Fermentation - Alcohol fermentation, Pasteur Effect

Fermentation occurs in the absence of aerobic or anaerobic respiration.  Here, since no external electron acceptor is available and no electron transport chain is happening, NADH is not oxidized back to NAD+. The NADH produced in the glycolytic pathway during the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate must still be oxidized back to NAD+ to make sure that the glycolytic pathway continues.  If NAD+ is not regenerated, the oxidation of glyceraldehyde 3-phosphate will cease, and glycolysis will stop.  This will lead to cessation of energy production.  A solution to this problem by many microorganisms is Fermentation.  They slow down or stop pyruvate dehydrogenase activity and use pyruvate or its derivatives as an electron and hydrogen acceptor for the reoxidation of NADH to NAD+ so that ATP production is continued.

Some chemoorganoheterotrophs do not carry out respiration even when oxygen or another exogenous acceptor is available, and carry out fermentation.  There are many types of fermentations, often characteristic of particular microbial groups.   Common to all microbial fermentations are the following points

1. NADH is oxidized to NAD+

2. Electron acceptor is often either pyruvate or a pyruvate derivative

3. The substrate is partially oxidized

4. ATP is formed by substrate-level phosphorylation only

5. oxygen is not needed.

Different types of fermentations

Alcohol Fermentation (Ethanol Fermentation) – Sugar is converted into ethanol and carbon dioxide. Examples are Saccharomyces cerevisiae (brewer's and baker's yeast), Zymomonas mobilis (bacterium).

This is an anaerobic process carried out by certain microorganisms, such as yeast (Saccharomyces cerevisiae) and some bacteria (Zymomonas mobilis). Sugar like glucose, fructose, and sucrose is metabolized to produce energy (ATP), with ethanol and carbon dioxide as the primary end products.

C₆​H₁₂​O_6​  → 2C₂​H₅​OH + 2CO₂​ + 2 ATP

(https://images.app.goo.gl/Hz7pFEGq4qPtz4ii7)

Lactic Acid Fermentation:

Glucose is converted into lactic acid by Lactic Acid Bacteria.   

Homolactic Fermentation - One molecule of glucose is converted into two molecules of lactic acid. Organisms performing homolactic fermentation convert glucose to pyruvate through EMP pathway or glycolysis, and then pyruvate is directly reduced to lactate by lactate dehydrogenase.  Examples are many Lactobacillus species (Lactobacillus acidophilus), Streptococcus, and Lactococcus species.  This process is used for producing most yogurts, cheeses, and many fermented dairy products. In human muscle cells, it occurs during intense exercise.

                        C₆​H₁₂​O₆​ →  2CH₃​CHOHCOOH (Lactic Acid)

Heterolactic Fermentation - One molecule of glucose is converted into lactic acid, along with other products such as ethanol and carbon dioxide. Pentose Phosphate Pathway roduces different intermediates that lead to a wider range of end products. Examples are some Lactobacillus species (e.g., Lactobacillus brevis), Leuconostoc species.This is used to contributes to the unique flavors and textures of certain fermented foods like sauerkraut, kimchi, etc.

C₆​H₁₂​O_6​ → CH₃​CHOHCOOH (Lactic Acid) + C₂​H₅​OH (Ethanol) + CO₂​ (Carbon Dioxide)

Acetic Acid Fermentation - Aerobic oxidation of ethanol to acetic acid and acetic acid (vinegar) is formed.

Butyric Acid Fermentation - Anaerobic fermentation of carbohydrates by Clostridium species to produce Butyric acid, H₂​, CO₂​, butanol and acetone.

Propionic Acid Fermentation - Fermentation of sugars or lactic acid by Propionibacterium to produce Propionic acid, acetic acid, and CO₂​.

Formic acid fermentation - Many bacteria, of the family Enterobacteriaceae, can metabolize pyruvate to formic acid and other products in a process sometimes called the formic acid fermentation. Formic acid may be converted to H₂ and CO₂ by formic hydrogenase.  There are two types of formic acid fermentation - Mixed acid fermentation  and butanediol fermentation.

Mixed acid fermentation results in the production of ethanol and a complex mixture of acids, particularly acetic, lactic, succinic, and formic acids. If formic hydrogenlyase is present, the formic acid will be degraded to H2 and CO2. Examples are Escherichia, Salmonella, Proteus, etc.

