Environ. Sci. Technol. 2008, 42, 2401–2407
Thermodynamic Evaluation on H2 Production in Glucose Fermentation HYUNG-SOOL LEE,* MICHAEL B. SALERNO, AND BRUCE E. RITTMANN Center for Environmental Biotechnology, The Biodesign Institute, Arizona State University, P. O. Box 875701, Tempe, Arizona 85287-5701
Received October 16, 2007. Revised manuscript received December 29, 2007. Accepted January 9, 2008.
The normal maximum H2 yield in mesophilic biohydrogen (bioH2) fermentation is ∼2 mol of H2/(mol of glucose). Thermodynamics could be the most fundamental control for bioH2 formation, since proton reduction is strongly energy consuming (+79.4 kJ/(mol of H2)). However, most of the electron equivalents in glucose do not accumulate in H2 but in a range of organic acids and alcohols. Thus, evaluating the hypothesisofthermodynamiccontrolrequiresthefullstoichiometry of the fermentation. We carried out batch bioH2 reactions with a range of pH values that yielded H2 yields from 0 to ∼2 mol of H2/(mol of glucose). We constructed complete electron equivalent (e- equiv) balances for high or low H2 yield by measuring all e- sinks. The highest H2 yield occurred with pH ∼ 4 and was coincident with major butyrate accumulation; ethanol or lactate correlated to reduced H2 yields at pH 7 and 10, respectively. Although the Gibb’s free energies for all overall reactions were similar (-10.6 to -11.2 kJ/(e- equiv)), thermodynamics controlled the H2-producing reaction coupled to ferredoxin; this reaction was favorable at acidic pH but thermodynamically blocked at pH 10. Also, butyrate formation was the most thermodynamically favorable reaction that produced ATP after glycolysis.
Introduction Increasing global demand for limited fossil fuel reserves and the need to decrease CO2 additions to the atmosphere drive a search for renewable, carbon-neutral energy sources. H2 can become one of the alternatives to fossil fuels in the future, due to its high electrical-conversion efficiency (≈55% in a H2 fuel cell) and carbon-free nature, if H2 is captured from nonfossil sources (1, 2). However, present technologies for H2 production mostly depend on fossil fuel. For example, steam methane re-forming accounted for at least 95% of annual H2 production in 2006 in the USA (3), underscoring that H2 is not a renewable fuel at this time. For H2 to become sustainable, it must be produced from renewable sources, such as biomass. Biological H2 production is divided into three categories: direct photolysis, photofermentation, and dark fermentation (also called fermentative bioH2). The first two methods depend on sunlight energy, which varies with the season and with the weather. Photosynthetic systems also require efficient means for capturing sunlight energy and distributing * Corresponding author phone: +1-480-727-0849; fax: +1-480727-0889; e-mail:
[email protected]. 10.1021/es702610v CCC: $40.75
Published on Web 03/05/2008
2008 American Chemical Society
it in reactors. Furthermore, photosynthetic H2-production rates are relatively low, from 0.07 to 0.16 mmol of H2/(L · h) (4). In contrast, fermentative bioH2 production rates reach 120 mmol of H2/(L · h) (4, 5), and the reactor configuration is simple, similar to a conventional anaerobic digester. For these reasons, the greatest attention has been paid to fermentative bioH2. Today, the practical H2 yield in fermentative bioH2 production in mesophilic conditions is considered too small to be used as an energy source by our society (5). The production of reduced compounds other than H2 is the main factor that limits H2 yield in fermentative bioH2, since their accumulation diverts electron equivalents from H2. To improve H2 yield, we must understand the biochemical principles of fermentation to H2 and the range of organic products. Figure 1 illustrates the major catabolic pathways involved in glucose fermentation in mixed cultures (6). Because fermentation involves no respiratory electron acceptor, energy conservation occurs only through substrate-level phosphorylation. Still, the microorganisms must generate reducing power in the form of intracellular electron carriers (e.g., NADH2). Figure 1 shows where ATP and NADH2 are generated in the reactions that ferment glucose to the usual products; here, NADH2 is short for NADH + H+. NADH2 and ATP are coproduced during the initial glycolysis step (reaction 1). ATP also is synthesized in formation