Next Article in Journal
Experiment and Simulation of Medium-Duty Tactical Truck for Fuel Economy Improvement
Next Article in Special Issue
Affects of Mechanical Milling and Metal Oxide Additives on Sorption Kinetics of 1:1 LiNH2/MgH2 Mixture
Previous Article in Journal
Comparison of Ferry Boat and Highway Bridge Energy Use
Previous Article in Special Issue
Recent Progress in Metal Borohydrides for Hydrogen Storage
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:

Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy

Biological Systems Engineering Department, 210-A Seitz Hall, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
Institute for Critical Technology and Applied Sciences (ICTAS) Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
DOE BioEnergy Science Center (BESC), Oak Ridge, TN 37831, USA
Gate Fuels Inc. 3107 Alice Drive, Blacksburg, VA 24060, USA
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA
Author to whom correspondence should be addressed.
Energies 2011, 4(2), 254-275;
Received: 15 December 2010 / Revised: 8 January 2011 / Accepted: 28 January 2011 / Published: 31 January 2011
(This article belongs to the Special Issue Hydrogen Storage)


The hydrogen economy presents an appealing energy future but its implementation must solve numerous problems ranging from low-cost sustainable production, high-density storage, costly infrastructure, to eliminating safety concern. The use of renewable carbohydrate as a high-density hydrogen carrier and energy source for hydrogen production is possible due to emerging cell-free synthetic biology technology—cell-free synthetic pathway biotransformation (SyPaB). Assembly of numerous enzymes and co-enzymes in vitro can create complicated set of biological reactions or pathways that microorganisms or catalysts cannot complete, for example, C6H10O5 (aq) + 7 H2O (l) → 12 H2 (g) + 6 CO2 (g) (PLoS One 2007, 2:e456). Thanks to 100% selectivity of enzymes, modest reaction conditions, and high-purity of generated hydrogen, carbohydrate is a promising hydrogen carrier for end users. Gravimetric density of carbohydrate is 14.8 H2 mass% if water can be recycled from proton exchange membrane fuel cells or 8.33% H2 mass% without water recycling. Renewable carbohydrate can be isolated from plant biomass or would be produced from a combination of solar electricity/hydrogen and carbon dioxide fixation mediated by high-efficiency artificial photosynthesis mediated by SyPaB. The construction of this carbon-neutral carbohydrate economy would address numerous sustainability challenges, such as electricity and hydrogen storage, CO2 fixation and long-term storage, water conservation, transportation fuel production, plus feed and food production.

Graphical Abstract

1. Introduction

The hydrogen economy is a hypothetical future energy system, where hydrogen will be used as an energy storage carrier for end users, especially in the transport sector. Electrochemist John O’M. Bockris coined the term “hydrogen economy” in his speech given at General Motors (GM) Technical Center in 1970 [1]. Two of the major advantages of the hydrogen economy are (i) much higher energy conversion efficiency from chemical energy to electricity through fuel cells compared to internal combustion engines, whose maximum efficiencies are restricted by the Second Law of thermodynamics, and (ii) nearly no pollutants generated at the sites of end users [2,3]. In addition, hydrogen can be produced by a number of approaches, and multiple energy sources from fossil fuels (e.g., natural gas, oil, coal) to renewable energy sources (e.g., biomass, solar energy, wind energy) in the future. Implementation of the hydrogen economy must address several challenges—low-cost sustainable hydrogen production, high density hydrogen storage, affordable hydrogen distribution infrastructure, durable and affordable fuel cell systems, and eliminating safety concern [4]. Among them, hydrogen storage is the most important challenge that we must solve [2,3,4,5,6,7,8].
For the transport sector, light-duty passenger vehicles account for ~60% of transportation energy consumption in the USA [9]. Ideal transportation fuel must be stored in both high gravimetric and volumetric densities for a sufficient driving range before refilling. Gravimetric and volumetric energy densities for liquid fuels, solid fuels, hydrogen storage, and batteries are presented in Figure 1. Clearly, liquid fuels, such as diesel, gasoline, and jet fuels, are the most energy-dense fuels followed by liquid biofuels, such as biodiesel, butanol, and ethanol (Figure 1a). High energy density liquid (bio)fuels bring several advantages, such as, long travelling distance, fast refilling (MJ/min), easy storage, and convenient distribution of liquid fuels.
Hydrogen has a very high energy density on a mass basis (143 MJ/kg) but it has very low volumetric densities because it is gas at ambient conditions. Increasing hydrogen gas pressure can improve the volumetric energy density but the weight of hydrogen containers decreases their apparent gravimetric density. For example, 350 bar and 700 bar hydrogen gas containers have gravimetric densities of 2.8–3.7 and 2.5–4.3 H2 wt%, respectively (Figure 1b). The most common on-board hydrogen storage method in today's demonstration fuel cell vehicles is compressed hydrogen gas at pressures of roughly 700 bars. Also, compressing hydrogen consumes a significant fraction of hydrogen energy by about 10–17% [4]. Alternatively, hydrogen can be liquefied at −253 °C with a volumetric density of 71 kg of H2/m3 [2]. Considering the weight of the well-insulated cryogenic containers, the apparent gravimetric and volumetric densities are ~4.5–6.5 H2 wt% and ~30–40 kg of H2/m3, respectively (Figure 1b). A third approach is to absorb hydrogen into ultra-large surface solid storage materials, such as metal organic framework (MOF), nanostructured carbons (including carbon nanotubes), and clathrate hydrate [5,6,7,10] but their densities are not high enough (Figure 1b). Distinct from direct hydrogen storage, hydrogen can be stored as a chemical hydride, a light metal, or in a hydrogen-containing compound [2,3,6,11]. At the point of end users, hydrogen gas can be generated from their decomposition or reaction with water. In addition to their low hydrogen densities, cost-effective regeneration of hydride or light metals must be addressed.
Figure 1. Comparison of gravimetric densities and volumetric densities for liquid fuels, solid fuels, hydrogen storage means, and batteries.
Figure 1. Comparison of gravimetric densities and volumetric densities for liquid fuels, solid fuels, hydrogen storage means, and batteries.
Energies 04 00254 g001
It is very challenging to find out a possible practical solution to meet the US Department of Energy’s ultimate hydrogen storage goals— ≥8 H2 wt% and ≥70 kg of H2/m3 (Figure 1b). To advertise and incentivize hydrogen research, the 2009-11 Hydrogen Prize will be awarded in the area of storage materials in mobile systems for light-duty vehicles [12]. The basic energy density requirements are 7.5% reversible H2 mass at conditions between −40 to +85 °C and between 1.5 to 150 bars of H2 pressure and 70 kg of total releasable hydrogen/m3 [3].
Another alternative to address the hydrogen storage challenges is use of high energy density hydrogen carriers, such as hydrocarbon, biodiesel, methanol, ethanol, ammonia and carbohydrate [3,13,14,15] (Figure 1a). But end users must have a reformer that can convert hydrogen carriers to hydrogen and purify the hydrogen stream before entering proton exchange membrane (PEM) fuel cells. The bulky size of reformers , difficult and complicated controls, plus the must need for product purification may prohibit their applications in small-size passenger vehicles [2].
Future hydrogen distribution infrastructure is closely linked with efficient hydrogen storage technology. Although natural gas and oil can be transported energy-efficiently through pipeline, it is costly for hydrogen because energy densities of compressed hydrogen based on volume is much lower than those of natural gas [16]. In addition, special pipeline materials and liners are required to avoid hydrogen embrittlement of steel. Local hydrogen stations far from a hydrogen pipeline must get the supply through hydrogen tanks, compressed hydrogen tube trailers, liquid hydrogen trailers, liquid hydrogen tank trucks or satellite on-site hydrogen producers. It is estimated that at least several hundred billions of dollars will be spent for constructing the infrastructure of the hydrogen economy based on the use of gaseous hydrogen in the US alone [17,18].
In this opinion article, we presented a new concept—the carbon-neutral carbohydrate economy based on the use of renewable carbohydrate (cellulose or starch) as a high energy density hydrogen carrier (Figure 1a), discussed great potential of the carbohydrate economy, and highlighted future R&D priorities for its innovation.