Butanediol fermentation (2,3-Butanediol Fermentation) is characteristic of Enterobacter, Serratia, Erwinia, and some species of Bacillus. Pyruvate is converted to acetoin, which is then reduced to 2,3-butanediol with NADH. A large amount of ethanol is also produced, together with smaller amounts of ethanol, lactic acid, and formic acid, and significant amounts of CO₂​ and H₂​.

Propane-1,3-diol Fermentation - Certain bacteria, such as Clostridium butyricum and Lactobacillus reuteri, can ferment glycerol into propane-1,3-diol, along with acetic acid, butyric acid, etc.

Succinic Acid Fermentation - Some anaerobic bacteria, such as Anaerobiospirillum succiniciproducens and certain species of Mannheimia and Actinobacillus, can ferment carbohydrates to produce succinic acid often with minor amounts of acetic acid, formic acid, or ethanol.

Malolactic Fermentation  -  Carried out by lactic acid bacteria (primarily Oenococcus oeni) in wine after primary alcoholic fermentation. It converts malic acid into lactic acid (a softer, monocarboxylic acid) and carbon dioxide.

Substances other than sugars are also fermented by microorganisms. Some members of the genus Clostridium ferment mixtures of amino acids. Proteolytic clostridia, such as the pathogens C. sporogenes and C. botulinum will carry out the Stickland reaction in which one amino acid is oxidized and a second amino acid acts as the electron acceptor.   Alanine is oxidized and glycine is reduced to produce acetate, CO2, and NH3. Some ATP is formed by substrate-level phosphorylation, and fermentation is quite useful for growing in anaerobic, protein-rich environments.

 

Alcohol Fermentation (Ethanol Fermentation) – Sugar is converted into ethanol and carbon dioxide by organisms such as Saccharomyces cerevisiae or Zymomonas mobilis.  Alcohol fermentation is a two-step process.

Glycolysis

This is the initial breakdown of glucose, a six-carbon sugar, into two molecules of pyruvate, a three-carbon compound. This process occurs in the cytoplasm of the cell and does not require oxygen.

• One molecule of glucose (C₆​H₁₂​O₆​) is broken down to 2 molecules of pyruvate (2CH₃COCOOH).
• Two molecules of ATP are utilised in the initial phosphorylation steps.
• Four molecules of ATP are produced through substrate-level phosphorylation, resulting in a net gain of 2 ATP molecules.
• Two molecules of NAD+ are reduced to 2 NADH.

Conversion of Pyruvate to Ethanol and Carbon Dioxide

·       In the absence of oxygen, the pyruvate molecules produced from glycolysis undergo further reactions to regenerate NAD+ (NAD+ is essential for glycolysis to continue) and produce ethanol and carbon dioxide.

·       Each pyruvate molecule loses a carboxyl group, which is released as carbon dioxide (CO2​) to produce 2 molecules of acetaldehyde.  This step is catalyzed by pyruvate decarboxylase.

·       The NADH molecules generated during glycolysis donate their electrons to acetaldehyde, regenerating NAD+  and acetaldehyde is reduced to ethanol. This reaction is catalyzed by alcohol dehydrogenase.

The most common and widely used microorganism for alcohol fermentation is yeast, particularly Saccharomyces cerevisiae (brewer's yeast or baker's yeast). Some bacteria, like Zymomonas mobilis, can also perform alcohol fermentation.

Alcohol fermentation has numerous significant applications across various industries for the production of Alcoholic Beverages such as Wine, Beer, Whiskey, Rum, Vodka, Brandy, etc, in Baking to produce bread, for Biofuel Production, for production of Industrial Chemicals and Pharmaceuticals and for Food Preservation and flavor Enhancement.

 

Pasteur effect

The Pasteur effect demonstrates the remarkable metabolic adaptability of many facultative anaerobes like yeast to their environment and highlights the efficiency difference between anaerobic and aerobic energy production.

The Pasteur effect was discovered in 1857 by Louis Pasteur. He observed that when yeast (Saccharomyces cerevisiae) was grown under anaerobic conditions, it consumed much more sugar and produced more ethanol.  But when it was provided with oxygen, its rate of sugar consumption significantly decreased, and the production of ethanol and carbon dioxide (via fermentation) also reduced.  This phenomenon of a decrease in the rate of sugar catabolism that occurs when microorganisms are switched from anaerobic to aerobic conditions is termed as Pasteur effect.

