Biological Utilization of Thermolytic Substrates by Bacteria and Microalgae: Addressing Toxicity of Substrate Contaminants

PI：JarboeProposal编号：1133319智力价值通过将热化学和生物化学步骤相结合的混合过程将生物质转化为生物燃料具有降低加工成本的潜力，但研究不足。这项拟议的研究旨在利用纤维素生物质快速热解产生的底物来提高微生物生产生物燃料的能力。生物质的快速热解，包括在没有氧气和催化剂的情况下快速加热材料，产生一种复杂的混合物，称为生物油。生物油的发酵是复杂的，因为除了有用的底物外，它还含有许多抑制(污染物)化合物，包括呋喃和酚。这项拟议研究的总体目标是提高两种模式微生物--大肠杆菌(乙醇)和莱茵衣藻(生物柴油的脂类)--对生物油中发现的抑制性污染物的耐受性，并提高这些微生物将生物油馏分发酵成生物燃料的能力。代谢进化将被用来增加这些模式微生物对污染物的稳健性，这在很大程度上是因为生物油是复杂的，而且抑制机制尚不清楚。该研究计划有三个目标。第一个目标是利用代谢进化来提高产乙醇的大肠杆菌对富含糖的生物油部分中抑制性污染物的耐受性。在富含糖的生物油馏分中，脱水糖左旋葡聚糖是最丰富的底物，但它被大多数微生物代谢。因此，一株产乙醇的大肠杆菌将被改造成利用左旋葡聚糖作为碳源和能源。第二个目标是利用代谢进化来利用异养、产脂的莱茵梭菌富含乙酸酯的生物油部分，从而提高莱茵梭菌对抑制性污染物的耐受性，以及利用富醋酸酯的生物油部分来提高其对醋酸酯本身的耐受性。第三个目标是通过对基因组序列和转录组数据的分析，对目标1下培育的耐污染大肠杆菌菌株进行反向工程，以确定能够耐受污染物的重要突变。广泛影响拟议的教育活动将培养两名化学工程和食品科学学科的研究生，并聘请三名本科生协助拟议的研究。艾奥瓦州立大学(ISU)的三门课程将引入这一研究课题的核心概念：二年级的材料和能量平衡课程，研究生水平的代谢工程课程，以及研究生水平的生物质热化学处理课程。关于K-12的推广，PI将为ISU Science Bound计划开发一个为期半天的生物可再生化学品模块，该计划是一个周六的计划，目标是代表人数不足的中学生。学生们将有机会看到运行中的生物反应器，并讨论生物可再生化学品如何与他们的日常生活相关。然后，学生们将玩一个基于网络的游戏，介绍使用运动生物学实验室的新陈代谢进化的概念。

PI: JarboeProposal Number: 1133319Intellectual MeritConverting biomass into biofuels through hybrid processes that integrate thermochemical and biochemical steps has the potential to reduce processing costs, but is understudied. This proposed research seeks to improve the capacity of microorganisms to produce biofuels using substrates derived from the fast pyrolysis of cellulosic biomass. The fast pyrolysis of biomass, which involves rapidly heating the material in the absence of oxygen and in presence of catalysts, yields a complex mixture called bio-oil. Fermentation of bio-oil is complicated by the fact that in addition to useful substrates, it also contains many inhibitory (contaminant) compounds, including furans and phenols. The overall goals of this proposed research are to increase the tolerance of two model microorganisms, Escherichia coli (ethanol) and Chlamydomonas reinhardtii (lipids for biodiesel), to the inhibitory contaminants found in bio-oil, and to improve the capacity of these organisms to ferment bio-oil fractions into biofuel. Metabolic evolution will be used to increase the robustness of these model microorganisms to the contaminants, largely because the bio-oil is complex and the mechanisms of inhibition are not known. The research plan has three objectives. The first objective is to use metabolic evolution to increase the tolerance of ethanol-producing E. coli to the inhibitory contaminants in the sugar-rich bio-oil fraction. The anhydrosugar levoglucosan is the most abundant substrate in the sugar-rich bio-oil fraction, but it is metabolized by most microorganisms. Therefore, an ethanol producing strain of E. coli will be engineered to utilize levoglucosan as a carbon and energy source. The second objective is use metabolic evolution to utilize the acetate-rich fraction of bio-oil by a heterotrophic, lipid-producing strain of C. reinhardti, and then increase the tolerance of C. reinhardtii to the inhibitory contaminants as well as acetate itself with the acetate-rich bio-oil fraction. The third objective is to reverse engineer the contaminant-tolerant E. coli strain developed under objective 1 through analysis of genome sequence and transcriptome data in order to identify the important mutations that enable contaminant tolerance.Broader ImpactsThe proposed education activities will train two graduate students from the disciplines of chemical engineering and food science, and engage three undergraduates to assist with the proposed research. Core concepts from the research topic will be introduced into three courses at Iowa State University (ISU): a sophomore-level Material and Energy Balances course, a graduate-level Metabolic Engineering, course, and a graduate-level course on the Thermochemical Processing of Biomass. With respect to K-12 outreach, the PI will develop a half-day module on bio-renewable chemicals for the ISU Science Bound program, a Saturday program that targets underrepresented middle school students. Students will have the opportunity to see an operating bioreactor and discuss how bio-renewable chemicals relate to their everyday life. Students will then play a web-based game that introduces the concept of metabolic evolution using the Biology in Motion Lab.