Energy & Fuels 2000, 14, 197-201
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Enzymatic Hydrogen Production: Conversion of Renewable Resources for Energy Production Jonathan Woodward,* Kimberley A. Cordray, Robert J. Edmonston, Maria Blanco-Rivera, Susan M. Mattingly, and Barbara R. Evans Chemical Technology Division, Oak Ridge National Laboratory,† Oak Ridge, Tennessee 37831-6194 Received June 16, 1999. Revised Manuscript Received October 4, 1999
Using the enzymes glucose dehydrogenase (GDH) and hydrogenase, we have shown that a variety of sugars that are components of renewable resources can be enzymatically converted to molecular hydrogen. The rates at which hydrogen was evolved paralleled the substrate specificity of GDH. The highest rate of hydrogen production measured was 97.8 µmol/h, and the stoichiometric yield of hydrogen was 98% with 50 mM glucose as the substrate. Lactose, sucrose, cellulose, xylan, steam-exploded aspen wood, and starch also served as substrates for hydrogen production when the corresponding enzymes were included in the reaction mixture to generate the appropriate monosaccharide for which GDH has specificity. The data obtained are discussed in the context of the rate-limiting steps of hydrogen production from renewable sugar and the possible applications of enzymatic hydrogen production.
Introduction and Background The enzymes Thermoplasma acidophilum glucose dehydrogenase (GDH) and Pyrococcus furiosus hydrogenase have been shown to be capable of converting glucose to molecular hydrogen and gluconic acid. The key step in the feasibility of the reaction is the ability of hydrogenase to accept electrons from the reduced cofactor of GDH (NADPH).1 The main component of renewable resources such as cellulose or starch is glucose; lactose and sucrose are composed of 50% glucose. It is conceivable, therefore, that all these sugars could be sources of glucose for this reaction. When agricultural biomass is considered as an energy source, it must be remembered that it contains three main ingredients: cellulose (50%); hemicellulose, primarily xylan (25%); and lignin (25%). Lactose and sucrose contain equimolar amounts of glucose and galactose or fructose, respectively.2,3 The specificity of GDH from sources such as T. acidophilum and Haloferax mediterranei is not restricted to glucose. They have been shown to have high activity on galactose and xylose as well.3,4 Although fructose can be oxidized by fructose dehydrogenase, NADP+ is not the cofactor for this enzyme.5 Fructose, however, could participate in this reaction if it were converted to glucose by glucose isomerase.6 Therefore, * Author to whom correspondence should be addressed. † Managed by Lockheed Martin Energy Research Corp. under contract DE-AC05-96OR22464 with the U.S. Department of Energy. (1) Woodward, J.; Mattingly, S. M.; Danson, M.; Hough, D.; Ward, N.; Adams, M. Nature Biotechnol. 1996, 14, 872-874. (2) Cowling, E. B.; Kirk, T. K. Biotechnol. Bioeng. Symp. 1976, 6, 95-123. (3) Smith, L. D.; Budgen, N.; Bungard, S. J.; Danson, M. J.; Hough, D. W. Biochem. J. 1989, 261, 973-977. (4) Bonete, M. J.; Pin, C.; Llorca, F. I.; Camacho, M. L. FEBS Lett. 1996, 383, 227-229. (5) Marcinkeviciene, J.; Johansson, G. FEBS Lett. 1993, 318, 23-26.
in effect, cellulose, xylan, lactose, and sucrose should be substrates for hydrogen evolution by this pathway if they are first hydrolyzed to their constituent sugars by cellulase, xylanase, lactase (β-glucosidase), and sucrase (β-fructofuranosidase or invertase), respectively. The evolution of molecular hydrogen from glucose in the GDH/hydrogenase system was observed to be immediate after the addition of NADP+, which initiated the reaction.1 This confirmed NADPH to be the substrate for P. furiosus hydrogenase.7 This study extends our original work by determining the specificity of the system for different renewable sugars and compares the kinetics of hydrogen production as a function of the concentration of glucose, galactose, and xylose. It is also demonstrated that cellulose, xylan, starch, sucrose, and lactose can be used to produce hydrogen when their constituent monosaccharides are generated by using the appropriate hydrolases. Experimental Section Enzyme Preparation and Substrates. T. acidophilum recombinant GDH (2381 units/mL) and Rhizopus mold amyloglucosidase (5 units/mg solid) were purchased from Sigma Chemical Company, St. Louis, Missouri. P. furiosus hydrogenase (630 units/mL) was purchased from the University of Georgia, Athens, Georgia. These enzymes were used as supplied. Pulpzyme HAJ, a source of cellulase and xylanase, was a generous gift of Novo Nordisk Bioindustrials, Inc., Danbury, Connecticut. The enzyme was desalted on a column of Sephadex G-25 (Pharmacia, Piscataway, New Jersey) prior to use. The crude enzyme possessed a cellulase activity of 193 units/ mL, based upon its activity on microcrystalline cellulose (Avicel7) and a xylanase activity of 14.3 units/mL, based upon (6) Industrial Enzymology, 2nd ed.; Godfrey, T., West, S., Eds.; Stockton Press: New York, 1996. (7) Ma, K.; Zhou, Z. H.; Adams, M. W. W. FEMS Microbiol. Lett. 1994, 122, 245-250.
