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High-Yield Hydrogen Production from Biomass by in Vitro Metabolic Engineering: Mixed Sugars Coutilization and Kinetic Modeling

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High-Yield Hydrogen Production from Biomass by in Vitro Metabolic Engineering: Mixed Sugars Coutilization and Kinetic Modeling High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling Joseph A. Rollina,b, Julia Martin del Campoa, Suwan Myunga,c, Fangfang Sunb, Chun Youa,b, Allison Bakovicd, Roberto Castroe, Sanjeev K. Chandrayanf, Chang-Hao Wuf, Michael W. W. Adamsf,g, Ryan S. Sengera, and Y.-H. Percival Zhanga,b,c,h,1 aDepartment of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061; bCell-Free Bioinnovations, Blacksburg, VA 24060; cInstitute for Critical Technology and Applied Science, Virginia Tech, Blacksburg, VA 24061; dDepartment of Physics and Chemistry, Milwaukee School of Engineering, Milwaukee, WI 53202; eDepartment of Chemical and Natural Gas Engineering, Texas A&M University, Kingsville, TX 78363; fDepartment of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602; gDepartment of Energy BioEnergy Science Center, Oak Ridge, TN 37831; and hTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China Edited by Alexis T. Bell, University of California, Berkeley, CA, and approved March 10, 2015 (received for review September 13, 2014) The use of hydrogen (H2) as a fuel offers enhanced energy conver- generation technologies, such as microbial fermentation, gasifi- sion efficiency and tremendous potential to decrease greenhouse cation, steam reforming, and aqueous phase reforming (6, 10– gas emissions, but producing it in a distributed, carbon-neutral, 13), suffer from low product yields. For example, the hydrogen low-cost manner requires new technologies. Herein we demon- yield from microbial fermentations is limited to 4 H2/mol of strate the complete conversion of glucose and xylose from plant glucose. However, much higher hydrogen yields (>4H2/glucose) biomass to H2 and CO2 based on an in vitro synthetic enzymatic are critical to achieve their production economics because sugars pathway. Glucose and xylose were simultaneously converted to H2 usually account for more than half of biofuel production costs (14). with a yield of two H2 per carbon, the maximum possible yield. In vitro metabolic engineering is the construction of synthetic Parameters of a nonlinear kinetic model were fitted with experi- enzymatic pathways without cell membranes to carry out com- mental data using a genetic algorithm, and a global sensitivity plex biochemical reactions. In addition to its fundamental role in ENGINEERING analysis was used to identify the enzymes that have the greatest elucidating complicated cellular metabolism and regulation (15), impact on reaction rate and yield. After optimizing enzyme load- this field is emerging as a promising alternative biomanufactur- ings using this model, volumetric H2 productivity was increased ing platform, especially for the production of low-value and high- · −1· −1 3-fold to 32 mmol H2 L h . The productivity was further en- impact biofuels and biochemicals (16–18). In vitro metabolic · −1· −1 hanced to 54 mmol H2 L h by increasing reaction temperature, engineering has a number of advantages over whole-cell bio- substrate, and enzyme concentrations—an increase of 67-fold systems: high product yield without the formation of by-products compared with the initial studies using this method. The produc- or the synthesis of cell mass (19, 20); fast reaction rates, due to SCIENCES tion of hydrogen from locally produced biomass is a promising elimination of transport across a cell membrane (21, 22); easy APPLIED BIOLOGICAL means to achieve global green energy production. product separation (21, 23); tolerance of compounds that would be toxic to intact cells (24); broad reaction conditions (25); and hydrogen | biomass | in vitro metabolic engineering | metabolic network modeling | global sensitivity analysis Significance nlocking the tremendous potential of the hydrogen (H2) Hydrogen (H2) has great potential to be used to power pas- Ueconomy depends on the availability of low-cost, distributed senger vehicles. One solution to these problems is to distribute hydrogen produced in a carbon-neutral manner. Approximately and store renewable carbohydrate instead, converting it to 50 million metric tons of hydrogen produced from fossil fuels hydrogen as required. In this work more than 10 purified en- annually are used mainly for oil refining and ammonia synthesis zymes were combined into artificial enzymatic pathways and a (1). However, current thermochemical production options are high yield from both glucose and xylose to hydrogen was prohibitively expensive, especially for distributed production sys- achieved. Also, gaseous hydrogen can be separated from aque- tems, when