2. Hydrogen Production from Carbohydrate by Cell-Free Enzymatic Technology

The production of hydrogen from low-cost renewable biomass (e.g., $60 per dry ton or $3.60 per GJ) [2] is appealing because biomass is an enriched, collectable chemical energy source and its production would be scalable. A number of chemical and biological approaches as well as their combinations have been investigated for the production of hydrogen from biomass carbohydrate. Chemical catalysis approaches include pyrolysis [19], gasification [20], and aqueous phase reforming [21]. All of the chemical catalysis approaches still suffer from low hydrogen yields, poor hydrogen selectivity, and poor energy efficiencies. Microbial anaerobic fermentation can produce up to four moles of hydrogen per mole of glucose along with two moles of acetate, called the Thauer limit [22]. In practice, much lower hydrogen yields (e.g., ~1–3.2 moles of hydrogen per mole of glucose) are usually obtained in dark anaerobic fermentation [23,24,25]. Thermophilic hydrogenesis microbes can produce nearly four moles of hydrogen per glucose unit [26]. The production of hydrogen by purple bacteria or microalgae are difficult to scale up due to low energy concentration insolation (e.g., ~200 W/m2) and low photon capture densities by the cells [27]. The bioelectrochemically-assisted microbial fuel cell reactor has been shown to convert two moles of acetate to up to eight moles of hydrogen with the help of a little electricity, resulting in the overall hydrogen yields of approximately nine moles per mole of glucose [28]. A combinatorial biological and chemical catalysis is ethanol microbial fermentation followed by ethanol partial oxidation reforming, resulting in an overall yield of ~9 moles of hydrogen per mole of glucose [14,29].
The production of nearly 12 moles of hydrogen generated from glucose unit was a long-term dream [30]. This goal was not achieved until a new biocatalysis technology—cell-free synthetic enzymatic pathway biotransformation (SyPaB) was developed. SyPaB can implement complicated biochemical reaction networks by assembling a number of purified enzymes and coenzymes [31,32,33]. Microorganisms have complicated biochemical reaction network (Figure 2a) but only a small fraction of enzymes (e.g., less than 1%) are responsible for bioconversion of a substrate to desired product while most of them are responsible for self-duplication, self-repairing, and non-product-related metabolisms. SyPaB is an open system that can be controlled and regulated easily based upon the inputs, such as temperature, substrate, pressure, and so on (Figure 2c). Breaking the cellular membrane of one microorganism and utilization of intracellular enzyme mixture for complicated biochemical reactions have been used to implement cell-free ethanol fermentation [31] or cell-free protein synthesis [34] (Figure 2b). But SyPaB has more flexibility as compared to the previous cell-free systems since we can assemble numerous enzymes from different sources and adjunct together (Figure 2c). One of the most important features of SyPaB can reach nearly theoretical product yields because selecting only required enzymes eliminates unnecessary pathways that consume a significant fraction of carbon and energy sources [35,36]. In addition, cell-free systems has usually much higher reaction rates than microbial fermentations because of faster mass transfer without cellular membrane and higher biocatalyst density without dilution effects from other biomacromolecules and their side reactions [2,3,31,37,38].
Figure 2. System complexity comparison of in vivo microbial fermentation (a), cell-free fermentation using the cell lysate from one organism by breaking its cellular membrane (b), and cell-free synthetic enzymatic pathway biotransformation that assembles the purified stable enzymes from different organisms (c).
Figure 2. System complexity comparison of in vivo microbial fermentation (a), cell-free fermentation using the cell lysate from one organism by breaking its cellular membrane (b), and cell-free synthetic enzymatic pathway biotransformation that assembles the purified stable enzymes from different organisms (c).
Energies 04 00254 g002
To break the Thauer limit of hydrogen-producing microbes, the synthetic enzymatic pathways have been designed to split water by using the chemical energy in carbohydrate [39,40] as
C6H10O5 (aq) + 7 H2O (l) → 12 H2 (g) + 6 CO2 (g)
These non-natural synthetic catabolic pathways are comprised of 13–14 enzymes in one pot (Figure 3a). The pathways contain four biocatalytic modules: (i) a chain-shortening phosphorylation reaction for producing glucose-1-phosphate (G-1-P) catalyzed by glucan phosphorylase (Equation 2); (ii) generation of glucose-6-phosphate (G-6-P) from G-1-P catalyzed by phosphoglucomutase (Equation 3); (iii) generation of 12 NADPH from G-6-P through a pentose phosphate pathway and some enzymes in the glycolysis and gluconeogenesis (Equation 4); and (iv) generation of hydrogen from NADPH catalyzed by NADP-dependent hydrogenase (Equation 5).
(C6H10O5)n + Pi ↔ (C6H10O5)n−1 + G-1-P
G-1-P ↔ G-6-P
G-6-P + 12 NADP+ + 7 H2O ↔ 12 NADPH + 12 H+ + 6 CO2 + Pi
12 NADPH + 12 H+ ↔ 12 H2 +12 NADP+
The entire process can be described as oxidization of glucose unit by utilization of water rather than oxygen as an electron receptor. The overall reaction (Equation 1) from polysaccharides and water is spontaneous and endothermic (i.e., ΔGo = ~−50 kJ/mol and ΔH° = +598 kJ/mol) [39,40]. These entropy-driven chemical reactions can generate output of chemical energy in the form of hydrogen more than the input of chemical energy in polysaccharides by absorbing ambient-temperature waste energy because of phase changes from more orderly to less orderly [39,40]. But such catabolism does not occur in any microorganism because it cannot produce any energy currency (i.e., ATP) for microorganism’s growth and duplication.
When polysaccharides and water are mixed, no reaction occurs as everyone observes. When the enzyme cocktail is added, hydrogen and carbon dioxide is generated spontaneously (Figure 3b,c). Unidirectional reactions occur because the removal of gaseous products—H2 and CO2 from the aqueous phase under mild reaction conditions (<100 °C and ~1 atm). We have shown that the production rates of H2 by SyPaB are higher than those of photobiological systems and comparable to those reported for dark fermentations and electrohydrogensis [41,42]. It is expected that enzymatic hydrogen generation rates can be further increased greatly by several orders of magnitude for different applications in the near future [2,3].
Due to essentially 100% chemical selectivity, mild reaction conditions, and high-purity hydrogen, we propose the use of renewable carbohydrates as a high-density hydrogen carrier. Polysaccharides have a chemical formula of C6H10O5 with a reaction of C6H10O5 (aq) + 7 H2O (l) → 12 H2 (g) + 6 CO2 (g). As a result, hydrogen storage density in polysaccharides is 24/162 = 14.8 H2 mass%, when water can be recycled from PEM fuel cells. Without water recycling from fuel cells, the sugar/water slurry has a hydrogen density of 24/(162 + 18 × 7) = 8.33% H2 mass%. Dry starch and amorphous cellulose have specific densities of ~0.7 and ~1.4 kg/L, respectively [43]. Therefore, carbohydrates as a hydrogen carrier would meet the ultimate DOE’s goals (Figure 1b).
Figure 3. The cell-free synthetic enzymatic pathway (a), high-yield generation of hydrogen from starch (b) [40] or soluble cellodextrin (c) [39]. The enzymes are: GNP, glucan phosphorylase; PGM, phosphoglucomutase; G6PDH, G-6-P dehydrogenase; 6PGDH, 6-phosphogluconate dehydrogenase; R5PI, phosphoribose isomerase; Ru5PE, ribulose 5-phosphate epimerase; TKL, transketolase; TAL, transaldolase; TIM, triose phosphate isomerase; ALD, aldolase; FBP, fructose-1, 6-bisphosphatase; PGI, phosphoglucose isomerase; and H2ase, hydrogenase. The metabolites and chemicals are: g1p, glucose-1-phosphate; g6p, glucose-6-phosphate; 6pg, 6-phosphogluconate; ru5p, ribulose-5-phosphate; x5p, xylulose-5-phosphate; r5p, ribose-5-phosphate; s7p, sedoheptulose-7-phosphate; g3p, glyceraldehyde-3-phosphate; e4p, erythrose-4-phosphate; dhap, dihydroxacetone phosphate; fdp, fructose-1,6-diphosphate; f6p, fructose-6-phosphate; and Pi, inorganic phosphate.
Figure 3. The cell-free synthetic enzymatic pathway (a), high-yield generation of hydrogen from starch (b) [40] or soluble cellodextrin (c) [39]. The enzymes are: GNP, glucan phosphorylase; PGM, phosphoglucomutase; G6PDH, G-6-P dehydrogenase; 6PGDH, 6-phosphogluconate dehydrogenase; R5PI, phosphoribose isomerase; Ru5PE, ribulose 5-phosphate epimerase; TKL, transketolase; TAL, transaldolase; TIM, triose phosphate isomerase; ALD, aldolase; FBP, fructose-1, 6-bisphosphatase; PGI, phosphoglucose isomerase; and H2ase, hydrogenase. The metabolites and chemicals are: g1p, glucose-1-phosphate; g6p, glucose-6-phosphate; 6pg, 6-phosphogluconate; ru5p, ribulose-5-phosphate; x5p, xylulose-5-phosphate; r5p, ribose-5-phosphate; s7p, sedoheptulose-7-phosphate; g3p, glyceraldehyde-3-phosphate; e4p, erythrose-4-phosphate; dhap, dihydroxacetone phosphate; fdp, fructose-1,6-diphosphate; f6p, fructose-6-phosphate; and Pi, inorganic phosphate.
Energies 04 00254 g003

