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.
Award No.: 1321528 Prepared by: PI: Dr. Zhiguang Zhu, CTO Cell-Free Bioinnovations Incorporated, Blacksburg, VA 24060 The hydrogen economy presents a clean and sustainable energy future and will feature enhanced energy efficiency. Currently, hydrogen is mainly produced from natural gas and crude oil in large scale facilities. This process makes satellite production facilities cost-prohibitive, and expensive gas distribution infrastructure is needed. To solve these issues, a novel idea of producing a theoretical amount of hydrogen from widely available renewable sugar feedstocks via enzyme biocatalysts has been proposed. Such production of enzymatic hydrogen from renewable sugars is regarded as the beginning stage of a cheap, green and high-yield hydrogen production. Additionally, the use of sugar as a high-density hydrogen storage carrier would solve problems associated with high-density hydrogen storage, costly infrastructure, and safety issues. The biocatalysis converting sugar molecules to hydrogen is through an enzyme cocktail containing a variety of enzymes. The complicated biochemical reactions have been performed to demonstrate the theoretical production yield of hydrogen from sugar units. However, several obstacles still prohibit the commercialization of this sugar-to-hydrogen technology, including low hydrogen production rate, low stability of enzyme catalysts, high production cost, and lack of demonstration in a large scale. The goal of this NSF STTR project is to develop a prototype mobile enzymatic hydrogen reactor that can power a fuel cell used as the portable power source. It will meet different needs such as mobile electricity generators for portable electronic devices and lighting, especially in the emergence conditions (e.g., hurricane prone areas, remote areas). In this project, we produced nearly all thermostable enzymes by using high-cell density fermentation and purified most of them by heat precipitation, constructed several synthetic enzyme complexes, significantly increased hydrogen production rates by using mathematic modeling, optimizing enzyme ratios and increasing reaction rate, and accomplished nearly theoretical yields of hydrogen production from pretreated corn stover for the first time. Four papers or manuscripts have been published or in revision. However, we also found several problems to be solved in the future. It was found out that not all the enzymes had been produced in high-cell-density fermentation or purified by heat precipitation. The production and purification of recombinant hydrogenase remained challenging. Relatively high enzyme production and preparation costs prevented its scale-up at this stage. Whether synthetic enzyme complexes exhibited substrate challenging depended on a lot of factors, such as enzymes chosen, their ratios, etc. Although an increase in reaction temperature from 50 to 60 oC increased hydrogen generation rates, the hydrogen yields at 60oC drastically decreased. Therefore, we failed to develop a prototype large-size enzymatic hydrogen reactor that can be used for fuel cells. What we learned and accomplished will help further development of this important technology. We will focus on large-size stationary hydrogen bioreactors that can convert local biomass sugars to hydrogen instead of mobile hydrogen generators in the near future.
