three ways of reducing the cost of enzymatic hydrolysis have so far been identified: minimizing the costs of producing the enzymes 1, 5, increasing the specific activity of the enzymes 6, 7, or recycling the enzymes for multiple rounds of hydrolysis 8, 9. The research about Bioenergy in China is very fundamental and still in its infancy, so this is the area that I want to study in. One of the challenges that human beings are facing is that the earth is running out of petroleum. We have to find renewable energy, and bioenergy is renewable. More importantly, we can find biomass almost everywhere and convert it into bioenergy. This would help companies such as pulp mill to earn more money and it is to be one of the leading techniques in the future. According to its history, the Forestry in UBC is initiative and has done lots of work in this area for many years.The Experimental Methods and Data Analysis Methods1 Assessing adsorption and activity profiles of specific Trichoderma reesei enzymes on steam pretreated corn stover (SPCS)This work will investigate the distribution and adsorption profiles of different enzyme components present in a commercial enzyme mixture during hydrolysis of SPCS. 2 Enzyme interaction with different lignocellulosic substratesSubstrate characteristics such as types and amounts of lignin, surface area, and hemicellulose distribution are likely to influence adsorption profiles of enzymes and eventually determine the suitability and efficiency of enzyme recycling strategies. Substrates derived from corn stover, a hardwood species, and a softwood species will be produced by steam pretreatment according to the protocols optimized for each biomass. A portion of the resulting substrates will be further treated to remove the lignin using the chlorite delignification method. A model substrate (pure cellulose) will also be included as a control. A number of techniques will be employed to determine substrate characteristics such as types of lignin (lignin charge and hydrophobicity) and surface area (fiber length and internal surface area). The 7 substrates will be hydrolyzed using different protein loadings of enzymes to determine the minimum amount of enzymes needed to achieve at least 80% conversion within 48 h at 50oC and 2 % solid consistency. Once the protein loadings have been specified, the substrates will be hydrolyzed for 48 h at 50oC, and samples will be taken at time 0, 1, 3, 5, 24, and 48 h to recover enzymes from the liquid and solid phases. Similar techniques as in study 1 will be used to assess adsorption profiles of individual enzymes in the mixture. 3 Efficiency of re-adsorption to fresh substrate as an enzyme recycling strategy Enzyme re-adsorption has been identified as a promising enzyme recycling strategy 9, but more work is needed to determine the efficiency of this strategy with regards to the types of enzymes that get recycled and the types of substrates and to identify potential improvements to this strategy. To simplify the study, based on the result from study 2, two substrates that show different enzyme adsorptivity will be chosen for this study. The substrates will be hydrolyzed at 5% consistency for 24 h at 50oC after which the enzymes will be recovered from the liquid phase through centrifugation and from the solid phase through desorption using fresh 0.05 M Na-acetate pH 4.8 buffer. These fractions will be assayed for their activities (FPA, CMCase, PNPG (-glucosidase), PNPCase, and xylanase) and protein profiles (Ninhydrin assay, SDS-PAGE, and zymogram). Glucan and xylan conversion will also be determined using HPLC. Fresh substrate will be added to the enzymes recovered from the solid and liquid phases, and both fractions will be incubated at 4oC for 2 h to re-adsorb the enzymes. Non re-adsorbed enzymes will be recovered and assayed for their activities and protein profiles. These assays will give information on the types of enzymes not recovered through re-adsorption. After the re-adsorption step, the solid substrates will be collected to which fresh buffer and fresh -glucosidase will be added. The whole mixture will then be incubated for another 24 h for 50oC. After hydrolysis, the same assays will be conducted to assess enzymatic activities and protein profiles of solid- and liquid-phase fractions. The same steps will be taken to re-adsorb and repeat the hydrolysis for a total of 5 rounds of hydrolysis. 4 Comparison of the efficiency of enzyme supplementation vs enzyme re-adsorption as an enzyme recycling strategyEnzyme supplementation to replenish activity that gets lost along with the liquid fraction during recycling has been identified in a recent paper as a promising way to reduce the amounts of enzymes required for multiple rounds of hydrolysis 14. This study, therefore, seeks to compare the efficiency of this strategy with enzyme re-adsorption.Hydrolysis will be done on one of the substrates used in chapter 3 which shows better conversion efficiency. Enzymes present in the liquid fraction will not be recycled. After determining the enzyme activities lost during recycling, fresh cellulase enzymes will be used to replenish these lost activities. Methods will be based on a paper by Yang et al. 14. Recycling will be done for 5 rounds of hydrolysis. Research Schedule and Time Plan(1) 1st year: Refreshing and learning some knowledge especially in the potential of enzyme re-adsorption in an enzyme recycling strategy as a means of reducing the cost of hydrolysis, reading some relevant articles to get some ideas. (2) 2nd year: Manage the data from the reading articles and work out the draft and discuss the feasibility of the program with professor. (3) 3rd year: conduct the experiment research and achieve the expected results. (4) In the final stage, Miss Dou will be required to finish the thesis and papers that are ready to be submitted to the international journals. The Study/Work Plan After Returning to China, etcWhen Miss Xiaoli Dou finishes the PhD study in the University of British Columbia, she will continue to engage in the research of bioresources especially in means of reducing the cost of hydrolysis in China.Reference1 Wilson DB. Cellulases and biofuels. Curr Opin Biotechnol 2009;3:295-9.2 Zheng Y, Pan Z, Zhang R, Jenkins BM. Kinetic Modeling for Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass. Biotechnol Bioeng 2009;6:1558-69.3 Banerjee G, Scott-Craig JS, Walton JD. Improving Enzymes for Biomass Conversion: A Basic Research Perspective. Bioenergy Research 2010;1:82-92.4 Somerville C. The billion-ton biofuels vision. Science 2006;5778:1277.5 Merino ST, Cherry J. Progress and challenges in enzyme development for Biomass utilization. Biofuels 2007:95-120.6 Darias R, Villalonga R. Functional stabilization of cellulase by covalent modification with chitosan. Journal of Chemical Technology and Biotechnology 2001;5:489-93.7 Boer H, Koivula A. The relationship between thermal stability and pH optimum studied with wild-type and mutan