3. The Carbon-Neutral Carbohydrate Economy

Terrestrial plants are natural solar energy collectors that can utilize intermittent low-energy concentration solar energy and fix CO2 for the production of biomass carbohydrate. Natural photosynthesis suffers from low solar energy conversion efficiencies (4.6–6%, theoretical; 0.2–1.0%, practical) mainly due to narrow utilization of insolation spectrum (called photo-synthetically active radiation), low efficiency of carbohydrate synthesis, and respiration loss of living plants [16,44,45,46]. At the same time, plants consume water largely for transpiration but not for carbohydrate synthesis [47,48]. In spite of its low efficiency, approximately ~150 billion tons of dry phytomass is generated by terrestrial plants because plants are widely distributed and growth in large area terrestrial lands [49,50]. If 10% of phytobiomass (i.e., 15 billion tons of dry biomass) was utilized, we would greatly decrease our reliance on fossil fuels.
The carbon-neutral carbohydrate economy is suggested as shown in Figure 4, where carbohydrate has a central role as an energy carrier for end users. Currently carbohydrate from terrestrial plants is used as food and animal feed, sources of renewable materials (e.g., wood, paper, polylactatic acid) and liquid biofuels (e.g., ethanol and butanol), accounting for only 1.5% of NPP [51]. In the future, we envision that carbohydrate would have two new functions—a high-energy density hydrogen carrier and an electricity storage compound (~11–14 MJ electricity/kg carbohydrate). For hydrogen users, the delivered solid carbohydrate or slurry can be converted to hydrogen at the points of end users for numerous applications, such as ammonia synthesis, oil refineries or biorefineries, pharmaceutical industry, or hydrogen fuel cell vehicles through local sugar-to-hydrogen stations. For high-power electricity users, carbohydrate can be converted to hydrogen followed by PEM fuel cells, such as sugar fuel cell vehicles or distributed electricity generators [3]. For low-power electricity users, sugars can be converted to electricity through enzymatic fuel cells, such as battery rechargers or portable electronics [3,38].
Figure 4. The carbon-neutral carbohydrate economy linked with its current and future productions and applications.
Figure 4. The carbon-neutral carbohydrate economy linked with its current and future productions and applications.
Energies 04 00254 g004
Solar energy is not really free because of the high hidden costs for its conversion, scale-up, and storage [52,53,54]. Efficient storage of solar electricity or solar fuel (e.g., hydrogen) on a large scale is one of the most important challenges for energy utilization, storage, and transport [55,56]. Professors Lewis and Nocera [56] suggested “store solar-converted energy in the form of chemical bonds, … such as hydrogen, methane, methanol, or other hydrocarbon species.” Here we argue that carbohydrate is a perfect energy storage compound, much better than methane, methanol, and hydrogen. To produce solar starch, we propose a scalable novel concept—artificial photosynthesis system including commercial solar cells (η = ~18–42%), mature water electrolysis (η = ~80–85%), or their combination—direct solar hydrogen, and a novel starch synthesis from hydrogen and carbon dioxide mediated by a non-natural cell-free synthetic enzymatic pathway (η = ~78%, projected) (Figure 4). The overall efficiency of starch synthesis from insolation could range from 12 to 28% and this system consumes only one thousandth of water of that of natural photosynthesis [46]. Implementation of the carbon-neutral carbohydrate economy would solve a number of challenges associated with sustainability, such as electricity and hydrogen storage, CO2 fixation, water conservation, transportation fuel production, feed and food production, and so on [46].