Aerobic respiration is much more effective for energy production than fermentation since more ATP are produced via electron transport and oxidative phosphorylation. So when moved from anaerobic to aerobic conditions, microbes reduce their rate of sugar catabolism as less sugar must be degraded to obtain the same amount of ATP than under anaerobic process.  The presence of oxygen under aerobic condition allows the complete oxidation of glucose, as oxygen acts as the final electron acceptor in the electron transport chain. This makes the entire process highly efficient. Without oxygen, electron transport chain and Krebs cycle shut down, forcing the cell to rely solely on fermentation for ATP.

ATP yield under aerobic and anaerobic conditions

Anaerobic Fermentation - One molecule of glucose yields a net of 2 ATP molecules.  The primary purpose of fermentation is to regenerate NAD+ from NADH so that glycolysis (which requires NAD+) can continue, providing a small but continuous supply of ATP.

Aerobic Respiration - One molecule of glucose yields approximately 30-32 ATP molecules (glycolysis, Krebs cycle, and electron transport chain). Glucose is completely oxidized to carbon dioxide and water.

 

 

Anaerobic respiration with special reference to dissimilatory nitrate reduction

Anaerobic respiration with special reference to dissimilatory nitrate reduction

Microorganisms usually use one of three sources of energy - Phototrophs capture radiation energy from the sun. Chemoorganotrophs oxidize organic molecules to liberate energy, while chemolithotrophs employ inorganic nutrients as energy sources.  Microorganisms also vary in the electron acceptors used by chemotrophs.  Three major kinds of acceptors are employed and as per the different modes are Fermentation, aerobic respiration and anaerobic respiration.

In fermentation, the energy substrate is oxidized and degraded without the participation of an exogenous or externally derived electron acceptor. Usually, the catabolic pathway produces an intermediate such as pyruvate that acts as the electron acceptor. Fermentation normally occurs under anaerobic conditions, but also occurs sometimes when oxygen is present.

When the energy-yielding metabolism make use of exogenous or externally derived electron acceptors, the metabolic process is called respiration and is divided into two - aerobic respiration and anaerobic respiration.  In aerobic respiration, the final electron acceptor is oxygen and in anerobic respiration, the final electron acceptor is a different exogenous acceptor, mostly an inorganic molecule such as (NO₃^_, SO₄^2_, CO₂, Fe₃^_, SeO₄^2_, etc), or organic acceptors such as fumarate.

The amount of energy is different for fermentation and respiration.  Since the electron acceptor in fermentation is at the same oxidation state as the original nutrient and there is no overall net oxidation of the nutrient, only a limited amount of energy is made available. The acceptor in respiratory processes has reduction potential much more positive than the substrate and thus considerably more energy will be released during respiration.

Fermentation - an energy-yielding process in which organic molecules serve as both electron donors and acceptors.

Respiration - an energy-yielding process in which the acceptor is an inorganic molecule, either oxygen (aerobic respiration) or another inorganic acceptor (anaerobic respiration).

Anaerobic respiration is a fascinating and essential process in various ecosystems, particularly where oxygen is scarce or absent. It allows organisms to generate energy (ATP) by breaking down organic molecules and instead of using oxygen, it utilizes other inorganic molecules such as nitrate, sulfate, and CO2, metals, etc or sometimes organic compounds as the final electron acceptor. 

Key Characteristics of anaerobic respiration:

• Absence of Oxygen: The defining feature is the lack of oxygen as the final electron acceptor.
• Electron Transport Chain: Unlike fermentation (which also occurs without oxygen but doesn't use an electron transport chain), anaerobic respiration utilizes an electron transport chain to generate a proton motive force, which drives ATP synthesis via chemiosmosis.
• Lower ATP Yield: Compared to aerobic respiration, anaerobic respiration typically yields less ATP per molecule of glucose.
• Diverse Microorganisms: It is carried out by prokaryotes that inhabit anaerobic environments like sediments, wetlands, deep subsurface environments, and even certain host-microbe interactions.
• Ecological Importance: Anaerobic respiration plays crucial roles in global biogeochemical cycles, particularly the nitrogen, sulfur, and carbon cycles, by transforming various compounds. It's also vital in bioremediation and wastewater treatment.