of acetate (reaction 3) and butyrate (reaction 14). Although it has been reported that ATP can be synthesized through propionate production, the amount of ATP per mole of propionate is inconsistent and often negligible (7); thus, reaction 5 does not show ATP formation in Figure 1. The reactions that produce lactate, propionate, acetaldehyde, ethanol, and butyrate (reactions 4, 5, 8, 9, and 11-14, respectively) consume NADH2 and release NAD+. The NADH2 needed for these reactions must come from reaction 1 or from conversion of pyruvate to acetyl-CoA (reaction 2A). The production and consumption of NADH2 among these reactions must be balanced. H2 production can occur in two catabolic steps. One is the decarboxylation of pyruvate into acetyl-CoA (reaction 2), which generates reduced ferredoxin (Fdred), a direct edonor for proton reduction to H2 gas; for example, Clostridium sp. utilize this pathway for producing H2 (8). Since reactions 2A,B compete for Fdred, generation of H2 eliminates the generation of NADH2, or vice versa. The other is formate cleavage (reaction 7), which is the dominant mechanism for H2 generation in facultative anaerobes, such as Enterobacter and Klebsiella (9). Fermenting bacteria produce different distributions of reduced products in response to environmental conditions, of which pH is significant (10–13). Fdred generated in reaction 2 can lead to NADH2 or H2 (15–18). The competition for Fdred between NAD+ and H+ must be a primary control over H2 yields. It seems likely that low pH stimulates the coupling reaction of Fdred and H+ (18) to form H2. On the other hand, coupling oxidation of Fdred to NAD+ generates NADH2, which is essential for biomass synthesis, as well as for driving the many reactions in Figure 1 that consume NADH2 (8). Consumption of NADH2 for these other functions may pull electron equivalents away from reaction 2B. Lactate and propionate often are dominant products at conditions close to neutral pH (14), which can be important at acidic pH, depending on substrate types or inocula (19), and they come directly from reduction of pyruvate (reactions VOL. 42, NO. 7, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Catabolic pathways of mixed-acid fermentation from glucose. The pathways illustrate the electron flows, key fermentation products, and formations of NADH2, NAD+, ADP, and ATP: Fdox, oxidized form of ferredoxin; Fdred, reduced form of ferredoxin; 2A, Fdred/Fdox oxidation coupled to NAD+/NADH2 reduction; 2B, Fdred/Fdox oxidation coupled to H+/H2 reduction. NADH2 is NADH + H+. NADH2 (or NAD+) and ATP yields are based on 1 mol of the reaction product. Table 3 shows standard Gibb’s free energy for individual reactions, and Table S1 in the Supporting Information describes the stoichiometry for the reactions. 4 and 5); propionate can be produced from another pathway (i.e., the methylmalonyl-CoA pathway), but we do not show it in Figure 1, since we focus on final e- sinks and thermodynamics. Competing reactions from the pyruvate node produce acetyl-CoA and Fdred (reaction 2) or formate (reaction 6). Since production of lactate and propionate prevents formation of Fdred and formate, both of which lead to H2 generation (reactions 2B and 7), and also consume NADH2, their formation (at neutral pH) ought to lower H2 generation. Ethanol and butyrate become significant at acidic pH; normally, ethanol is abundant at around pH 4–4.5, and butyrate is predominant at slightly higher acidic pH than ethanol (12). Ethanol and butyrate are produced through reduction of acetyl-CoA (beginning with reactions 8 and 10, respectively), which consumes NADH2 and may lower H2 generation via reaction 2A. Ethanol production does not involve ATP synthesis, while ATP is generated in butyrate production. Because acetate is generated by hydrolysis of CoA from acetyl-CoA (reaction 3), acetate production does not involve NADH2 or H2 formation. Acetate is common for a wide range of pH (10, 11), presumably because the bacteria conserve chemical energy as ATP. In principle, glucose could be fermented into 12 mol H2 and 6 mol CO2, but acetate cannot be fermented in dark fermentation. Converting acetate into H2 requires exogenous energy, such as light (photofermentation) or electrical energy (microbial electrolytic cells) (20, 21). Thus, the commonly accepted maximum bioH2 yield is 4 mol of H2/(mol of glucose), when acetate is the only organic fermentation product (without considering biomass growth). However, most bioH2 research has shown actual H2 yields close to or 2402