4. SyPaB Challenges and Opportunities

Modifying living organisms with new functions is difficult for several reasons while cell-free systems can be modified and controlled more easily [31,32,33,57]. First, living biological entities are complex open systems that operate far from thermodynamic equilibrium. Second, we can control and define the components in vitro but this operation is much harder to do in vivo. Third, it is relatively straightforward to measure rates and equilibrium constants in vitro, but these quantities are generally not easy to ascertain in vivo [58]. Fourth, the number of some types of molecules in individual cells is small and variable such that stochastic effects may dominate behavior [59]. Fifth, we can control the specificity of molecular processes in vitro by simply omitting or adding components but we know little about in vivo spatial organization of intracellular enzymes in pathways [60,61].
The prejudices that SyPaB is impossible for the economically viable production of low-value biofuels are attributed to a fixed paradigm in most persons’ minds, resulting in a blind spot. The possible doubts include (i) enzyme instability, (ii) costly enzyme production, (iii) high cost for protein purification, (iv) high cost for protein immobilization, (v) labile costly cofactors, (vi) a lack of thermostable enzymes, (vii) complicated process operation, and (viii) high risk in scale-up.
Most enzymes in research laboratories are inactivated so rapidly that this observation leaves the impression that enzymes are not stable enough for long-time use. In reality, they may be very stable under the proper conditions by using correct enzymes, buffers, and conditions. For example, proteases in detergents have a shelf lifetime of several years at ambient temperature and keep their activities in up to 80–90 °C hot water for tens of minutes. The enzymes used in the blood sugar testing kits have a shelf lifetime of more than one year at ambient temperature. High fructose corn syrup, low-cost sucrose replacement, is made by stabilized glucose isomerase, which can work at ~60 °C for even about two years [62]. We estimated that the enzyme costs would be very small when every enzyme in the SyPaB cocktails has total turn-over number (TTN) values of more than 107–108 [31,33,35]. Based on our experience and data in the literature, it is relatively easy to obtain thermoenzymes meeting with such basic TTN requirement, for example, Clostridium themocellum phosphoglucomutase (PGM) [63], C. themocellum phosphoglucose isomerase (PGI) [64], Thermotoga martima 6-phosphogluconate dehydrogenase (6PGDH) [65], T. martima fructose bisphosphatase (FBP) [66], and immobilized glucose isomerase [62].
Costly enzymes purchased from Sigma-Aldrich and other biotechnology companies (e.g., New England Biolabs) give most researchers an impression that enzymes are very costly. Also, most academic researchers know little about the advances of industrial enzymes. In reality, bulk industrial enzymes can be produced and obtained at very low costs, for example, ~$5 per kg of crude protease produced by Bacillus subtilis, ~$10 per kg of cellulase produced by Trichoderma spp., and tens of dollars per recombinant proteins produced in E. coli [67,68,69,70]. For industrial enzyme manufacturers, high-cell density fermentations are usually conducted. For example, Trichderma spp. can produce more than 100 gram of dry weight cellulase per liter of the culture [68], and Bacillus spp. can produce more than 30 gram of protease per liter of the culture [70]. But it is very difficult for academic laboratories to produce Trichoderma cellulase at even one tenth industrial levels. Systematic efforts are usually needed to increase recombinant protein expression levels. For example, expression levels of a recombinant hyperthermophilic T. maritima 6-phosphogluconate dehydrogenase in E. coli have been increased by ~500-fold by optimization of codon usage, expression plasmid and host, inducer type, concentration, and adding time, and so on [65]. Typical values of recombinant enzyme yield based on sugar (YP/S) may be up to 0.2 g of protein/g of sugar consumed [35], based on several well-known facts of (i) cell mass yield based on sugar in aerobic fermentation (YX/S) = ~0.5–0.6 g cell mass/g sugar [33,36,71], (ii) high intracellular cellular protein content = ~0.5–0.6 g protein/g cell mass [72], and (iii) high recombinant enzyme percentage in a total cellular protein = 50–70% [63,65,66,73]. Based on the rule of thumb of industrial fermentations, it is estimated that the production cost of recombinant protein may be $4.50–7.5/kg of protein = $0.30/kg sugar/(0.2 g protein/g sugar) × 3–5 (a cost adjustment coefficient due to other nutrient costs, labor, etc.).
In academic laboratory, most enzymes are purified through chromatographic techniques so that they are costly and hard to scale up. In reality, several low-cost scalable protein purification approaches are available, for example, simple centrifugation for secretory enzymes, adsorption/desorption on low-cost cellulosic materials [74,75] (Figure 5), heat precipitation for thermostable enzymes [65,76] (Figure 6), and ammonia precipitation [77] (Figure 6). Therefore, the purification costs for bulk enzymes would become relatively minor.
Figure 5. Scalable protein purification based on affinity adsorption of cellulose-binding module (CBM)-tagged protein on low-cost cellulosic absorbent followed by intein self-cleavage [74].
Figure 5. Scalable protein purification based on affinity adsorption of cellulose-binding module (CBM)-tagged protein on low-cost cellulosic absorbent followed by intein self-cleavage [74].
Energies 04 00254 g005
Figure 6. Simple purification of a thermoenzyme—Thermotoga maritima fructose-1,6-bisphosphate aldolase by heat precipitation and ammonia precipitation. M, protein marker; SN, supernatant of the cell lysate; HP, the supernatant after heat precipitation; SNA, the precipitate after heat treatment containing the E. coli heat-labile cellular proteins; AP, precipitated aldolase by using ammonia sulfate.
Figure 6. Simple purification of a thermoenzyme—Thermotoga maritima fructose-1,6-bisphosphate aldolase by heat precipitation and ammonia precipitation. M, protein marker; SN, supernatant of the cell lysate; HP, the supernatant after heat precipitation; SNA, the precipitate after heat treatment containing the E. coli heat-labile cellular proteins; AP, precipitated aldolase by using ammonia sulfate.
Energies 04 00254 g006
Enzyme immobilization is a relative mature industrial operation. The aforementioned example of glucose isomerase stability is only possible due to its immobilization permitting it to function at 60 °C for more than two years before it must be replaced [62]. Low-cost enzyme immobilization technologies are becoming widely-adopted, for example, cross-linking enzyme aggregate (CLEA) [78,79,80] and cellulose-binding module-tagged (CBM-tagged) protein immobilization [74,81,82,83,84]. The CBM-tagged enzymes can be purified and immobilized in one step, which greatly decreases processing costs and operation complexity (Figure 7).
Figure 7. One-step protein purification and immobilization of a CBM-tagged protein on a cellulosic absorbent.
Figure 7. One-step protein purification and immobilization of a CBM-tagged protein on a cellulosic absorbent.
Energies 04 00254 g007
With regard to costly labile cofactors, efficient NAD(P)H recycling through immobilization of the cofactor linked to polymers has been developed [85]. The highest turn-over number (TTN) value for NAD recycling reported in the literature is >1,000,000 [86]. The pharmaceutical industry nearly stops further efforts in increasing TTN values of ~10,000–100,000 [87,88] due to high selling values of chiral compounds plus the additional regulatory requirements if the current process is modified. But it is vital that TTN values of NAD should be higher than 106 for the economically viable production of biocommodities [31]. Alternatively, the labile cofactor issue can be addressed by the use of low-cost more stable NAD biomimetic cofactors. But this research area is in its infancy [89,90] because there has not been a sufficiently large market/product opportunity to support further R&D before the invention of SyPaB. Several redox enzymes have been engineered for better performance on biomimetic coenzymes [91,92,93].
For similar reasons, there is a lack of thermostable carbohydrate-metabolized enzymes for the SyPaB projects before the invention of SyPaB. Some thermostable enzymes having clear applications, such as DNA polymerases, glucose isomerases, proteases, amylases, and hemicellulases, have been exploited intensively [94,95,96]. Since cellulase has great potential in the production of cellulosic biofuels, several companies such as Megazyme, NZYtech are selling recombinant cellulases for research purpose. To understand crystalline structure of the proteins, Japanese scientists have undertaken the Thermus thermophiles HB8 Whole Cell Project aimed at expressing all the genes in E. coli. Consequently, more than 1400 T. thermophilus HB8 genes have been successfully over-expressed [97], but most of recombinant proteins are still not commercially available. With the fast explosion of thermophilic enzyme genomic DNA sequence databases and bioinformatics tools [98,99,100,101,102,103], the discovery of thermoenzymes is becoming a low-risk project. A combination of robotic automation and high throughput cloning makes large-size discovery of thermoenzymes feasible. In the biopharmaceutical industry, screening a large enzyme library (e.g., ~500 to thousands of enzymes) for identification of one desired enzyme for the specific biocatalytic reaction is becoming a routine operation. An alternative approach for stabilizing highly-active enzymes from mesophilic sources is to use directed evolution or rational design [104,105,106,107]. Also, it is possible to let one host co-express several enzymes [37]. As a result, a combination of the enzyme cocktails from several hosts would greatly decrease fermentation burden and purification requirement. With the great potential of SyPaB technology, one might expect the construction of large library of thermoenzymes within a short time. In the future, we would expect a market to develop for numerous thermostable enzymes as basic building blocks just like purchasing computer parts, and each enzyme would have different optimal working conditions (e.g., pH, temperature, cofactor, buffer, etc.)
SyPaB seems to have more complicated process operation involving protein production through microbial fermentation, cell lysis, protein purification and/or immobilization, and cocktailing (Figure 8b) as compared to microbial fermentation (Figure 8a). Although SyPaB requires protein purification and immobilization as well as cocktailing, SyPaB has a great advantage—a properly engineered SyPaB enzymatic system should function much longer (e.g., one or two orders of magnitude) than (resting-cell) microbial fermentation because each enzyme of the enzyme cocktails has been stabilized.
Figure 8. Process complexity comparison of microbial (ethanol) fermentation involving two steps—microorganism growth and ethanol fermentation (a) and a typical SyPaB involving protein production by microbial fermentation, protein purification and/or immobilization, and biotransformation by using the stabilized enzyme mixture (b).
Figure 8. Process complexity comparison of microbial (ethanol) fermentation involving two steps—microorganism growth and ethanol fermentation (a) and a typical SyPaB involving protein production by microbial fermentation, protein purification and/or immobilization, and biotransformation by using the stabilized enzyme mixture (b).
Energies 04 00254 g008
There is no obvious technical obstacle to SyPaB implementation because all of the recombinant enzymes, including the complicated nickel-iron hydrogenase [108], can be produced by E. coli; neither (labile) membranes nor (costly) membrane proteins are needed; neither CoA nor ATP are involved; no costly substrate is needed; the sugar-to-hydrogen reaction is conducted under strictly anaerobic conditions even when trace amount of oxygen at the beginning can be self-refreshed by the gaseous products; and low-temperature heat sources are available from PEM fuel cells or the environment.
The ultimate hydrogen production cost is estimated based on the substrate costs, catalyst costs, product separation and purification costs, and other consumable chemicals. For the production of commodities by mature technologies, the feedstock usually account for ~50–80% of the final product price, for example, petroleum vs. gasoline, vegetable oil vs. biodiesel, corn kernels vs. ethanol [31,109]. Given a price of $0.18/kg carbohydrate (i.e., $10.6/GJ) [2,44], the hydrogen production cost could range from $1.52 to $2.43/kg of hydrogen based on the above rule of thumb. The detailed estimate is based on (i) $0.18/kg carbohydrate, (ii) all enzymes have production costs of $40 or $400 per kg of enzyme, and (iii) low-cost biomimetic cofactors used. Figure 8a shows the enzyme total turn-over number (TTN, mol product/mol enzyme) effects on hydrogen production costs. The production costs decrease rapidly with increasing TTN of the enzymes in SyPaB, and then levels off when all enzymes regardless of their production costs have TTN values of more than 108–9. When all enzymes have TTN values of 3 × 107 and each one has production costs of ~$40/kg, hydrogen production cost is anticipated to be $1.87/kg H2, where carbohydrate ($0.18/kg carbohydrate) accounts for approximately 65% of hydrogen production cost. When TTN values of the enzymes are further enhanced to 108 or 109, the ultimate cost of hydrogen would be as low as $1.30/kg H2. Such hydrogen production costs by SyPaB would be lower than that from natural gas (e.g., ~$2.00 per kg of hydrogen). Considering potential benefits, such as nearly zero carbon footprint, evenly-distributed renewable resource, biohydrogen produced by SyPaB represents a beginning stage of low-cost, green, hydrogen production [110].
Our research showed that the gas produced by SyPaB contain 67% H2 and 33% CO2 [39,40]. Hydrogen and carbon dioxide can be separated by membrane technology, pressure swing adsorption, or a hybrid of both [111]. Separation cost of H2 and CO2 is estimated to be very low [36]. Since high-purity hydrogen is mixed with an inert gas—CO2, this H2/CO2 mixture can be directly used by PEM fuel cells with ~1% slight loss in fuel cell efficiency [36].
Figure 9b shows typical TTN values of the enzymes in industrial applications and obtained in our laboratory. The relatively high cost of biomass saccharification is mainly due to very low TTN values for cellulase, for example, compared to amylase, which has at least 10-fold higher TTN values allowing amylase costs to be much lower in starch ethanol biorefineries. For industrial fructose production, ultra-stable immobilized thermophilic glucose isomerase with TTN of ~109 leads enzyme cost to a near negligible level. In our laboratory, we have obtained a few thermostable enzymes with TTN values of more than 107, for example, C. thermocellum phosphoglucomutase [63], T. maritima fructose-1,6-bisphosphatase [66], and T. maritima 6-phosphogluconate dehydrogenase [65]. We have found out that free C. thermocellum phosphoglucose isomerase has low TTN values but it becomes ultra-stable (more than 109) after simple immobilization through adsorption on cellulose surface by using cellulose-binding module [64], as shown in Figure 7. Clearly, it is highly operative to obtain numerous high-TTN non-membrane enzymes suitable for the SyPaB projects.
Figure 9. Cost analysis of hydrogen production in terms of TTN values of the enzymes (a) and typical total turn-over number (TTN) values of enzymes (b).
Figure 9. Cost analysis of hydrogen production in terms of TTN values of the enzymes (a) and typical total turn-over number (TTN) values of enzymes (b).
Energies 04 00254 g009
With developments in (i) engineered oxidoreductases that can use biomimetic NAD factors [91,92,93] and (ii) stable enzymes as building blocks of SyPaB [63,65,66,73], we estimate that ultimate hydrogen production costs may decrease to ~$1.30 per kg of hydrogen (Figure 9a), where carbohydrate accounts for ~95% of its production costs, in part because hydrogen has very low product separation and purification costs and the other chemicals in the reaction can be recycled.