10.1021/ef990126l CCC: $19.00 © 2000 American Chemical Society Published on Web 11/20/1999
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its activity on birchwood xylan.8 Invertase and β-galactosidase were, respectively, extracted from a sucrose- or lactose-induced pure bacterial strain, designated TOR-39, isolated from an Oak Ridge National Laboratory culture collection that originated in a 2.7-km-deep drill hole in Virginia. This strain was found to be 98% identical with that of Thermoanaerobacter ethanolicus.9 The cells were lysed using lysozyme followed by centrifugation at 14000 rpm for 30 min. The supernatant was subjected to fast protein liquid chromatography by ion exchange on a Mono P HR 5/20 column.10 The fractions containing invertase or β-galactosidase activity in 20 mM Bis-Tris buffer, pH 7.1, containing 0.35 M sodium chloride were pooled and possessed 0.48 and 1.25 units/mL activity, respectively. Activity of these enzymes was determined by measuring sucrose or lactose hydrolysis to glucose. The sugars glucose, galactose, xylose, mannose, 2-deoxyglucose, fructose, and lactose were purchased from Sigma Chemical Company, St. Louis, Missouri. Sucrose was purchased from Mallinckrodt, Inc., and starch was purchased from MCB Manufacturing Chemists, Inc., Cincinnati, Ohio. Avicel PH105 was a generous gift from FMC Corporation, Philadelphia, Pennsylvania. Steam-exploded aspen wood containing 60% cellulose was a gift from Michael Himmel of the National Renewable Energy Laboratory, Golden, Colorado. Apparatus: Hydrogen Detection. A continuous-flow system was constructed to measure the production of hydrogen as described in detail previously.1,11 The hydrogen detection system consisted of a Figaro semiconductor tin oxide gas sensor, model TGS 822 (Bio-Gas Detector Corporation, Okemos, Michigan), which was calibrated with an in-line electrolysis cell and Faraday’s law of electrochemical equivalence. The current was provided by a Keithley 220 programmable current source (Cleveland, Ohio). Data were recorded by a PC microcomputer after analog-to-digital conversion of the hydrogen signal and analyzed using ASYST Technologies, Inc., 4.0 Analysis Software (Rochester, New York). Hydrogen Evolution from Sugar. The kinetics of hydrogen evolution from sugar were carried out as a function of substrate specificity and concentration as well as temperature, pH, and enzyme concentration. The details are given in the legends to the figures and tables. The basic reaction mixture in a 2.0-mL volume consisted of a given concentration of substrate, T. acidophilum GDH (12.5 units), P. furiosus hydrogenase (50 units), 1 µmol of NADP+, and 50 mM sodium phosphate buffer, pH 7.5, at 50 °C. The reaction was always initiated by the addition of NADP+. It should be noted that hydrogen produced was swept to the hydrogen sensor by the helium carrier gas.
Results and Discussion Kinetics of Hydrogen Production from Monosaccharides. Figure 1 plots the evolution of hydrogen from the monosaccharidessglucose, galactose, xylose, and mannosesusing an initial concentration of these sugars of 50 mM in the reaction mixture. Hydrogen production was immediate on the addition of NADP+ and, as expected, the highest rates achieved were based upon the sugar specificity of glucose dehydrogenases.3,4 The evolution of hydrogen was highest (97.8 µmol/h) with glucose as the substrate (Table 1). Galactose, xylose, and 2-deoxyglucose were also substrates for this reaction, and hydrogen evolution rates of 29.0, 7.0, and 3.6 µmol/h (8) Evans, B. R.; Margalit, R.; Woodward, J. Appl. Biochem. Biotechnol. 1995, 51/52, 225-239. (9) Liu, S. V.; Zhou, J.; Zhang, C.; Cole, D. R.; GajdarziskaJosifovska, M. Science 1997, 277, 1106-1109. (10) Offord, D. A.; Lee, N. E.; Woodward, J. Appl. Biochem. Biotechnol. 1991, 28/29, 377-386. (11) Greenbaum, E. Photobiochem. Photophys. 1984, 8, 323-332.
