Fermentation Processes: Emerging and Conventional Technologies. Группа авторов
decarboxylation of pyruvate to acetyl coenzyme A (acetyl‐CoA), a major fuel of the tricarboxylic acid cycle (Ferrier 2017).
During glycolysis, two ATP molecules are initially consumed to phosphorylate glucose, which thus receives an essential energy supply to continue the catabolic pathway. Subsequently, two molecules of NAD+ oxidize phosphorylated sugar and are reduced to NADH + H+. During these reactions, which will lead to the synthesis of two molecules of pyruvic acid from a glucose molecule, a part of the energy released allows the direct synthesis of four molecules of ATP. Considering that two molecules of ATP are consumed and four are produced, glycolysis presents a net balance of two molecules of ATP for each molecule of oxidized glucose.
Figure 1.2 Glycolysis and citric acid cycle pathways.
1.2.2.2 Citric Acid Cycle
Pyruvic acid is first decarboxylated (release of CO2) and then oxidized by a molecule of NAD+ (reduced to NADH + H+) to form the two carbon molecules of acetyl‐CoA. As two molecules of pyruvic acid are produced from a glucose molecule, two molecules of CO2, NADH + H+, and acetyl‐CoA are produced. In prokaryotic cells (e.g. bacteria), the citric acid cycle (or Krebs cycle) occurs in the cytosol, while in eukaryotic organisms (e.g. yeast), it takes place in the mitochondrial matrix.
Overall, two molecules of CO2 are produced and three molecules of NAD+ are reduced to NADH + H+ during a turn of the citric acid cycle. Besides, two electrons and two protons released from pyruvic acid are used to reduce a molecule of flavin adenine dinucleotide (FAD) into FADH2, similar to that of NAD+. Finally, there is a release of chemical energy, which allows the synthesis of an ATP molecule. To summarize, for each molecule of glucose oxidized, four molecules of CO2, six molecules of NADH + H+, two molecules of FADH2, and two molecules of ATP are generated.
1.2.2.3 Electron Transport Chain and Oxidative Phosphorylation
In a process called oxidative phosphorylation, the energy stored in the reduced coenzymes (NADH and FADH2) is released to produce ATP molecules. During this process, each coenzyme is oxidized and gives its two energy‐rich electrons to an electron transport chain, or respiratory chain, located in the plasma membrane in prokaryotes and the inner membrane of mitochondria in eukaryotes. The chain consists of complex organic acceptors that transfer electrons through a series of redox reactions (Figure 1.3). As the electrons are transferred in this chain, the energy that they contain is released by stages and is used to activate chemiosmosis. This creates a gradient of H+ protons whose energy is reinvested, through the enzyme ATP synthetase, in the production of three molecules of ATP per molecule of oxidized NADH and two molecules of ATP per molecule of oxidized FADH2. At the end of the transport chain, the electrons exhausted of their energy bond with O2 and H+ ions to form a water molecule. To summarize, for each glucose molecule that enters glycolysis, 34 molecules of ATP will be produced by oxidative phosphorylation (i.e. 30 molecules coming from NADH and 4 coming from FADH2). Further reading about the electron transport chain could be found in Campbell (2015).
Figure 1.3 The electron transport chain showing the respiratory complexes.
1.2.3 Anaerobic Respiration
Some microbial species use anaerobic respiration to obtain their energy in the absence of O2. During this process, the electrons that are removed from organic nutrients such as glucose follow the same pathways as in aerobic respiration, except that the final acceptor is not O2, but another inorganic molecule (e.g. sulfate, nitrate, etc.). The sulfate ion is generally reduced to hydrogen sulfide, while nitrate ion can be reduced to nitrite, nitrogen oxide, or molecular nitrogen. Some bacteria reduce carbonate to methane. The number of ATP molecules produced by anaerobic respiration varies from one organism to another and from one metabolic pathway to another. This number is generally less than the 38 mol of ATP generated by aerobic respiration, the energy yield is lower, and anaerobic microorganisms usually grow slower than aerobic ones.
1.2.4 Fermentation
Some microbial species can obtain their energy in the absence of O2 through the catabolic pathway of fermentation. The only difference compared to respiration is in the final electron acceptor (Angelidaki et al. 2011; Dunford 2012; Madigan et al. 2015). In this case, ATP is produced without the Krebs cycle or an electron transport chain involved. This metabolic pathway does not require O2, because ATP comes exclusively from glycolysis, and the last electron acceptor is an organic molecule such as pyruvic acid (or a derived molecule). Figure 1.4 summarizes the metabolic pathway of fermentation.
As shown in Figure 1.4, glucose is oxidized during glycolysis to form two molecules of pyruvate. The electrons and protons released during this pathway are captured by the NAD+ to be reduced to NADH + H+. As shown above, two molecules of ATP are produced during glycolysis. To regenerate the NAD+, the NADH + H+ must be reoxidized; otherwise, the oxidation of glucose will stop and glycolysis too. During this oxidation, electrons and protons are directly transferred to pyruvate or one of its derivatives. The reduction of these final electron acceptors results in the formation of many different compounds, which provide a great variety of types of fermentation. At the same time, the NAD+ is regenerated and can engage in another round of glycolysis. The goal is to provide an uninterrupted supply of NAD+, which allows uninterrupted oxidation of glucose.
During fermentation, all ATP is produced solely by glycolysis, which implies a much lower energy yield compared to aerobic respiration (2 mol of ATP against 38 in prokaryotes). Considering that glucose oxidation is partial, a large part of the energy originally contained in glucose remains stored in the chemical bonds of the final fermentation product (e.g. ethanol, lactic acid, etc.). Fermentation microorganisms must, therefore, compensate for this shortfall by the oxidation of a larger quantity of substrate.
Figure 1.4 Schematic representation of fermentation and energy generation.
Different microorganisms can metabolize organic substrates (e.g. monosaccharides, amino acids, glycerol, etc.) (Angelidaki et al. 2011) to produce organic products such as acids, alcohols, and gases (Wilkins and Atiyeh 2012). This transformation occurs when all essential conditions and factors (e.g. temperature, pH, sugar concentration, culture medium, dissolved O2, and other micronutrients) required to the growth of microorganisms are provided (Smith 2009). Besides, the microorganism used must be viable, genetically stable, and able to resist several factors including the high concentrations of substrate, salt, and product, and sometimes to the presence of inhibitors, especially when using an industrial by‐product, or a lignocellulosic hydrolyzate.
Under anaerobic conditions, pyruvate is fermented to a wide range of fermentation products; most of them are of industrial importance (Figure 1.5).