5. Conclusion

The depletion of petroleum and the concerns of climate change, national energy security, and wealth transfer are driving the world to seek alternative transportation fuel sources and increase energy utilization efficiency. Due to special requirements for the transport sector, such as high power-to-weight ratio for engines, high energy storage density [2,3,36], the International Energy Agency, along with numerous experts, predicts that hydrogen fuel cell powertrain systems will become dominant, more acceptable than electric vehicles, in the future [112,113]. The use of renewable carbohydrate as a hydrogen storage carrier not only solves low-cost sustainable hydrogen production challenge but also address the challenges associated with storage, safety, distribution, and infrastructure in the future hydrogen economy [2,3,40]. International collaborative research is urgently needed to remove the remaining technical obstacles to a new carbon-neutral carbohydrate economy.


This work was supported mainly by the Air Force Office of Scientific Research, and partially by DOE Bioenergy Science Center (BESC) and USDA Biodesign and Bioprocess Center. The BioEnergy Science Center is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. We appreciated the valuable suggestions and comments by Brian Davison. We also thanked Jian-Jiang Zhong for the expression plasmid encoding T. maritima fructose-1,6-bisphosphate aldolase.


  1. Bockris, J.O.M. Energy, the Solar Hydrogen Alternative; Halsted Press: New York, NY, USA, 1975. [Google Scholar]
  2. Zhang, Y.-H.P. A sweet out-of-the-box solution to the hydrogen economy: Is the sugar-powered car science fiction? Energy Environ. Sci. 2009, 2, 272–282. [Google Scholar] [CrossRef]
  3. Zhang, Y.-H.P. Renewable carbohydrates are a potential high density hydrogen carrier. Int. J. Hydrogen Energy 2010, 35, 10334–10342. [Google Scholar] [CrossRef]
  4. DOE. Basic Research Needs for the Hydrogen Economy. Available online: (accessed on 28 January 2011).
  5. Graetz, J. New approaches to hydrogen storage. Chem. Soc. Rev. 2009, 38, 73–82. [Google Scholar] [CrossRef] [PubMed]
  6. Orimo, S.i.; Nakamori, Y.; Eliseo, J.R.; Zuttel, A.; Jensen, C.M. Complex hydrides for hydrogen storage. Chem. Rev. 2007, 107, 4111–4132. [Google Scholar] [CrossRef] [PubMed]
  7. Struzhkin, V.V.; Militzer, B.; Mao, W.L.; Mao, H.k.; Hemley, R.J. Hydrogen storage in molecular clathrates. Chem. Rev. 2007, 107, 4133–4151. [Google Scholar] [CrossRef] [PubMed]
  8. Wang, P.; Ma, L.; Fang, Z.; Kang, X.; Wang, P. Improved hydrogen storage property of Li–Mg–B–H system by milling with titanium trifluoride. Energy Environ. Sci. 2009, 2, 120–123. [Google Scholar] [CrossRef]
  9. Lynd, L.R.; de Brito Cruz, C.H. Make way for ethanol. Science 2010, 330, 1176. [Google Scholar] [CrossRef] [PubMed]
  10. Dillon, A.C.; Heben, M.J. Hydrogen storage using carbon adsorbents: Past, present and future. Appl. Phys. A Mater. Sci. Process. 2001, 72, 133–142. [Google Scholar] [CrossRef]
  11. Sakintuna, B.; Lamari-Darkrim, F.; Hirscher, M. Metal hydride materials for solid hydrogen storage: A review. Int. J. Hydrogen Energy 2007, 32, 1121–1140. [Google Scholar] [CrossRef]
  12. Homepage of H-prize. Available online: (accessed on 28 January 2011).
  13. Christensen, C.H.; Johannessen, T.; Sørensen, R.Z.; Nørskov, J.K. Towards an ammonia-mediated hydrogen economy? Catalysis Today 2006, 111, 140–144. [Google Scholar] [CrossRef]
  14. Deluga, G.A.; Salge, J.R.; Schmidt, L.D.; Verykios, X.E. Renewable hydrogen from ethanol by autothermal reforming. Science 2004, 303, 993–997. [Google Scholar] [CrossRef] [PubMed]
  15. Olah, G.A. Beyond Oil and Gas: The Methanol Economy. Angew. Chem. Int. Ed. 2005, 44, 2636–2639. [Google Scholar] [CrossRef]
  16. Smil, V. Energy in Nature and Society; MIT Press: Cambridge, MA, USA, 2008. [Google Scholar]
  17. Moore, R.B.; Raman, V. Hydrogen infrastructure for fuel cell transportation. Int. J. Hydrogen Energy 1998, 23, 617–620. [Google Scholar] [CrossRef]
  18. Grant, P.M.; Starr, C.; Overbye, T.J. A power grid for the hydrogen economy. Sci. Am. 2006, 295, 76–83. [Google Scholar] [CrossRef] [PubMed]
  19. Rezaiyan, J.; Cheremisinoff, N.P. Gasification Technologies: A Primer for Engineers and Scientist; CRC press: Boca Raton, FL, USA, 2005; pp. 119–145. [Google Scholar]
  20. Navarro, R.M.; Pena, M.A.; Fierro, J.L.G. Hydrogen Production Reactions from Carbon Feedstocks: Fossil Fuels and Biomass. Chem. Rev. 2007, 108, 3952–3991. [Google Scholar] [CrossRef]
  21. Cortright, R.D.; Davda, R.R.; Dumesic, J.A. Hydrogen from catalytic reforming of biomass-derived hydrocarbons in liquid water. Nature 2002, 418, 964–967. [Google Scholar] [CrossRef] [PubMed]
  22. Thauer, K.; Jungermann, K.; Decker, K. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 1977, 41, 100–180. [Google Scholar] [PubMed]
  23. Adams, M.W.W.; Stiefel, E.I. Biological hydrogen production: Not so elementary. Science 1998, 282, 1842–1843. [Google Scholar] [CrossRef] [PubMed]
  24. Hallenbeck, P.C.; Benemann, J.R. Biological hydrogen production: Fundamentals and limiting processes. Int. J. Hydrogen Energy 2002, 27, 1185–1193. [Google Scholar] [CrossRef]
  25. Kleerebezem, R.; van Loosdrecht, M.C.M. Mixed culture biotechnology for bioenergy production. Curr. Opin. Biotechnol. 2007, 18, 207–212. [Google Scholar] [CrossRef] [PubMed]
  26. Chou, C.-J.; Jenney, F.E., Jr.; Adams, M.W.W.; Kelly, R.M. Hydrogenesis in hyperthermophilic microorganisms: Implications for biofuels. Metab. Eng. 2008, 10, 394–404. [Google Scholar] [CrossRef] [PubMed]
  27. Lardon, L.; Helias, A.; Sialve, B.; Stayer, J.P.; Bernard, O. Life-Cycle Assessment Of Biodiesel Production From Microalgae. Environ. Sci. Technol. 2009, 43, 6475–6481. [Google Scholar] [CrossRef] [PubMed]
  28. Logan, B.E.; Regan, J.M. Microbial fuel cells—Challenges and applications. Environ. Sci. Technol. 2006, 40, 5172–5180. [Google Scholar] [CrossRef] [PubMed]
  29. Haryanto, A.; Fernando, S.; Murali, N.; Adhikari, S. Current status of hydrogen production techniques by steam reforming of ethanol: A review. Energy Fuels 2005, 19, 2098–2106. [Google Scholar] [CrossRef]