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Figure 1. Kinetics of hydrogen production from monosaccharides. The reaction mixture contained 100 µmol of the monosaccharide in a 2.0-mL reaction volume. Refer to Experimental Section for additional details. Table 1. Sugar Specificity of Thermoplasma acidophilum GDH in Hydrogen Production substrate (100 mM)
maximum rate µmol h-1
hydrogen yield (% theor max)
glucose galactose xylose mannose 2-deoxyglucose fructose
97.8 29.0 7.0 0.8 3.6 0.0
98.0 93.9 52.8 9.8 34.9 0.0
were observed, respectively. In effect, mannose and fructose were not oxidized by GDH. The only difference between glucose and mannose is in the configuration of the asymmetric carbon at position 2. Fructose is a ketose sugar and lacks an asymmetric carbon atom at position 2.5 Because galactose and xylose have the same configuration as glucose at carbon 2 and are also substrates for T. acidophilum GDH, this configuration appears to be important to the specificity of GDH. Even though 2-deoxyglucose lacks an hydroxyl group at carbon 2, it is still oxidized by GDH, albeit at a much lower rate than glucose. There are several possible reasons for the decline in the rate of hydrogen evolution after the maximum rate has been reached. One explanation is a decline in substrate concentration over time during the experiment. Previous work has shown that further addition of substrate, when the rate has fallen to zero, results in a restoration of the maximum rate of hydrogen evolution.1 This work also determined the effect of initial substrate concentration on the rates and yields of hydrogen production (Table 2). For glucose, galactose,
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Table 2. Comparison of Rates and Yields of Hydrogen Production from Sugars as a Function of Concentrationa glucose (mM) max rate (µmol h-1) yieldb (µmol H2) % theor yieldc final pH
galactose (mM)
xylose (mM)
mannose (mM)
5
50
250
5
50
250
5
50
250
50
4.1
97.8
42.6
10.3
29.0
16.8
3.1
7.0
13.1
0.81
7.65
98.0
95.9
10
93.9
103.2
10
52.8
79.4
9.8
76.5
98.0
19
100
93.9
20.6
100
52.8
15.9
9.8
7.1
6.3
6.2
4.0
6.7
5.9
7.4
4.1
7.1
7.1
The reaction mixture (2.0 mL) contained a specified concentration of sugar in 50 mM sodium phosphate buffer, 1 µmol of NADP+, Thermoplasma acidophilum GDH (12.5 units), and Pyrococcus furiosus hydrogenase (50 units) at 50 °C. b Calculated after hydrogen evolution had ceased. c Based on stoichiometry of 1 mol of H2 per mol of sugar. a
and xylose, the maximum rate of hydrogen evolution increased when the substrate concentration was increased from 5 to 50 mM. At glucose and galactose concentrations of 250 mM, the maximum rate was lower and could be explained by substrate inhibition since the Km of T. acidophilum GDH for glucose is, reportedly, 10 mM.3 This, however, remains to be determined. No inhibition in the rate of hydrogen evolution was observed as the concentration of xylose increased, suggesting that GDH possesses a higher Km value for this substrate than for glucose. Consideration must also be given to other possible causes for the decline in rate of hydrogen evolution. The maximum stoichiometric yield of hydrogen from glucose, galactose, and xylose, using the GDH/hydrogenase enzyme couple, is 1 mol of hydrogen per mol of sugar, with 1 mol of the corresponding sugar acid as the byproduct. High stoichiometric yields of hydrogen were obtained from these sugars at the two lowest concentrations used (Table 2). Such high yields are possible because the system for measuring hydrogen evolution is not closed and, therefore, the hydrogen produced is swept away from the reaction mixture and never approaches equilibrium which could result in lower yields. At glucose and galactose concentrations of 250 mM, the yields of hydrogen were only 19 and 21%, respectively. The molar yields of hydrogen from these sugars were the same amount as those obtained from an initial concentration of 50 mM. This phenomenon may be explained as follows. During the reaction, the pH of the reaction mixture drops because of the production of acid (e.g., gluconic acid) that forms during the oxidation of glucose by GDH. With initial concentrations of 50 and 250 mM glucose, the pH values of the reaction mixture at the end of the experiment were 6.27 and 4.13, respectively, with hydrogen amounts of 98.0 and 95.9 µmol, respectively. These data could be explained as follows. Unlike hydrogenase (Figure 2), GDH retains significant activity below pH 6.0 and, therefore, although glucose is capable of being oxidized to gluconic acid by GDH with the resulting fall in pH, the ability of hydrogenase to oxidize NADPH would be diminished severely below pH 6.0. Also, NADPH autooxidizes faster at more acidic pH values.12 It should also be noted that maximum rates of hydrogen evolution were always reached prior to a significant drop in the reaction pH. Also, with xylose and mannose as substrates, it took longer amounts of time for hydrogen evolution to reach completion. (12) Woodward, J.; Orr, M. Biotechnol. Prog. 1998, 14, 897-902.
