Butyric Acid Fermentation: Process, Industrial Applications, and Health Benefits Explained

Table of Contents

• Introduction to Butyric Acid Fermentation
• Butyric Acid Chemical and Physical Properties
• What is Fermentation?
• Butyric Acid Fermentation
• Butyric Acid Fermentation Principle
• Butyric Acid Fermentation Steps
• Limiting Factors in Butyric Acid Fermentation
• Butyric Acid Applications
• Human Health and Butyric Acid
• Conclusion
• References

Introduction to Butyric Acid Fermentation

Butyric acid, a short-chain fatty acid (SCFA) with four carbon atoms, is a volatile compound represented by the formula CH₃CH₂CH₂COOH. It is naturally produced by bacteria under anaerobic conditions. This colorless liquid has an oily texture and a characteristic unpleasant odor, reminiscent of rancid butter. Despite its strong smell, butyric acid has significant applications in various industries, including food, chemicals, animal feed, and pharmaceuticals. It plays a vital role in microbial activity within the mammalian gut and is commercially valuable for producing flavoring agents and plastics, such as cellulose butyrate.

Industrially, butyric acid is primarily synthesized through chemical processes, such as the oxidation of butyraldehyde. Also referred to as butanoic acid, it is a four-carbon fatty acid with a terminal carboxylic functional group. It is often found in esterified forms in plant oils and animal fats.

In humans, butyric acid is naturally present in breast milk, body odor, and the colon, where it is produced by gut bacteria fermenting carbohydrates and dietary fibers. It also contributes significantly to the flavor and aroma of commercial products like parmesan cheese and rancid butter.

Butyric Acid Chemical and Physical Properties

• Chemical Formula: CH₃CH₂CH₂COOH
• Molecular Weight: 88.11 g/mol
• Corrosive Nature: Can corrode both tissues and metals.
• Appearance: A colorless liquid with an oily texture.
• Taste: Has a buttery flavor.
• Solubility: Readily dissolves in water, alcohol, propylene glycol, and most oils.

What is Fermentation?

Fermentation is one of the oldest biotechnological processes, historically used to create various commercial products such as yogurt, wine, beer, cheese, and more. The term "fermentation" originates from the Latin word fervere, meaning "to boil," referencing the release of carbon dioxide during the process.

In biological systems, fermentation serves as an alternative energy-generating pathway in the absence of oxygen. Microorganisms such as yeast, bacteria, and fungi perform fermentation under anaerobic conditions, converting sugars into ATP, water, alcohol, lactic acid, and carbon dioxide.

Fermentation is classified into two main types:

• Lactic Acid Fermentation: In this process, lactose serves as the substrate, and lactic acid is the primary end product.
• Alcoholic Fermentation: Here, glucose is the substrate, and the end products are alcohol and carbon dioxide.

Fermentation is widely utilized in the production of food items like bread, yogurt, cheese, and fermented vegetables such as kimchi.

Butyric Acid Fermentation

Butyric acid is widely used in the food and chemical industries. In the food sector, it acts as a flavor enhancer to produce butter-like tastes, while its esters are employed to add aromas and fruity flavors. To overcome challenges in large-scale butanol production, food manufacturers prefer fermentation as an efficient biotechnological method.

Butyric Acid Fermentation Principle

The principle behind large-scale butyric acid production via fermentation involves the glycolysis of glucose by bacterial strains. Glucose is oxidized to pyruvate and further converted into acetyl-CoA by the enzyme pyruvate ferredoxin oxidoreductase. Some acetyl-CoA molecules form acetic acid, while the remaining acetyl-CoA is transformed into acetoacetyl-CoA, which is reduced to butyryl-CoA and eventually converted into butyric acid and ATP, along with carbon dioxide.

The end products of this process include butyric acid, 3 ATP molecules, water, CO₂, and a small quantity of ethanol.

Butyric Acid Fermentation Steps

1. Microorganisms and Inoculum Preparation

Several bacterial strains, including species from Clostridium, Eubacterium, Butyribacterium, Megasphera, Sarcina, and Fusobacterium, can anaerobically produce butyric acid.

Clostridium species are particularly used in commercial applications due to their characteristics such as chemo-organotrophy, gram-positive nature, spore formation, and strict anaerobiosis.

2. Media Preparation

The medium for fermentation includes chemicals like ammonium sulfate [(NH₄)₂SO₄], manganese II sulfate (MnSO₄), potassium dihydrogen phosphate (KH₂PO₄), and ferrous sulfate (FeSO₄), with yeast extract dissolved in sterile distilled water. Glucose is added as a carbon source in varying concentrations. Clostridium butyricum is stored at 4°C in a medium containing calcium carbonate and medicinal oil to maintain anaerobic conditions.

3. Inoculum Preparation

10 ml of sterile medium is added to a test tube containing C. butyricum, incubated for 20 hours at 37°C, and calcium carbonate is added to maintain pH balance. The metabolic pathways in Clostridium species yield butyrate, butanol, and acetate, with small amounts of lactate and ethanol as byproducts.