  30. Chheda, J.; Huber, G.; Dumesic, J. Liquid-phase catalytic processing of biomass-derived oxygenated hydrocarbons to fuels and chemicals. Angew. Chem. Int. Ed. 2007, 46, 7164–7183. [Google Scholar] [CrossRef]
  31. Zhang, Y.-H.P. Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations: Challenges and opportunities. Biotechnol. Bioeng. 2010, 105, 663–677. [Google Scholar] [PubMed]
  32. Zhang, Y.-H.P. Using extremophile enzymes to generate hydrogen for electricity. Microbe 2009, 4, 560–565. [Google Scholar]
  33. Zhang, Y.-H.P.; Sun, J.-B.; Zhong, J.-J. Biofuel production by in vitro synthetic pathway transformation. Curr. Opin. Biotechnol. 2010, 21, 663–669. [Google Scholar] [CrossRef] [PubMed]
  34. Wang, Y.; Zhang, Y.-H.P. Cell-free protein synthesis energized by slowly-metabolized maltodextrin. BMC Biotechnol. 2009, 9, 58. [Google Scholar] [CrossRef] [PubMed]
  35. Wang, Y.; Huang, W.; Sathitsuksanoh, N.; Zhu, Z.; Zhang, Y.-H.P. Biohydrogenation from biomass sugar mediated by in vitro synthetic enzymatic pathways. Chem. Biol. 2011. accepted. [Google Scholar]
  36. Huang, W.D.; Zhang, Y.-H.P. Analysis of biofuels production from sugar based on three criteria: Thermodynamics, bioenergetics, and product separation. Energy Environ. Sci. 2011. [Google Scholar] [CrossRef]
  37. Zehentgruber, D.; Hannemann, F.; Bleif, S.; Bernhardt, R.; Lütz, S. Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2. ChemBioChem. 2010, 11, 713–721. [Google Scholar] [CrossRef] [PubMed]
  38. Cooney, M.J.; Svoboda, V.; Lau, C.; Martin, G.; Minteer, S.D. Enzyme catalysed biofuel cells. Energy Environ. Sci. 2008, 1, 320–337. [Google Scholar] [CrossRef]
  39. Ye, X.; Wang, Y.; Hopkins, R.C.; Adams, M.W.W.; Evans, B.R.; Mielenz, J.R.; Zhang, Y.-H.P. Spontaneous high-yield production of hydrogen from cellulosic materials and water catalyzed by enzyme cocktails. ChemSusChem. 2009, 2, 149–152. [Google Scholar] [CrossRef] [PubMed]
  40. Zhang, Y.-H.P.; Evans, B.R.; Mielenz, J.R.; Hopkins, R.C.; Adams, M.W.W. High-yield hydrogen production from starch and water by a synthetic enzymatic pathway. PLoS One 2007, 2, e456. [Google Scholar] [CrossRef] [PubMed]
  41. Das, D.; Veziroglu, T.N. Hydrogen production by biological processes: A survey of literature. Int. J. Hydrogen Energy 2001, 26, 13–28. [Google Scholar] [CrossRef]
  42. Cheng, S.; Logan, B.E. Sustainable and efficient biohydrogen production via electrohydrogenesis. Proc. Nat. Acad. Sci. USA 2007, 104, 18871–18873. [Google Scholar] [CrossRef] [PubMed]
  43. Zhang, Y.-H.P.; Lynd, L.R. Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems. Biotechnol. Bioeng. 2004, 88, 797–824. [Google Scholar] [CrossRef] [PubMed]
  44. Zhang, Y.-H.P. Reviving the carbohydrate economy via multi-product biorefineries. J. Ind. Microbiol. Biotechnol. 2008, 35, 367–375. [Google Scholar] [CrossRef] [PubMed]
  45. Zhu, X.-G.; Long, S.P.; Ort, D.R. What is the maximum efficiency with which photosynthesis can convert solar energy into biomass? Curr. Opin. Biotechnol. 2008, 19, 153–159. [Google Scholar] [CrossRef] [PubMed]
  46. Zhang, Y.-H.P. Artificial photosynthesis would unify the electricity-carbohydrate-hydrogen cycle for sustainability. Nature Precedings 2010. [Google Scholar] [CrossRef][Green Version]
  47. Hightower, M.; Pierce, S.A. The energy challenge. Nature 2008, 452, 285–286. [Google Scholar] [CrossRef] [PubMed]
  48. Dominguez-Faus, R.; Powers, S.E.; Burken, J.G.; Alvarez, P.J. The Water Footprint of Biofuels: A Drink or Drive Issue? Environm. Sci. Technol. 2009, 43, 3005–3010. [Google Scholar] [CrossRef]
  49. Berner, R.A. The long-term carbon cycle, fossil fuels and atmospheric composition. Nature 2003, 426, 323–326. [Google Scholar] [CrossRef] [PubMed]
  50. Falkowski, P.; Scholes, R.J.; Boyle, E.; Canadell, J.; Canfield, D.; Elser, J.; Gruber, N.; Hibbard, K.; Hogberg, P.; Linder, S.; Mackenzie, F.T.; Moore, B., III; Pedersen, T.; Rosenthal, Y.; Seitzinger, S.; Smetacek, V.; Steffen, W. The global carbon cycle: A test of our knowledge of earth as a system. Science 2000, 290, 291–296. [Google Scholar] [CrossRef] [PubMed]
  51. Hermann, W.A. Quantifying global exergy resources. Energy 2006, 31, 1685–1702. [Google Scholar] [CrossRef]
  52. Balzani, V.; Credi, A.; Venturi, M. Photochemical Conversion of Solar Energy. ChemSusChem. 2008, 1, 26–58. [Google Scholar] [CrossRef] [PubMed]
  53. Kalyanasundaram, K.; Graetzel, M. Artificial photosynthesis: Biomimetic approaches to solar energy conversion and storage. Curr. Opin. Biotechnol. 2010, 21, 298–310. [Google Scholar] [CrossRef] [PubMed]
  54. Lewis, N.S. Toward cost-effective solar energy use. Science 2007, 315, 798–801. [Google Scholar] [CrossRef] [PubMed]
  55. Gust, D.; Moore, T.A.; Moore, A.L. Solar Fuels via Artificial Photosynthesis. Acc. Chem. Res. 2009, 42, 1890–1898. [Google Scholar] [CrossRef] [PubMed]
  56. Lewis, N.S.; Nocera, D.G. Powering the planet: Chemical challenges in solar energy utilization. Proc. Nat. Acad. Sci. USA 2006, 103, 15729–15735. [Google Scholar] [CrossRef] [PubMed]
  57. Chin, J.W. Modular approaches to expanding the functions of living matter. Nat. Chem. Biol. 2006, 2, 304–311. [Google Scholar] [CrossRef] [PubMed]
  58. Srivastava, D.K.; Bernhard, S.A. Metabolite transfer via enzyme-enzyme complexes. Science 1986, 234, 1081–1086. [Google Scholar] [CrossRef] [PubMed]
  59. Taniguchi, Y.; Choi, P.J.; Li, G.-W.; Chen, H.; Babu, M.; Hearn, J.; Emili, A.; Xie, X.S. Quantifying E. coli Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells. Science 2010, 329, 533–538. [Google Scholar] [CrossRef] [PubMed]
  60. Conrado, R.J.; Varner, J.D.; DeLisa, M.P. Engineering the spatial organization of metabolic enzymes: Mimicking nature’s synergy. Curr. Opin. Biotechnol. 2008, 19, 492–499. [Google Scholar] [CrossRef] [PubMed]
  61. Agapakis, C.; Ducat, D.; Boyle, P.; Wintermute, E.; Way, J.; Silver, P. Insulation of a synthetic hydrogen metabolism circuit in bacteria. J. Biol. Eng. 2010, 4, 3. [Google Scholar] [CrossRef] [PubMed]
  62. Vasic-Racki, D. History of industrial biotransformations—Dreams and realities. In Industrial Biotransformations; Liese, A., Seebald, S., Wandrey, C., Eds.; Wiley-VCH: Weinheim, Germany, 2006; pp. 1–37. [Google Scholar]