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lower than 2 mol of H2/(mol of glucose) in mesophilic conditions (5, 11–13). Regulation, kinetics, bacterial community structure, thermodynamics, or a combination causes the bacteria to invest electron equivalents into products other than H2 and acetate. Among them, perhaps the most fundamental is thermodynamics. In a general sense, generation of H2 can be thermodynamically unfavorable, since (∆G°)′ (pH ) 7) for H+ reduction to H2 is +79.4 kJ/(mol of H2) (+39.9 kJ/(e- equiv)). Thus, electron flow to protons could be an energetic drain. However, the thermodynamics can be made favorable when the H2 concentration is low enough (22). Up to now, no studies have provided a thorough analysis of the thermodynamics in fermentative bioH2. One reason is the lack of definition of the stoichiometry of the fermentative reactions. An electron-equivalent (e- equiv) balance is essential for constructing stoichiometric reactions and evaluating the actual thermodynamics. However, prior research has not established mass balance for all electron sinks. Thus, our goal is to overcome this limitation so that we can establish the stoichiometry and thermodynamic relationships in fermentative bioH2. Our first step is to identify experimentally all soluble organic products associated with high or low H2 yield. From this, we can construct a complete e- equiv balance and assess what pathways, among those illustrated in Figure 1, are emphasized with high or low H2 yield. Some claim that ethanol/acetate fermentation produces the most H2 (12, 19, 23, 24), while others claim that butyrate/acetate fermentation is best (11, 13, 25–29). Our e- equiv balances allow us to make an objective assessment of which condition produces the most H2 and where the other electrons go. Our second step is to evaluate the thermodynamic feasibility for the overall reactions and each intermediate step for high or low H2 yield. From this analysis, we can identify thermodynamic roadblocks to high H2 production, if they exist.
Materials and Methods Inoculum. We took waste activated sludge from the Mesa Northwest Water Reclamation Plant (Mesa, AZ), settled the sludge for 24 h at 4 °C, and used it as the inoculum. The total suspended solids (TSS) concentration of the settled sludge was 16000 to 21000 mg/L. Batch Experiments. We carried out batch tests of bioH2 fermentation using serum bottles with a liquid volume of 100 mL and a total volume of 160 mL. Glucose was the electron donor, and its starting concentration was constant at 1 e- equiv glucose/L (7.5 g of glucose/L or 8 g of oxygen demand/L) in a mineral-nutrient medium at different initial pH values. The composition of the nutrient solution (listed per liter of deionized water) was as follows: 530 mg of NH4Cl, 200 mg of Na2HPO4, 200 mg of KH2PO4, 52 mg of KCl, 72 mg of MgCl2 · 6H2O, 10 mg of CaCl2 · 2H2O, 0.8 mg of MnCl2 · 4H2O, 1.2 mg of CoCl2 · 6H2O, 0.2 mg of H3BO3, 0.1 mg of CuCl2 · 2H2O, 0.1 mg of NaMoO4 · 2H2O, 0.1 mg of ZnCl2, and 25 mg of FeCl2 · 4H2O. We adjusted the initial pH to 5.8, 7.3, or 11 with 2 N H2SO4 or NaOH. For some experiments, we added buffer to reduce the pH change during the experiments: 40 mM 2-(N-mopholino)ethanesulfonic acid monohydrate (MES) for pH 5.8; 40 mM phosphate buffer (KH2PO4/Na2HPO4) for pH 7.3; or 40 mM boric acid buffer (Na2B4O7/H3BO3) for pH 11. Once the medium and inoculum (7 mL) were added to the serum bottles, we filled them to 100 mL with 18 MΩ deionized water, capped the bottles with rubber serum stoppers and aluminum caps, purged them with N2 gas for 1 min to remove O2, and placed them in a 37.5 °C incubator shaker (160 rpm, C25KC, New Brunswick Scientific). We measured the volume of gas produced following the biochemical methane potential (BMP) protocol (30) using a frictionfree glass syringe of 10, 20, 50, or 100 mL volume (Popper
TABLE 1. Fractions of Electron Sinks at Different pH Conditions in Fermentative BioH2 average percentage of each electron sink of initial glucose for given initial pH [final pH] without buffer compound
5.8[3.5]
7.3 [4.0]
with buffer 11 [4.2]
5.8 [4.0]
7.3 [6.8]
11 [10]
acetate n-butyrate propionate isobutyrate ethanol formate lactate res-glucoseb biomassc H2 total error
10 54 ( 1.2 1 1 4
