STTR Phase I: High-Yield Hydrogen Production from Biomass Sugars by Cell-Free Biosystems for Mobile Electricity Generation

这个小型企业创新研究第一阶段项目将扩大麦芽糊精和水在无细胞酶生物系统介导下的高产量氢气生产，并开发移动发电机原型(MEGS)。用于生物制造的无细胞生物系统(CFB2)通过在体外组装三种以上的酶和/或辅助因子，在一个锅中实现复杂的生化反应。弗吉尼亚理工大学的Co-Pi已经证明了使用CFB2从糖(包括己糖和戊糖)和水中以CH2O(糖)+H2O 2H2+CO2的形式生产氢气的产量几乎是理论上的。在这个STTRI项目中，无细胞生物创新公司将把酶法制氢从2毫升的生物反应器(目前的实验室规模)扩大到5-L生物反应器，并将这一过程与质子交换膜燃料电池组集成，以实现高效发电。这些集成系统将为众多便携式电子设备充电，并以低成本提供应急电力。具体目标是(I)通过高密度发酵放大重组嗜热酶的生产，(Ii)发现和生产更高活性和超热稳定的酶，(Iii)构建易于纯化和快速反应的合成酶复合体(代谢物)，(Iv)进一步提高产氢速率三倍，以及(V)原型MEGS的演示。该项目更广泛的影响/商业潜力是扩大酶法生产氢气的规模，氢气主要由天然气和原油生产。它的卫星生产设施及其分销并不广泛，而且成本太高。未来，从当地可再生的生物质糖生产低成本的绿色氢气将在生物经济中创造生物制造和农业就业机会，降低氢经济的基础设施成本，减少对有限化石燃料的依赖，并减少温室气体净排放。在大规模生产具有经济竞争力的糖氢之前，建议开发几个高端应用来进一步完善CFB2平台，例如MEGS、酶燃料电池和手性化合物合成。以糖为基础的MEGs将有一些特殊的市场。此外，与全细胞发酵相比，新的生物技术平台CFB2具有独特的优势，如更高的产品产量(即既不形成副产物也不合成细胞)，更大的工程灵活性，更快的反应速度，更广泛的反应条件(如高温、低pH、有机溶剂、有毒化合物)，以及更容易操作和控制。据信，CFB2平台未来可用于生产喷气燃料和长链醇、储存电力和固定二氧化碳。

This Small Business Innovation Research Phase I project will scale up high-yield hydrogen production from maltodextrin and water mediated by cell-free enzymatic biosystems and develop prototype mobile electricity generators (MEGs). Cell-free biosystems for biomanufacturing (CFB2) implement complicated biochemical reactions in one pot by the in vitro assembly of more than three enzymes and/or cofactors. The Co-PI at Virginia Tech has demonstrated the production of nearly theoretical yields of hydrogen from sugars (including hexoses and pentoses) and water as CH2O (sugar) + H2O 2H2 + CO2 using CFB2. In this STTR I project, Cell-Free Bioinnovations Inc. will scale up enzymatic hydrogen production from a 2-mL bioreactor (current laboratory scale) to a 5-L bioreactor and integrate this process with a proton exchange membrane fuel cell stack for the high-efficiency generation of electricity. These integrated systems will charge numerous portable electronics and provide emergency power at low costs. The specific objectives are (i) scale-up of recombinant thermophilic enzyme production through high-cell density fermentation, (ii) discovery and production of more high-activity and ultra-thermostable enzymes, (iii) construction of synthetic enzyme complexes (metabolons) for easy purification and fast reaction rates, (iv) further enhancement of hydrogen generation rate by three fold, and (v) demonstration of prototype MEGs. The broader impact/commercial potential of this project is the scale-up of enzymatic production of hydrogen, which is mainly produced from natural gas and crude oil. Its satellite production facilities and its distribution are not widely available and are too costly. In the future, the production of low-cost, green hydrogen from local, renewable biomass sugars would create biomanufacturing and agricultural jobs in the bioeconomy, lower infrastructure costs for the hydrogen economy, decrease reliance on finite fossil fuels, and reduce net greenhouse gas emissions. Prior to large-scale production of economically competitive sugary hydrogen, several high-end applications are suggested for development to further improve the CFB2 platform, for example, MEGs, enzymatic fuel cells, and chiral compound synthesis. MEGs based on sugars will have some special markets. In addition, the new biotechnology platform CFB2 has unique advantages, such as higher product yields (i.e., neither by-product formation nor cell mass synthesis), greater engineering flexibility, faster reaction rates, broader reaction conditions (e.g., high temperature, low pH, organic solvent, toxic compound), and easier operation and control, compared with whole-cell fermentation. It is believed that the CFB2 platform could be used to produce jet fuels and long chain alcohols, store electricity, and fix CO2 in the future.