infrastructure costs are considered (1, 2). Water elec- ous substrates easily, greatly decreasing product separation costs, trolysis is scalable and potentially renewably powered, but this and avoid reconcentrating sugar solutions. This study describes technology remains expensive (3). Water splitting powered by solar high-yield enzymatic hydrogen production from biomass sugars energy (4, 5) suffers from low solar-to-chemical energy conversion and an engineered reaction rate increase achieved through the efficiencies and low volumetric productivities and is far from use of kinetic modeling. Distributed hydrogen production based practical applications (5, 6). For the hydrogen economy to become on evenly distributed less-costly biomass could accelerate the reality, new technologies must be developed to meet increasing implementation of the hydrogen economy. needs while keeping both production costs and capital costs low and simultaneously reducing greenhouse gas emissions (7). Author contributions: J.A.R., R.S.S., and Y.-H.P.Z. designed research; J.A.R., J.M.d.C., S.M., Biomass is the most abundant renewable resource and is far F.S., C.Y., A.B., R.C., S.K.C., and C.-H.W. performed research; C.Y., S.K.C., C.-H.W., M.W.W.A., and R.S.S. contributed new reagents/analytic tools; J.A.R., R.S.S., and Y.-H.P.Z. analyzed data; more evenly distributed worldwide than fossil fuels. A 2011 and J.A.R., M.W.W.A., R.S.S., and Y.-H.P.Z. wrote the paper. biomass resource report suggested that over 1 billion tons of dry Conflict of interest statement: Y.-H.P.Z. and J.A.R. are cofounders and officers of Cell-Free biomass could be harvested sustainably in the United States by Bioinnovations. Y.-H.P.Z. is the coinventor of the enzymatic hydrogen production tech- 2050 and that farm gate prices of $60 per dry ton are feasible (8). nology (US patent 8211681). The predominant biomass sugars are glucose (C6) and xylose This article is a PNAS Direct Submission. (C5), accounting for more than 90% of the fermentable sugars in Freely available online through the PNAS open access option. plant cell walls. Microbial cofermentation of C6 and C5 sugars 1To whom correspondence should be addressed. Email: [email protected]. usually results in diauxic growth due to carbon catabolite re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pression, resulting in low volumetric productivity (9). Hydrogen 1073/pnas.1417719112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1417719112 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 nonnatural reactions, such as the enzymatic transformation of has been used to identify the key operating bottlenecks of kinetic cellulose to starch (23). If the half-life times of all enzymes in an and agent-based models (30, 31). Importantly, the predictions in vitro enzymatic pathway were similar to that of industrial made in this research were validated experimentally, and an glucose isomerase, which can produce more than 1,000,000 kg of improved volumetric hydrogen productivity rate was achieved. product per kilogram of enzyme, the catalyst cost of this bio- transformation would be a small fraction of product price, en- Results abling the production of biofuels and biochemicals at very low Two approaches were used to demonstrate (i) simultaneous, costs (16, 26). high-yield utilization of C5 and C6 biomass sugars and (ii) ra- In this study, we demonstrate for the first time to our knowl- tional metabolic engineering of the system for increased reaction edge the production of hydrogen directly from pretreated plant rate through the use of a kinetic model. For high-yield biomass biomass containing C5 and C6 sugars via in vitro metabolic en- utilization, one mesophilic enzyme, polyphosphate glucokinase gineering. In addition, a kinetic model was formulated using (PPGK) (discussed below), had to be used as a thermophilic multiple experimental datasets and was used to optimize the source and was not available. Because activity of this enzyme overall reaction rate and hydrogen yield. The relative simplicity significantly decreases above 40 °C (32), biomass utilization ex- of in vitro synthetic enzymatic pathways compared with whole periments were conducted at 40 °C to ensure complete conver- cells makes the overall system far easier to simulate and use sion. For reaction rate increase experiments, a simplified path- for predictions (15). Kinetic models for enzymatic hydrogen way was used, where phosphorylation enzymes were eliminated production have been used previously to optimize the reactor and glucose-6-phosphate was used as the substrate. Due to the design; however, experimental validation had not yet been use of only thermophilic enzymes in this pathway, the reaction performed (27). The problem of fitting kinetic parameters of temperature was 50 °C for these experiments.
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