4. Batch Fermentation Extraction Process

Fermentation takes place in a double-walled cylinder with a sample tube and stirrer, all sterilized before adding the materials. The working inoculum volume is 225 ml (10% v/v) in a sterilized medium. The sterilized materials are mixed with the organic phase, containing sucrose (the sole carbon source), amines, and emulsifiers like oleyl alcohol. Anaerobic conditions are achieved by sparging carbon dioxide into the medium. The fermentation is carried out at 37°C, with regular sampling for analysis. Butyric acid is extracted from the organic phase into an aqueous solution, and its concentration is measured using gas chromatography.

4. Fed-Batch Fermentation

The procedure for fed-batch fermentation is similar to the batch method, but sucrose or glucose is added at regular intervals. Sterile conditions must be maintained to avoid pathogen growth. The optimum pH of 6.5 is maintained for bacterial growth and butyric acid production. Compared to batch fermentation, fed-batch fermentation has shown a 36.65% increase in butyric acid production due to better substrate utilization.

Limiting Factors in Butyric Acid Fermentation

• Osmotic Pressure: High sugar and salt concentrations can lead to osmotic pressure on bacterial strains, disrupting cell membrane phospholipids and causing cell death.
• Butyric Acid Inhibition: Increased acid production may affect cell membrane integrity and metabolic processes, leading to arrested fermentation and lower butyric acid yields.
• pH and Nutrients: pH levels must be carefully controlled to ensure optimal bacterial growth and enzyme activity. The use of inappropriate nutrients can reduce butyric acid productivity. Corn steep liquor (CSL), rich in amino acids, peptides, and trace elements, is often used to enhance yields.

Butyric Acid Applications

Butyric acid has numerous industrial uses due to its unique biochemical properties:

• As a Bioplastic: Butyric acid is used in the production of cellulose acetate butyrate (CAB), which is more resistant to degradation than cellulose acetate and is used in coatings, paints, and tools.
• In Fragrance and Flavoring: Esters of butyric acid, such as methyl butyrate, provide pleasant aromas used in perfumes, disinfectants, and flavorings.
• As a Flavoring Agent: Butyric acid is used in the flavoring of cheese, beer, and wine.
• As Fishing Bait: Due to its potent odor, butyric acid is used in fishing bait to attract fish.

Human Health and Butyric Acid

• Butyrate, a circulating form of butyric acid, plays a crucial role in regulating immune homeostasis and promoting the differentiation of regulatory T-cells.
• In the gut, butyrate helps inhibit pro-inflammatory cytokines and maintains the integrity of the intestinal epithelial barrier. 
• Produced by colonic bacteria, butyric acid helps prevent conditions like colon cancer and ulcerative colitis by enhancing energy production and inhibiting excessive cell proliferation.

Conclusion

Butyric acid is a volatile, short-chain fatty acid (SCFA) with a four-carbon backbone and a carboxylic group at the fourth carbon. In humans, it is produced anaerobically by bacterial strains in the gut and colon, helping prevent inflammatory diseases. Due to its distinct taste, flavor, and aroma, butyric acid is industrially mass-produced through fermentation, with batch and fed-batch techniques being the main methods. Research shows that the fed-batch method offers improved butyric acid production. Industrial applications include bioplastic production, flavoring agents in food and beverages, and esters for perfumes and disinfectants.

References

• Jiang, Ling, et al. “Butyric acid: Applications and recent advances in its bioproduction.” Biotechnology Advances 36.8 (2018): 2101-2117.
• The Future of Butyric Acid in Industry – https://www.hindawi.com/journals/tswj/2012/471417/
• Butyric Acid – https://www.britannica.com/science/butyric-acid
• Qureshi, Nasib, Siqing Liu, and Badal C. Saha. “Butyric Acid Production by Fermentation: Employing Potential of the Novel Clostridium tyrobutyricum Strain NRRL 67062.” Fermentation 8.10 (2022): 491.
• Butyric Acid – https://pubchem.ncbi.nlm.nih.gov/compound/Butyric-acid
• Fermentation: Humanity’s Oldest Biotechnological Tool – https://kids.frontiersin.org/articles/10.3389/frym.2021.568656
• Butyric Acid Fermentation- Definition, Principle, Procedure, Uses – https://microbenotes.com/butyric-acid-fermentation/
• Zigová, Jana, et al. “Butyric acid production by Clostridium butyricum with integrated extraction and pertraction.” Process Biochemistry 34.8 (1999): 835-843.
• The Difference Between Batch, Fed-batch and Continuous Processes – https://www.infors-ht.com/en/blog/the-difference-between-batch-fed-batch-and-continuous-processes/
• He, Guo-qing, et al. “Batch and fed-batch production of butyric acid by Clostridium butyricum ZJUCB.” Journal of Zhejiang University-SCIENCE B 6.11 (2005): 1076-1080.
• Butanoic acid – C4H8O2 – https://byjus.com/chemistry/butanoic-acid/