Figure 2. Effect of pH on the activity of GDH and hydrogenase. GDH assays were carried out in a reaction mixture (1.0 mL of 50 mM sodium phosphate buffer, 55 °C) containing 10 µmol of glucose, 1 µmol of NADP+, and 0.13 units of GDH. Reactions were initiated by the addition of NADP+, and the rate of NADPH formation was monitored at 340 nm. Data for hydrogenase are from ref 14. It should be noted that the optimum temperature for hydrogenase is at least 90 °C.
Studies on the effect of temperature on the rate of hydrogen evolution indicated that the maximum rate was observed at 50 °C, above which it decreased (data not shown). Hydrogenase has an optimum temperature of 85 °C when NADPH is the electron donor.7 However, the GDH used in these experiments has an optimum temperature of 50 °C and is inherently unstable above this temperature because of dissociation of its tetrameric structure.3 Experiments have shown that the heat stability (56 °C) of GDH decreases at low protein concentrations, and that heating GDH in the presence of bovine serum albumin stabilizes its catalytic activity. We have also found that T. acidophilum GDH is unstable when stored at 23 °C at a concentration of 3 µg/mL, losing all activity within 60 min (data not shown). However, under our experimental conditions, it appears that GDH is stable at 60 °C for at least 24 h and, therefore, does not dissociate into inactive subunits. The reason for this stability of GDH is not understood at present, but is probably due to the higher protein concentration in the reaction mixture. In experiments where hydrogen yields are less than 100% of maximum, we showed that after the rate of hydrogen evolution has declined to zero, further addition of GDH or hydrogenase to a reaction mixture did not restore hydrogen
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Table 3. Rates and Yields of Hydrogen Production from Renewablesa renewable
rate (µmol h-1)
yield (%)
maximum yield (Y/N)
lactoseb sucroseb cellulose/xylanc starchc steam-exploded aspenc
6.3 2.8 4.5 8.1 3.4
37.2 25.5 22.3 16.0 39.2
N N N Y Y
a Reaction mixture conditions standard and included 0.63 units of lactase, 0.48 units of invertase, 38.6 units of avicelase, and 10 units of amyloglucosidase for the hydrolysis of lactose, sucrose, cellulose/xylan, steam-exploded aspen, and starch, respectively. b Substrate concentration used 100 µmol in 2.0 mL reaction volume. c Substrate concentration used 111 µmol glucose/xylose equivalent.
