Lactic acid fermentation

Lactate fermentation or Lactic acid fermentation

Lactic acid fermentation is an anaerobic process where bacteria convert sugars into lactic acid. The primary goal is the generation of ATP (adenosine triphosphate) for cellular energy in the absence of oxygen and the regeneration of NAD+ from NADH to maintain the functionality of glycolysis. Lactic acid fermentation plays a crucial role in the food and beverage industry and is employed in the production of cheese, yogurt, fermented vegetables, etc.

Lactic acid fermentation, a process used by certain bacteria, can be categorized into homofermentative and heterofermentative pathways based on their end products. 

Lactic acid bacteria (LAB) are divided into two major groups: The homofermentative group produce lactic acid as the sole product of the fermentation of sugars and the heterofermentative group produce lactic acid as well as ethanol and CO2. Homofermentative lactic acid bacteria convert the glyceraldehyde 3-phosphate to lactic acid.   Heterofermentative lactic acid bacteria receive five-carbon xylulose 5 phosphate from the Pentose pathway and this is converted to glyceraldehyde 3-phosphate which in turn get converted to lactic acid.  The two carbon acetyl phosphate molecule form the the Pentose pathway is converted to ethanol.

(https://images.app.goo.gl/bgGodqaQZBPxMku1A)

 

Homofermentative pathway

In this pathway, LAB converts glucose into lactic acid as the main product through the Embden-Meyerhof-Parnas (EMP) pathway or glycolysis.  One molecule of glucose is converted into two molecules of lactic acid and two molecules of ATP. 

Lactate dehydrogenase (LDH) is the essential enzyme that catalyzes the reduction of pyruvate to lactic acid and the reoxidation of NADH to NAD. This pathway predominantly yields lactic acid, with minimal or no other byproducts, producing two ATP molecules per glucose molecule.

C₆​H_12​O₆​  →  2CH₃​CHOHCOOH

Glucose  → Lactic acid

Steps involved

Glycolysis (Embden-Meyerhof-Parnas Pathway):

• Glucose Uptake and Phosphorylation: Glucose, a six-carbon sugar, is phosphorylated to glucose-6-phosphate (using ATP) by enzymes like glucokinase or hexokinase.
• Isomerization: Glucose-6-phosphate is isomerized to fructose-6-phosphate.
• Second Phosphorylation: Fructose-6-phosphate is phosphorylated again (using ATP) to become fructose-1,6-bisphosphate.
• Cleavage: Fructose-1,6-bisphosphate is cleaved into two three-carbon molecules: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). DHAP is then converted to G3P.
• Glyceraldehyde-3-Phosphate (G3P) undergo oxidation and phosphorylation by the enzyme Glyceraldehyde-3-phosphate dehydrogenase to form 1,3-Bisphosphoglycerate (1,3-BPG).  NADH is formed.
• Substrate-Level Phosphorylation (First ATP Generation) - 1,3-BPG is converted to 3-Phosphoglycerate (3-PG) and ATP by the enzyme Phosphoglycerate kinase.  
• Isomerization - Phosphoglycerate mutase convert 3-Phosphoglycerate  to 2-Phosphoglycerate
• Dehydration – The enzyme Enolase catalyse production of Phosphoenolpyruvate (PEP) from 2-Phosphoglycerate.
• Substrate-Level Phosphorylation (Second ATP Generation) – PEP is converted by the enzyme Pyruvate kinase to pyruvate with the release of a molecule of ATP.
• Net ATP and NADH from Glycolysis: At this stage, glycolysis yields a net of 2 ATP molecules and 2 NADH molecules per glucose molecule.

Pyruvate Reduction to Lactic Acid:

• The enzyme lactate dehydrogenase (LDH) catalyzes the reduction of two molecules of pyruvate to Lactic Acid.  The reduction of pyruvate to lactic acid reoxidizes NADH back to NAD+.
• This regeneration of NAD+ allows glycolysis to continue, as NAD+ is required for earlier step in glycolysis.

Examples of Organisms Performing Homolactic Fermentation:

• Many species of Lactic Acid Bacteria (LAB), such as Lactobacillus delbrueckii, Streptococcus thermophilus, Lactococcus lactis, Pediococcus species, Lactobacillus acidophilus, Lactobacillus bulgaricus.
• Animal muscle cells under anaerobic conditions.

Homolactic acid fermentation is used for fast acidification in dairy products like yoghurt and cheese, contributing to preservation and texture.

 

Heterofermentative pathway

Heterofermentative LAB produce lactic acid along with other end products, such as ethanol and carbon dioxide, through the phosphoketolase pathway.  One molecule of glucose is converted into one molecule of lactic acid, one molecule of ethanol, and one molecule of carbon dioxide. 