  63. Wang, Y.; Zhang, Y.-H.P. A highly active phosphoglucomutase from Clostridium thermocellum: Cloning, purification, characterization, and enhanced thermostability. J. Appl. Microbiol. 2010, 108, 39–46. [Google Scholar] [CrossRef] [PubMed]
  64. Myung, S.; Zhang, X.-Z.; Zhang, Y.-H.P. Ultra-stable phosphoglucose isomerase through immobilization of cellulose-binding module-tagged thermophilic enzyme on low-cost high-capacity cellulosic adsorbent. Biotechnol. Prog. 2011. submitted. [Google Scholar]
  65. Wang, Y.; Zhang, Y.-H.P. Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration. Microb. Cell Fact. 2009, 8, 30. [Google Scholar] [CrossRef] [PubMed]
  66. Myung, S.; Wang, Y.R.; Zhang, Y.-H.P. Fructose-1,6-bisphosphatase from a hyper-thermophilic bacterium Thermotoga maritima: Characterization, metabolite stability and its implications. Process Biochem. 2010, 45, 1882–1887. [Google Scholar] [CrossRef]
  67. Taylor, L.E.I.; Dai, Z.; Decker, S.R.; Brunecky, R.; Adney, W.S.; Ding, S.-Y.; Himmel, M.E. Heterologous expression of glycosyl hydrolases in planta: A new departure for biofuels. Trends Biotechnol. 2008, 26, 413–424. [Google Scholar] [CrossRef] [PubMed]
  68. Zhang, Y.-H.P.; Himmel, M.; Mielenz, J.R. Outlook for cellulase improvement: Screening and selection strategies. Biotechnol. Adv. 2006, 24, 452–481. [Google Scholar] [CrossRef] [PubMed]
  69. Sathitsuksanoh, N.; Zhu, Z.; Templeton, N.; Rollin, J.; Harvey, S.; Zhang, Y.-H.P. Saccharification of a potential bioenergy crop, Phragmites australis (common reed), by lignocellulose fractionation followed by enzymatic hydrolysis at decreased cellulase loadings. Ind. Eng. Chem. Res. 2009, 48, 6441–6447. [Google Scholar] [CrossRef]
  70. Zhang, X.-Z.; Zhang, Y.-H.P. One-step production of biocommodities from lignocellulosic biomass by recombinant cellulolytic Bacillus subtilis: Opportunities and challenges. Eng. Life Sci. 2010, 10, 398–406. [Google Scholar] [CrossRef]
  71. Shuler, M.; Kargi, F. Bioprocess Engineering: Basic Concepts, 2nd ed.; Prentice-Hall: Englewood Cliffs, NJ, USA, 2001. [Google Scholar]
  72. Zhang, Y.-H.; Lynd, L.R. Quantification of cell and cellulase mass concentrations during anaerobic cellulose fermentation: Development of an ELISA-based method with application to Clostridium thermocellum batch cultures. Anal. Chem. 2003, 75, 219–227. [Google Scholar] [CrossRef] [PubMed]
  73. Ye, X.; Rollin, J.; Zhang, Y.-H.P. Thermophilic α-glucan phosphorylase from Clostridium thermocellum: Cloning, Characterization and Enhanced thermostability. J. Mol. Cat. B Enzymatic 2010, 65, 110–116. [Google Scholar] [CrossRef]
  74. Hong, J.; Wang, Y.; Ye, X.; Zhang, Y.-H.P. Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage. J. Chromatogr. A 2008, 1194, 150–154. [Google Scholar] [CrossRef] [PubMed]
  75. Hong, J.; Ye, X.; Wang, Y.; Zhang, Y.-H.P. Bioseparation of recombinant cellulose binding module-protein by affinity adsorption on an ultra-high-capacity cellulosic adsorbent. Anal. Chim. Acta 2008, 621, 193–199. [Google Scholar] [CrossRef] [PubMed]
  76. Iyer, R.; Wang, J.; Bachas, L. Cloning, expression, and characterization of the gsdA gene encoding thermophilic glucose-6-phosphate dehydrogenase from Aquifex aeolicus. Extremophiles 2002, 6, 283–289. [Google Scholar] [CrossRef] [PubMed]
  77. Scopes, R.K. Protein Purification: Principles and Practice, 3rd ed.; Springer-Verleg: New York, NY, USA, 1993. [Google Scholar]
  78. Cao, L.; Langen, L.V.; Sheldon, R.A. Immobilised enzymes: Carrier-bound or carrier-free? Curr. Opin. Biotechnol. 2003, 14, 387–394. [Google Scholar] [CrossRef] [PubMed]
  79. Mateo, C.; Palomo, J.M.; van Langen, L.M.; van Rantwijk, F.; Sheldon, R.A. A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates. Biotechnol. Bioeng. 2004, 86, 273–276. [Google Scholar] [CrossRef] [PubMed]
  80. Sheldon, R. Cross-linked enzyme aggregates (CLEAs): Stable and recyclable biocatalysts. Biochem. Soc. Trans. 2007, 35, 1583–1587. [Google Scholar] [CrossRef] [PubMed]
  81. Ong, E.; Gilkes, N.R.; Warren, R.A.J.; Miller, R.C.; Kilburn, D.G. Enzyme immobilization using the cellulose-binding domain of a cellulomonas fimi exoglucanase. Nat. Biotechnol. 1989, 7, 604–607. [Google Scholar] [CrossRef]
  82. Tomme, P.; Boraston, A.B.; McLean, B.; Kormos, J.M.; Creagh, A.L.; Sturch, K.; Gilkes, N.R.; Haynes, C.A.; Warren, R.A.; Kilburn, D.G. Characterization and affinity applications of cellulose-binding domains. J. Chromatogr. B. 1998, 715, 283–296. [Google Scholar] [CrossRef]
  83. Shoseyov, O.; Shani, Z.; Levy, I. Carbohydrate binding modules: Biochemical properties and novel applications. Microbiol. Mol. Biol. Rev. 2006, 70, 283–295. [Google Scholar] [CrossRef] [PubMed]
  84. Chen, H.; Hsieh, Y.-L. Enzyme immobilization on ultrafine cellulose fibers via poly(acrylic acid) electrolyte grafts. Biotechnol. Bioeng. 2005, 90, 405–413. [Google Scholar] [CrossRef] [PubMed]
  85. Liu, W.; Wang, P. Cofactor regeneration for sustainable enzymatic biosynthesis. Biotechnol. Adv. 2007, 25, 369–384. [Google Scholar] [CrossRef] [PubMed]
  86. Kazandjian, R.; Klibanov, A. Regioselective Oxidation of Phenols Catalyzed by Polyphenol Oxidase in Chloroform. J. Am. Chem. Soc. 1985, 107, 5448–5450. [Google Scholar] [CrossRef]
  87. Moore, J.C.; Pollard, D.J.; Kosjek, B.; Devine, P.N. Advances in the enzymatic reduction of ketones. Acc. Chem. Res. 2007, 40, 1412–1419. [Google Scholar] [CrossRef] [PubMed]
  88. Wildeman, S.M.A.D.; Sonke, T.; Schoemaker, H.E.; May, O. Biocatalytic reductions: From lab curiosity to "first choice". Acc. Chem. Res. 2007, 40, 1260–1266. [Google Scholar] [CrossRef] [PubMed]
  89. Lutz, J.; Hollmann, F.; Ho, T.V.; Schnyder, A.; Fish, R.H.; Schmid, A. Bioorganometallic chemistry: Biocatalytic oxidation reactions with biomimetic NAD+/NADH co-factors and [Cp*Rh(bpy)H]+ for selective organic synthesis. J. Organomet. Chem. 2004, 689, 4783–4790. [Google Scholar] [CrossRef]
  90. Ansell, R.J.; Lowe, C.R. Artificial redox coenzymes: Biomimetic analogues of NAD+. Appl. Microbiol. Biotechnol. 1999, 51, 703–710. [Google Scholar] [CrossRef]
  91. Lo, H.C.; Fish, R.H. Biomimetic NAD+ Models for Tandem Cofactor Regeneration, Horse Liver Alcohol Dehydrogenase Recognition of 1,4-NADH Derivatives, and Chiral Synthesis. Angew. Chem. Int. Ed. 2002, 41, 478–481. [Google Scholar] [CrossRef]