Steps involved:

1. Glycolysis: Glucose is broken down into pyruvate, producing a small amount of ATP and NADH. This step is common to both aerobic and anaerobic respiration.
2. Krebs Cycle: Pyruvate is further oxidized, generating more NADH and FADH₂ (electron carriers).
3. Electron Transport Chain: The electrons from NADH and FADH₂ are passed down an electron transport chain. Instead of oxygen, the alternative inorganic electron acceptor is reduced at the end of chain.
4. Chemiosmosis: The movement of electrons through the electron transport chain pumps protons across a membrane, creating an electrochemical gradient or proton motive force. This gradient is then used by ATP synthase to produce ATP from ADP and inorganic phosphate.

Dissimilatory Nitrate Reduction (DNR)

Some bacteria can use nitrate as the electron acceptor at the end of their electron transport chain and produce ATP. This process is called dissimilatory nitrate reduction. This is termed "dissimilatory" because the nitrate is reduced for energy generation, not for assimilation into cellular biomass (In assimilatory nitrate reduction, nitrate is incorporated into organic molecules for growth).   Nitrate may be reduced to nitrite by nitrate reductase.

However, since a nitrate molecule will accept only two electrons, reduction of nitrate to nitrite is not a particularly effective way of making ATP, and thus a large amount of nitrate is required for growth.   Also the nitrite formed is quite toxic.

So nitrate is often is further reduced to nitrogen gas, and this process is known as denitrification. In this case, each nitrate will then accept five electrons, and the product will be nontoxic too.

Denitrification is a multistep process with four enzymes: nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase with stepwise reduction of nitrate (NO₃^−​) to nitrite (NO₂⁻​), nitric oxide (NO), nitrous oxide (N₂​O), and finally to dinitrogen gas (N₂​). This process releases gaseous nitrogen compounds into the atmosphere.  The four enzymes use electrons from coenzyme Q and c-type cytochromes to reduce nitrate and generate PMF.

• Nitrate Reductase: Reduces NO₃⁻​ to NO₂⁻​.   
• Nitrite Reductase: Reduces NO₂⁻​ to NO.  This is present in periplasmic space in gram-negative bacteria.
• Nitric Oxide Reductase: Reduces NO to N₂​O.  Nitric oxide reductase catalyzes the formation of nitrous oxide from NO and is a membrane-bound cytochrome bc complex.
• Nitrous Oxide Reductase: Reduces N₂​O to N₂​.  This enzymes is also periplasmic.

Two types of bacterial nitrite reductases catalyze the formation of NO in bacteria. One contains cytochromes c and d1 (e.g., Paracoccus and Pseudomonas aeruginosa), and the other is a copper protein (e.g., Alcaligenes).

Key Characteristics of Denitrification:

• The ultimate product, N2​ gas, is unreactive and returns to the atmosphere, effectively removing fixed nitrogen from the ecosystem.
• It is a crucial component of the global nitrogen cycle, returning fixed nitrogen to the atmosphere.
• Many denitrifying bacteria are facultative anaerobes, they can switch between aerobic respiration (when oxygen is available) and denitrification (when oxygen is absent or low). Examples are Pseudomonas denitrificans and Paracoccus denitrificans.
• Intermediate products like N₂​O (nitrous oxide) are potent greenhouse gases and can contribute to ozone depletion.
• Denitrification in anaerobic soil results in the loss of soil nitrogen and adversely affects soil fertility.

Dissimilatory Nitrate Reduction to Ammonium (DNRA) or Nitrate Ammonification

This is the reduction of nitrate (NO₃^−​) or nitrite (NO₂⁻​) directly to ammonium (NH₄⁺​). This process conserves bioavailable nitrogen within the ecosystem.

Here, the final product is a soluble, bioavailable form that can be readily utilized by plants and microorganisms.  DNRA and denitrification often compete for the same substrates (NO₃⁻and NO₂⁻​) in anaerobic environments.  Certain bacteria like Beggiatoa, Thioploca, and Shewanella species are known to perform DNRA.

 

Other mechanisms of anaerobic respiration employed by obligate anaerobes are as follows.

·       Methanogens use CO₂ or carbonate as a terminal electron acceptor and they reduce CO₂ to methane.   This is a significant process in anaerobic environments like wetlands and the guts of ruminants. 

·       Desulfovibrio use Sulfate as  the final acceptor and get reduced to sulfide (S^2_ or H₂S).