Stoichiometric analysis of biological hydrogen production by fermentative bacteria

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Abstract

In this study, biohydrogen production from glucose by two fermentative bacteria (Clostridium butyricum, a typical strictly anaerobic bacterium, and Klebsiella pneumoniae, a well-studied facultative anaerobic and nitrogen-fixing bacterium) are stiochiometrically analyzed according to energy (ATP), reducing equivalent and mass balances. The theoretical analysis reveals that the maximum yield of hydrogen on glucose by Clostridium butyricum is 3.26 mol/mol when all acetyl-CoA entering into the acetate pathway (α=1), which is higher than that by Klebsiella pneumoniae under strictly anaerobic conditions. In the latter case, the maximum yield by Klebsiella pneumoniae is 2.86 mol hydrogen per mol glucose when five sevenths of acetyl-CoA is transformed to acetate. However, under microaerobic condition the maximum yield of hydrogen on glucose by Klebsiella pneumoniae could reach 6.68 mol/mol if all acetyl-CoA entered into tricarboxylic acid (TCA) cycle (γ=1) and a quantity of 53% of the reducing equivalents generated in the metabolism were completely oxidized by molecular oxygen. On the other hand, the relationship between hydrogen production and biomass formation is distinct by Clostridium butyricum from that by Klebsiella pneumoniae. The former yield of hydrogen on glucose increases as biomass. In contrast, the latter one decreases as biomass in a certain range of molar fraction of acetate in total acetyl-CoA metabolism (5/7β0). Microaerobic condition is favorable for high hydrogen production with low biomass formation by Klebsiella pneumoniae in a certain range of the molar fraction of all reducing equivalents oxidized completely by molecular oxygen (0.53δ0.83).

Introduction

As favorable fuel, hydrogen has been investigated for over a century. After hydrogen combustion, the product is water, other than oxide pollutants, such as COx, NOx, SOx etc, emitted from the traditional fossil fuels, such as petroleum and coal. The heating value is 282.119 kJ per mol of H2O generated, which corresponds to a value much higher than that of traditional fossil fuels. Hydrogen is a fuel of the future mainly due to its high conversion efficiency, recyclability and nonpolluting nature. Hydrogen can be chemically or electrochemically produced as a by-product of oil or coal processing. Alternatively, biological hydrogen production has also been studied since 1800s [1] and recently draws much attention. Given that renewable resources can be utilized as substrates to produce hydrogen under mild operating conditions, biotechnological hydrogen production is an environmental, harmless and economically sustainable method. In contrast, not only are nonrenewable fossil fuels used, but also large amounts of energy are consumed and wastewater is produced in physical and chemical methods.

In general, there are two biosystems to produce hydrogen: photochemical production and fermentative production. The former is carried out by photosynthetic microorganisms, such as algae [2], cyanobacteria [3], and photosynthetic bacteria [4], while the latter consists of fermentative microorganisms including strictly and facultative anaerobic bacteria [5].

Hydrogen production by fermentative microorganisms is an expectable method compared with photosynthetic bacteria due to its high utilization of organic compounds or wastes as substrate to produce hydrogen day and night. In fermentations, the production of molecular hydrogen is used to maintain the electron balance, in which protons serve as electron acceptors and reducing equivalents act as electron donors [5], [6]. This process is generally associated with an intracellular iron–sulfur protein called ferredoxin, which is an electronegative electron carrier [5], [6]. The transfer of electrons from ferredoxin to H+ is catalyzed by the enzyme hydrogenase. Hydrogenases are widely distributed in anaerobic microorganisms and capable of either taking up or evolving H2. They can be classified into three classes: [Fe]-hydrogenase, [NiFe]-hydrogenase and metal-free hydrogenase [6], of which the former two classes are dominant. [NiFe]-hydrogenase is usually considered to derive reductant from hydrogen [7], whereas [Fe]-hydrogenase functions to remove excessive reducing equivalents during fermentation by strictly anaerobic bacteria, such as Clostridia [8]. The activity of hydrogenase will be dramatically repressed in the existence of oxygen [8].

Another enzyme of significant importance that is involved in the biosynthesis of H2 is nitrogenase. This enzyme consists of two separate proteins, i.e. dinitrogenase and dinitrogenase reductase [7]. Various substrates are reduced by nitrogenase as well as MgATP (2ATP/e-) and low-potential electrons derived from an electron carrier such as ferredoxin or flavodoxin [7]. In general, nitrogenase is employed to reduce N2 to NH3 in the nitrogen fixation. When N2 is absent, it can also reduce H+ to hydrogen by consuming 4 ATP per mol hydrogen generated. The existence of N2, O2 and NH4+ will significantly inhibit the production of hydrogen [7].

Two classes of fermentative bacteria are capable of producing hydrogen at a high rate and yield, including strictly anaerobic and facultative anaerobic bacteria. In the former category, a typical strictly anaerobic bacterium, largely utilized in the biotechnological hydrogen production belongs to the species Clostridium butyricum. In the latter category, a typical facultative anaerobic bacterium is the nitrogen fixing one Klebsiella pneumoniae. Both microorganisms have been utilized into a plethora of biotechnological applications which were mainly focused upon the production of high added-value chemicals namely 1,3-propanediol, 2,3-butanediol and butyric acid [9], [10], [11], [12], [13], [14]. The aim of the present investigation was to analyze stoichiometrically the glucose metabolism for hydrogen production according to ATP, reducing equivalents and mass balances, by the two aforementioned microorganisms, Clostridium butyricum and Klebsiella pneumoniae. Biochemical interpretations concerning the microbial metabolism were considered and discussed.

Section snippets

Strictly anaerobic bacteria

Clostridium butyricum, a typical strictly anaerobic bacterium, is known as a classical acid producer and usually ferments carbohydrates to butyrate, acetate, carbon dioxide and molecular hydrogen [9], [12], [13], [14], [15]. As the metabolic products are concerned, acetate and butyrate are the main products when NADH2 and ATP are generated in the bioprocess [16]. There are two pathways to produce hydrogen superficially, as shown in Fig. 1 [17]. One is the cleavage of pyruvate to acetyl-CoA, CO2

Stoichiometric analysis of Clostridium butyricum

The influences of α (molar fraction of acetate in total acetyl-CoA metabolism) on yields of H2, biomass, butyrate, ATP to glucose and yield of H2 to biomass in Clostridium butyricum are illustrated in Fig. 3. The yield of H2 to glucose increases as α. The maximum yield of H2 could theoretically reach 3.26 mol per mol of glucose when all glucose is metabolized on acetate pathway (α=1). The theoretical minimum yield is 1.71 mol H2 per mol of glucose on the butyrate pathway. So an increment of molar

Conclusion

Glucose metabolism by C. butyricum or K. pneumoniae to produce hydrogen is stiochiometrically analyzed according to energy (ATP), reducing equivalent and mass balance in this study. The maximum yield by C. butyricum is 3.26 mol hydrogen per mol glucose if all of the glucose is metabolized through the pathway of acetic acid biosynthesis under anaerobic conditions. The more glucose metabolized on acetate pathway, the more hydrogen and biomass could be produced. The maximum yield of hydrogen on

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