C₆​H₁₂​O₆​  →  CH₃​CHOHCOOH  +  C₂​H₅​OH  +  CO₂​

(Glucose) → (Lactic Acid) + (Ethanol) + (Carbon Dioxide)

Steps involved

Glucose Phosphorylation - Glucose is phosphorylated to glucose-6-phosphate by glucokinase and consumes 1 ATP. Glucose-6-phosphate is then isomerized to 6-phosphogluconate.

Oxidation and Decarboxylation - 6-Phosphogluconate is oxidized and decarboxylated to form ribulose-5-phosphate by the enzyme 6-phosphogluconate dehydrogenase. This step produces 1 NADH and 1 CO2​.

Isomerization - Ribulose-5-phosphate is then isomerized to xylulose-5-phosphate by the enzyme Phosphoketopentose epimerase

Phosphoketolase Cleavage - Xylulose-5-phosphate is cleaved by the enzyme phosphoketolase to Glyceraldehyde-3-phosphate (G3P) and Acetyl phosphate

Conversion of Glyceraldehyde-3-phosphate (G3P) to Lactic Acid (similar to homolactic fermentation)

•  G3P is oxidized and phosphorylated to 1,3-bisphosphoglycerate
•  1,3-bisphosphoglycerate is converted to 3-phosphoglycerate.       
•  3-phosphoglycerate is then converted to 2-phosphoglycerate, then to phosphoenolpyruvate (PEP).
•    PEP is converted to pyruvate.
•  Pyruvate is reduced to lactic acid by lactate dehydrogenase.

Conversion of Acetyl Phosphate to Ethanol or Acetic Acid  - Acetyl phosphate can follow one of two main routes:

To Ethanol (Standard Heterolactic)

Cells use this when they must recycle NADH to keep running.

Step 1: Acetyl phosphate + NADH ➔ Acetaldehyde + NAD+ + Pi - by the enzyme Acetaldehyde dehydrogenase, No ATP is made.

Step 2: Acetaldehyde + NADH ➔ Ethanol + NAD+ - by the enzyme Alcohol dehydrogenase.

To Acetic Acid (Extra Energy)

Cells use this only if they have an external pathway to recycle NADH.  

Acetyl phosphate + ADP ➔ Acetate + ATP (Enzyme: Acetate kinase).

Heterofermentative LAB utilize the phosphoketolase pathway, which involves the cleavage of glucose into glyceraldehyde-3-phosphate and acetyl phosphate.   The end products vary but typically include Lactic acid, Ethanol, Acetate, Carbon dioxide, etc.

Some bacteria known to use the heterofermentative pathway include Leuconostoc mesenteroides, Lactobacillus brevis, Lactobacillus fermentum, Oenococcus oeni, Some Weissella species, etc. 

Heterofermentative LAB are used in various industrial processes, including sourdough fermentation, and contribute to the flavor and texture of fermented foods such as Sauerkraut and kimchi. The mixed end products give these fermented foods a more complex and desired flavor than those produced by homolactic fermentation.  However, their ability to produce CO₂ and certain acids can also lead to spoilage in some type of food products. 

The heterofermentative pathway generally produces less ATP per glucose molecule compared to homolactic fermentation. 

Energy Yield

Homolactic Fermentation Summary

• ATP Invested: -2 ATP
• Glucose ➔ Glucose-6-phosphate (-1 ATP)
• Fructose-6-phosphate ➔ Fructose-1,6-bisphosphate (-1 ATP)
• ATP Generated: +4 ATP
• Two molecules of G3P are processed. Each yields 2 ATP via substrate-level phosphorylation.  
• Redox Balance (NADH): Net Zero
• +2 NADH generated during the oxidation of the two G3P molecules.
• -2 NADH consumed when pyruvate is reduced to lactic acid.
• Net Yield: +2 ATP and 2 Lactic Acid molecules.

 

Heterolactic Fermentation Summary (Ethanol Route)

Because the six-carbon glucose is broken into a three-carbon (G3P) and a two-carbon (acetyl phosphate) molecule, only half of the molecule goes through the high-yield ATP steps.  

• ATP Invested:  -1 ATP
• Glucose ➔ Glucose-6-phosphate (-1 ATP)
• ATP Generated: +2 ATP
• Only one molecule of G3P is generated per glucose. This single G3P generate 2 ATP
• Redox Balance NADH / NADPH: Net Zero
• +2 NADH or 1 NADPH + 1 NADH are generated during the early oxidation steps to make ribulose-5-phosphate.
• + 1 NADH is generated when the G3P is oxidized.
• -1 NADH is consumed when pyruvate is reduced to lactic acid.
• -2 NADH are consumed to reduce acetyl phosphate to ethanol.
• Net Yield: +1 ATP, 1 Lactic Acid, 1 Ethanol, and 1 CO2