Metabolic Pathways
The microorganisms used in this approach to produce alcohol fuels from CO, H2 and CO2 gas mixture are the type of anaerobic bacteria called acetogens. An acetogen is a general term used for microorganisms that generate acetate as a product of anaerobic respiration. This acetate-producing process follows the Wood-Ljungdahl metabolic pathway and historical perspective of this pathway, as well as metabolic versatilities of acetogens, are discussed in a classic review by Drake et al. [8]. In this pathway CO2 is reduced to CO, which is then converted to acetyl coenzyme A. Enzymes CO dehydrogenase and acetyl-CoA synthase are the enzymes involved in the end process, and the former catalyzes the reduction of the CO2 and the latter combines the resulting CO with a methyl group to give acetyl CoA [9]. In the conversion of syngas to ethanol, acetic acid is also generally produced as a co-product and two anabolic processes, acetogenesis and solventogenesis, are involved. Acetogenesis produces acetic acid, while solventogenesis produces ethanol. The two processes do not happen simultaneously, but rather acetogenesis precedes solventogenesis.
Acetyl-CoA pathway leading to the production of ethanol and acetic acids from CO, CO2 and hydrogen in the syngas is shown in Figure 12.1 [6,10-12]. This pathway is an irreversible, non-cyclic path that takes place under strictly anaerobic conditions and governs acetogenic bacterial fermentation. The net ATP formation to provide energy for the growth of cells is zero for this pathway. Furthermore, the proposed acetyl-CoA synthesis pathway consists of two branches as shown in Figure 12.1. The left branch is known as the methyl branch, whereas the right as the carbonyl branch.
Through these paths CO2 is reduced to methyl and carbonyl level via several enzyme-dependent reactions outlined in the scheme in Figure 12.1.
In the methyl branch of the acetyl-CoA pathway, CO2 is first reduced to formate (HCOO-). This reaction is reversibly catalyzed by the formate dehydrogenase (FDH) enzyme, whose function is to reduce CO2 to formate [12]. The generated formate is then the precursor for the methyl group synthesis of the acetyl-CoA pathway. The formate is activated by tetrahydrofolate (H4folate) to form 10-formyl-H4folate catalyzed by 10-formyl-H4folate synthetase [12]. The enzyme cyclohydrolase catalyze the further conversion of this intermediate to yield 5,10-methenyl-H4folate. In the next NADPH — dependent reduction, the 5,10-methenyl-H4folate is converted to 5,10-methylene-H4folate by the methylene-H4folate dehydrogenase enzyme. Then, the enzyme methylene-H4folate reductase reduces this intermediate to (6S)-5-methyl-H4folate. At the final stage of the methyl synthesis, CH3-H4folate is transferred to the cobalt center of the corrinoid/iron-sulfur protein. The corrinoid protein must be reduced to accept a methyl group from 5-methyl-H4folate. This reduction is carried out by reduced ferredoxin which may be generated using pyruvate and pyruvate-ferredoxin oxidoreductase or CO and CODH as shown in the equation below [12].
[Co3+-E] + 2 ferredoxinred ^ [Co+-E] + 2 ferredoxinox (12.2)
In the next step, the reduced corrinoid protein is methylated by transmethylase through the following reaction [12]:
[Co+-E] + CH3-H4folate ^ [CH3-Co-E] + H4folate (12.3)
Within the carbonyl branch of the acetyl-CoA pathway, a carbonyl group is produced which is then merged with the methyl group to produce acetyl-CoA.
The enzyme carbonyl dehydrogenase (CODH) plays a central role in the carbonyl branch, or the right branch, of the pathway. Ni-Dependent carbon monoxide dehydrogenase (Ni-CODH) is a key enzyme in the scheme, and its role can be classified as: (1) monofunctional CODH, which catalyzes the oxidation of CO to CO2, which as a result could be reduced to formate and then methyl group in acetyl-CoA pathway, and (2) bifunctional CODH, which reduces CO2 to CO as the carbonyl group in acetyl-CoA synthesis and also mediates the evolution of acetyl-CoA alongside the acetyl — CoA synthase (ACS) [13].
During the closing stage of acetyl-CoA synthesis, CO (carbonyl moiety) condenses with the Co-methyl group (methyl moiety) of the methylated corrinoid protein and coenzyme A to yield acetyl — CoA. This reaction is catalyzed in the presence of carbonyl dehydrogenase (CODH)/acetyl-CoA synthase (ACS) as shown in the reaction below [12,13].
[CH3-Co-E] + CO + HS-CoA ^ CH3COS-CoA + [Co-E] (12.4)
The acetyl-CoA produced is the perfect precursor for the synthesis of a number of cell materials including nucleotides, amino acids, carbohydrates, and lipids [12]. Acetyl-CoA can be used as a source of cellular carbon or cellular energy depending on anabolic or catabolic pathway involved in these processes. In the anabolic pathway, acetyl-CoA is carboxylated to pyruvate in the presence of pyruvate synthase. Then, the pyruvate is converted to phosphoenolpyru — vate, which is considered as an intermediate in the evolution of cell materials. In contrast, in catabolic pathway the acetyl-CoA undergoes some reaction to generate ATP and acetate.
The conversion of acetyl-CoA to acetate is catalyzed by phos — photransacetylase, and this reaction is carried out via formation of acetyl-phosphate (CH3COO — PO3 ) as the intermediate. In the next reaction, acetyl-phosphate is transformed to acetate, while a molecule of ADP is phosphorylated to ATP in the presence of acetate kinase. This phase of metabolism results in acetate production, and is frequently known as the acidogenic phase as well. Conversion of acetyl-CoA to acetate and ATP is carried out during the growth phase of the microorganism. While the evolution of ethanol and NADP is performed during the non-growth phase. Therefore the growth is slow and no ATP is evolved in the solventogenic phase where ethanol is produced from acetyl-CoA. In this phase of the fermentation process, the reducing potential in the form of NADPH is utilized by the organism to form acetaldehyde (CH3CHO) in the presence of enzyme acetaldehyde dehydrogenase. In the last step of the production of alcohol, the acetaldehyde generated is converted to ethanol by the enzyme alcohol dehydrogenase where NADPH is converted to NADP+ during the process as shown in the equation below.
CH3CHO + NADPH + H+ ^ CH3CH2OH + NADP+ (12.5)
In order to avoid the consumption of reducing equivalents by other metabolic pathways such as aerobic respiration, it is very important to maintain a strict anaerobic environment during the acetyl-CoA pathway shown in Figure 12.1. Furthermore, through the proposed metabolic pathway, intermediate acetyl-CoA performs two major roles: firstly, it acts as a precursor for the cell macromolecule, and secondly, it serves as an