Mixed-Acid Fermentation: A Complete Guide to This Microbial Metabolic Pathway

Mixed-acid fermentation is a core microbial metabolic process used by many bacteria especially members of the Enterobacteriaceae family. It results in the production of a variety of acidic and non-acidic end products such as lactic acid, acetic acid, formic acid, ethanol, CO₂, H₂, and succinate.

This metabolic strategy is essential in microbial ecology, pathogenesis, food microbiology, and industrial biotechnology.

This article explains the biochemical pathway, key organisms, regulation, products, and real-world applications.

What Is Mixed-Acid Fermentation?

Mixed-acid fermentation is an anaerobic metabolic pathway where bacteria convert glucose into multiple end products rather than a single dominant product.

Key features:

• Occurs under oxygen-limited or anaerobic conditions

• Produces several acids + ethanol

• Generates ATP through substrate-level phosphorylation

• Produces gases (CO₂ and H₂), useful for diagnostic tests

This process allows bacteria to maximize ATP yield while maintaining redox balance.

Microorganisms That Perform Mixed-Acid Fermentation

The main bacterial groups include:

 1. Enterobacteriaceae

• Escherichia coli

• Salmonella spp.

• Shigella spp.

• Proteus spp.

 2. Clostridia (some species)

Anaerobic conditions favor acid production.

 3. Certain anaerobic Gram-negative rods

Particularly in gut microbiota and soil bacteria.

E. coli is the most studied model organism for mixed-acid fermentation.

 Biochemical Pathway of Mixed-Acid Fermentation

Once glucose enters the cell and is metabolized via glycolysis, pyruvate becomes the central branching point for several metabolic fates.

Step 1: Glycolysis

• Glucose → pyruvate

• Produces 2 ATP and NADH

Step 2: Pyruvate Conversion to Multiple End Products

Through multiple enzymatic pathways:

1. Lactic Acid Pathway

Pyruvate → Lactate

Enzyme: Lactate dehydrogenase

2. Acetic Acid Pathway

Pyruvate → Acetyl-CoA → Acetate

Produces additional ATP.

3. Ethanol Production

Acetyl-CoA → Acetaldehyde → Ethanol

Regenerates NAD⁺.

4. Formic Acid (Formate) Production

Pyruvate → Formate + Acetyl-CoA

Enzyme: Pyruvate formate lyase (PFL)

Formate may be further broken down into:

• H₂

• CO₂

(via formate-hydrogen lyase complex)

5. Succinic Acid Pathway

Phosphoenolpyruvate (PEP) → Oxaloacetate → Succinate

Important for redox balance.

 End Products of Mixed-Acid Fermentation

The presence of multiple acids sharply lowers pH, a characteristic used in microbiological identification.

Mixed-Acid Fermentation vs. 2,3-Butanediol Fermentation

Some Enterobacteriaceae use 2,3-butanediol fermentation instead of mixed-acid fermentation. The Voges–Proskauer (VP) test can differentiate them.

Mixed-Acid Fermenters (VP-negative)

• E. coli

• Shigella

• Salmonella

2,3-Butanediol Fermenters (VP-positive)

• Enterobacter

• Klebsiella

• Serratia

Mixed-acid fermenters produce more acids → lower pH.

Environmental and Physiological Significance

1. Energy Production in Anaerobic Environments

Allows bacteria to survive in:

• Gut microbiota

• Sediments

• Wastewater systems

• Anaerobic culture environments

2. Redox Balance

NADH must be oxidized back to NAD⁺; mixed-acid pathways ensure this balance.

3. Adaptation During Low Oxygen Stress

Enteric bacteria switch from aerobic respiration to fermentation in hypoxic tissues or intestinal environments.

Industrial and Applied Importance

1. Food Microbiology

Mixed-acid fermentation shapes the characteristics of:

• Fermented vegetables

• Some cured meats

• Dairy-free fermented foods

Acids inhibit pathogens and act as natural preservatives.

2. Biofuel and Biochemical Production

Engineered bacteria can enhance production of:

• Ethanol

• Acetate

• Succinic acid (important for bioplastics)

3. Clinical Microbiology

Diagnostic tests rely on this metabolic pathway:

• Methyl Red (MR) Test: detects strong acid production

• Gas production in Durham tubes

• Biochemical identification of pathogens

4. Waste Treatment

Acidogenic bacteria use mixed-acid pathways during:

• Anaerobic digestion

• Organic waste degradation

• Biogas production (H₂ and CO₂)

Regulation of Mixed-Acid Fermentation

Bacterial metabolism is tightly controlled by:

• Oxygen levels

• FNR (fumarate nitrate reduction regulator)

• ArcA/ArcB two-component system

• pH changes

• Availability of electron acceptors

Under anaerobic and acidic conditions, mixed-acid pathways dominate.

 Future Perspectives and Biotechnology

• Metabolic engineering to increase succinate, ethanol, or acetate yields

• CRISPR-based editing in E. coli for industrial fermentation

• Mixed-acid pathways in synthetic microbial consortia

• High-value biochemicals from renewable feedstocks

Mixed-acid fermentation will remain a foundational process for next-generation sustainable bioprocessing.

Conclusion

Mixed-acid fermentation is a versatile, energy-efficient metabolic strategy that enables bacteria to thrive in anaerobic environments while producing a variety of valuable end products. Its relevance spans clinical microbiology, biotechnology, food science, and bioenergy.

Understanding this pathway is essential for optimizing industrial fermentation and for interpreting microbial behavior in both natural and clinical settings.