  92. Ryan, J.D.; Fish, R.H.; Clark, D.S. Engineering Cytochrome P450 Enzymes for Improved Activity towards Biomimetic 1,4-NADH Cofactors. ChemBioChem. 2008, 9, 2579–2582. [Google Scholar] [CrossRef] [PubMed]
  93. Campbell, E.; Wheeldon, I.R.; Banta, S. Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior. Biotechnol. Bioeng. 2010, 107, 763–774. [Google Scholar] [CrossRef] [PubMed]
  94. Adams, M.W.W.; Kelly, R.M. Finding and using hyperthermophilic enzymes. Trends Biotechnol. 1998, 16, 329–332. [Google Scholar] [CrossRef] [PubMed]
  95. Auernik, K.S.; Cooper, C.R.; Kelly, R.M. Life in hot acid: Pathway analyses in extremely thermoacidophilic archaea. Curr. Opin. Biotechnol. 2008, 19, 445–453. [Google Scholar] [CrossRef] [PubMed]
  96. Blumer-Schuette, S.E.; Kataeva, I.; Westpheling, J.; Adams, M.W.W.; Kelly, R.M. Extremely thermophilic microorganisms for biomass conversion: Status and prospects. Curr. Opin. Biotechnol. 2008, 19, 210–217. [Google Scholar] [CrossRef] [PubMed]
  97. WCPDB—Thermus thermophilus HB8 Whole Cell Project Database. Available online: (accessed on 30 January 2011).
  98. Cava, F.; Hidalgo, A.; Berenguer, J. Thermus thermophilus as biological model. Extremophiles 2009, 13, 213–231. [Google Scholar] [CrossRef] [PubMed]
  99. Henne, A.; Bruggemann, H.; Raasch, C.; Wiezer, A.; Hartsch, T.; Liesegang, H.; Johann, A.; Lienard, T.; Gohl, O.; Martinez-Arias, R.; Jacobi, C.; Starkuviene, V.; Schlenczeck, S.; Dencker, S.; Huber, R.; Klenk, H.-P.; Kramer, W.; Merkl, R.; Gottschalk, G.; Fritz, H.-J. The genome sequence of the extreme thermophile Thermus thermophilus. Nat. Biotechnol. 2004, 22, 547–553. [Google Scholar] [CrossRef] [PubMed]
  100. Nelson, K.E.; Clayton, R.A.; Gill, S.R.; Gwinn, M.L.; Dodson, R.J.; Haft, D.H.; Hickey, E.K.; Peterson, J.D.; Nelson, W.C.; Ketchum, K.A.; McDonald, L.; Utterback, T.R.; Malek, J.A.; Linher, K.D.; Garrett, M.M.; Stewart, A.M.; Cotton, M.D.; Pratt, M.S.; Phillips, C.A.; Richardson, D.; Heidelberg, J.; Sutton, G.G.; Fleischmann, R.D.; Eisen, J.A.; White, O.; Salzberg, S.L.; Smith, H.O.; Venter, J.C.; Fraser, C.M. Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga maritima. Nature 1999, 399, 323–329. [Google Scholar] [CrossRef] [PubMed]
  101. Kawarabayasi, Y.; Sawada, M.; Horikawa, H.; Haikawa, Y.; Hino, Y.; Yamamoto, S.; Sekine, M.; Baba, S.-I.; Kosugi, H.; Hosoyama, A.; Nagai, Y.; Sakai, M.; Ogura, K.; Otsuka, R.; Nakazawa, H.; Takamiya, M.; Ohfuku, Y.; Funahashi, T.; Tanaka, T.; Kudoh, Y.; Yamazaki, J.; Kushida, N.; Oguchi, A.; Aoki, K.-I.; Yoshizawa, T.; Nakamura, Y.; Robb, F.T.; Horikoshi, K.; Masuchi, Y.; Shizuya, H.; Kikuchi, H. Complete Sequence and Gene Organization of the Genome of a Hyper-thermophilic Archaebacterium, Pyrococcus horikoshii OT3. DNA Res. 1998, 5, 55–76. [Google Scholar] [CrossRef] [PubMed]
  102. Bult, C.J.; White, O.; Olsen, G.J.; Zhou, L.; Fleischmann, R.D.; Sutton, G.G.; Blake, J.A.; FitzGerald, L.M.; Clayton, R.A.; Gocayne, J.D.; Kerlavage, A.R.; Dougherty, B.A.; Tomb, J.-F.; Adams, M.D.; Reich, C.I.; Overbeek, R.; Kirkness, E.F.; Weinstock, K.G.; Merrick, J.M.; Glodek, A.; Scott, J.L.; Geoghagen, N.S.M.; Weidman, J.F.; Fuhrmann, J.L.; Nguyen, D.; Utterback, T.R.; Kelley, J.M.; Peterson, J.D.; Sadow, P.W.; Hanna, M.C.; Cotton, M.D.; Roberts, K.M.; Hurst, M.A.; Kaine, B.P.; Borodovsky, M.; Klenk, H.-P.; Fraser, C.M.; Smith, H.O.; Woese, C.R.; Venter, J.C. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 1996, 273, 1058–1073. [Google Scholar] [CrossRef] [PubMed]
  103. Deckert, G.; Warren, P.V.; Gaasterland, T.; Young, W.G.; Lenox, A.L.; Graham, D.E.; Overbeek, R.; Snead, M.A.; Keller, M.; Aujay, M.; Huber, R.; Feldman, R.A.; Short, J.M.; Olsen, G.J.; Swanson, R.V. The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 1998, 392, 353–358. [Google Scholar] [CrossRef] [PubMed]
  104. Liu, W.; Hong, J.; Bevan, D.R.; Zhang, Y.-H.P. Fast identification of thermostable beta-glucosidase mutants on cellobiose by a novel combinatorial selection/screening approach. Biotechnol. Bioeng. 2009, 103, 1087–1094. [Google Scholar] [CrossRef] [PubMed]
  105. Liu, W.; Zhang, X.-Z.; Zhang, Z.-M.; Zhang, Y.-H.P. Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability. Appl. Environ. Microbiol. 2010, 76, 4914–4917. [Google Scholar] [CrossRef] [PubMed]
  106. Arnold, F.H. Combinatorial and computational challenges for biocatalyst design. Nature 2001, 409, 253–257. [Google Scholar] [CrossRef] [PubMed]
  107. Zhang, X.-Z.; Zhang, Y.-H.P. Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis. Microb. Biotechnol. 2011, 4, 98–105. [Google Scholar] [CrossRef] [PubMed]
  108. Sun, J.; Hopkins, R.C.; Jenney, F.E.; McTernan, P.M.; Adams, M.W.W. Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production. PLoS One 2010, 5, e10526. [Google Scholar] [CrossRef] [PubMed]
  109. Lynd, L.R.; Wyman, C.E.; Gerngross, T.U. Biocommodity engineering. Biotechnol. Prog. 1999, 15, 777–793. [Google Scholar] [CrossRef] [PubMed]
  110. The Royal Society of the UK Synthetic biology: Call for views. Available online: (accessed on 28 January 2011).
  111. Yang, H.; Xu, Z.; Fan, M.; Gupta, R.; Slimane, R.B.; Bland, A.E.; Wright, I. Progress in carbon dioxide separation and capture: A review. J. Environ. Sci. 2008, 20, 14–27. [Google Scholar] [CrossRef]
  112. Melamu, R.; von Blottnitz, H. A comparison of environmental benefits of transport and electricity applications of carbohydrate derived ethanol and hydrogen. Int. J. Hydrogen Energy 2009, 34, 1126–1134. [Google Scholar] [CrossRef]
  113. Thomas, C.E. Fuel cell and battery electric vehicles compared. Int. J. Hydrogen Energy 2009, 34, 6005–6020. [Google Scholar] [CrossRef]

Share and Cite

MDPI and ACS Style

Zhang, Y.-H.P.; Mielenz, J.R. Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy. Energies 2011, 4, 254-275.

AMA Style

Zhang Y-HP, Mielenz JR. Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy. Energies. 2011; 4(2):254-275.

Chicago/Turabian Style

Zhang, Y.-H. Percival, and Jonathan R. Mielenz. 2011. "Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy" Energies 4, no. 2: 254-275.

Article Metrics

Back to TopTop