evolution to its maximum rate.12 However, further addition of NADP+ did restore the rate, indicating that the stability and, therefore, the effectiveness of the electron shuttle between GDH and hydrogenase are clearly an important rate-determining factor. Finally, experiments on the effect of initial pH of the reaction mixture on the rate and yield of hydrogen production showed that a pH between 6.5 and 7.5 resulted in the highest rates of hydrogen evolution, while the maximum yield occurred between pH 7.0 and 8.0 (data not shown). It should also be noted that at pH 8.0, 100% of the theoretical maximum yield of hydrogen was obtained; however, this yield occurred after a longer period of time as a result of the lower rate of hydrogen production. At an initial pH of 6.5, yields were lower, presumably because of the instability of hydrogenase at this pH. Conversion of Renewable Sugars to Hydrogen. The previous data demonstrate that the two primary components of renewable sources of energy such as biomasssglucose and xylosesare capable of oxidiation by GDH, resulting in hydrogen production if hydrogenase is present. Table 3 shows that renewable sugars (disaccharides and polysaccharides) can be a source of hydrogen but that the appropriate enzymes must be included in the reaction mixture to hydrolyze them to their monosaccharide components. Lactose (50 mM) was hydrolyzed to glucose and galactose by β-galactosidase (lactase) with a maximum theoretical hydrogen yield of 200 µmol possible. The actual yield after 24 h was 74 µmol, but the reaction was stopped before completion as the rate of hydrogen evolution had declined considerably at this point. The maximum theoretical yield of hydrogen from sucrose (50 mM) was 100 µmol since fructose is not a substrate for GDH, and no attempt was made to isomerize fructose to glucose as has been described.12 As with lactose as the substrate, the 25.5% yield of hydrogen was obtained before the reaction had gone to completion. Although the amounts of β-galactosidase and invertase used for the hydrolysis of lactose and sucrose in the reaction mixture were 0.63 and 0.48 units, respectively, it is not known whether a higher activity of these enzymes would have resulted in higher rates of hydrogen evolution and a 100% yield after 24 h. It should be noted that we determined these enzymes were compatible with GDH and hydrogenase with respect to their pH activity curves. However, the amyloglucosidase used in this study to hydrolyze starch to glucose has an optimum pH of ∼4.5, and the 16% yield
of the theoretical amount of hydrogen expected could be explained by the instability of amyloglucosidase at pH 7.5, where this enzyme would have little activity or stability. Hydrogen was also evolved using a mixture of pure cellulose (Avicel) and birchwood xylan or when steamexploded aspen wood was used as the substrates (Table 3). The rate-limiting step in the hydrolysis of insoluble polysaccharides, such as cellulose, appears to be the rate at which they are converted to monosaccharides. The turnover number of enzymes acting on soluble substrates is similar (about 104 min-1); however, that of the cellulase enzyme complex acting on insoluble cellulose is much lower (about 1-10 min-1), depending on the substrate.13 This difference appears to be the reason the rate of hydrogen production is much lower in a system containing cellulose, cellulase, GDH, and hydrogenase.1 Although the maximum rate of hydrogen evolution from these substrates was found to be similar to those obtained with lactose and sucrose, this was probably caused by the high and low levels of enzyme activity used for hydrolyzing the insoluble and soluble substrates, respectively. Theoretical Considerations with Respect to Yields and Applications of Products. Using the enzymatic pathway for hydrogen production described in this work, the theoretical yield of hydrogen from glucose, galactose, fructose (isomerized to glucose), and xylose is 1 mol of H2 per mol of sugar. Since the maximum stoichiometric yield of hydrogen from glucose, galactose, and fructose is 12 mol of H2 per mol and from xylose 10 mol per mol, the efficiencies of hydrogen production by this pathway are 8.3% and 10%, respectively. Equal molar yields of the oxidized sugars (gluconic, galactonic, and xylonic acids)sall of which have commercial valueswould also be obtained. For example, gluconic acid is used in the chelation of metals in cleaning, binding of hard-water ions, rust removal, and paint stripping. It also has application in the textile and pharmaceutical industries. Although it would be necessary to improve the stoichiometric yields of hydrogen from glucose for large-scale production of hydrogen, the amount of hydrogen produced in the reactions described would be sufficient for low-energy-requiring electronic devices powered by an appropriate fuel cell. Conclusions Monosaccharides such as glucose, galactose, and xylose can be oxidized by glucose dehydrogenase with the evolution of hydrogen gas in the presence of hydrogenase and the electron carrier NADP+. This is of interest in the context of hydrogen production from renewable sources of energy such as agricultural biomass, of which 70-80% is composed of cellulose and xylan, polymers of glucose and xylose, which can be generated by the appropriate hydrolytic enzymes. Starch, sucrose, and lactose are also potential substrates for hydrogen production after hydrolysis to their monosaccharide components. The broad substrate specificity of (13) Kostantinidis, K.; Marsden, I.; Sinnott, M. L. Biochem. J. 1993, 291, 883-888. (14) Park, J.-B. Ph.D. Dissertation, University of Georgia, Athens, GA, 1992.
Enzymatic Hydrogen Production
the glucose dehydrogenase used in this study is of key importance in the production of hydrogen from the primary sugars found in biomass. Acknowledgment. The authors thank H. M. O’Neill and N. P. Nghiem for comments and suggestions on the manuscript, L. W. Wagner for secretarial support, and M. K. Savage for editorial services. This work was supported by the Christopher Columbus Fellowship
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Foundation, the Office of Research and Development, the Defense Advanced Projects Agency, the Office of Basic Energy Sciences, the Office of Utility Technologies, and the U.S. Department of Energy. Oak Ridge National Laboratory is managed by Lockheed Martin Energy Research Corp. under contract DE-AC0596OR22464 with the U.S. Department of Energy. EF990126L
