Butyric Acid Fermentation- Definition, Principle, Procedure, Uses

Butyric Acid is a short-chain saturated fatty acid having a backbone of 4-C and is found in animal fats and plant oils in esterified form. It is a colorless liquid with an unpleasant smell having a flashpoint of 170 degrees Fahrenheit. Its function is as a metabolite of Mycoplasma genitalium and also as a human urinary metabolite.

Butyric Acid Fermentation
Butyric Acid Fermentation

Fermentation is defined as any metabolic process that releases energy from an organic molecule in absence of oxygen or an electron transport system and uses an organic molecule as the final electron acceptor. This process can occur in different natural ecosystems as well as in man-made environments such as industries. In these ecosystems, many microorganisms compete for the substrate and determine the metabolic pathways and hence the final product.

What is butyric acid fermentation?

Butyrate is produced by many fermentation processes which are performed by obligate anaerobic bacteria and spore-forming bacteria belonging to the genus Clostridium. Besides butyric acid, several other products are formed during fermentation such as acetic acid, ethanol, isopropanol, and acetone depending upon the species. 

For example bacteria like Clostridium bretylium, C. lactoacetophylum , C. pasteurianum produces butyric acid along with the acetic acid, while C. butylicum and C. acetobutylicum produce butyric acid, acetic acid, and isopropanol or acetone.

The butyric acid fermentation pathway was first discovered by Louis Pasteur in 1861.

Read Also: Fermentation of Vinegar

Principle of butyric acid fermentation

The general principle of butyric acid fermentation includes glycolysis in which bacteria use sugar and oxidize it to pyruvate. Which is later oxidized to acetyl-CoA using pyruvate ferredoxin oxidoreductase enzyme. One part of Acetyl-CoA later forms acetic acid and ATP and the rest generate acetoacetyl-CoA which is reduced to butyryl CoA, which is converted to butyric acid with ATP.

Total ATP produced is 3 molecules along with a small amount of ethanol and isopropanol is also produced during the fermentation.

Principle of butyric acid fermentation
Figure: Principle (Pathways) of butyric acid fermentation. Image Source: Ajay Kumar Jha et al. 2014.

The process is common in silage when the pH is not low for the growth and activity of lactic acid bacteria. The release of carbon dioxide during the process increases the pH of the silage, which enhances the production. 

Some bacteria such as Clostridium acetobutylicum, produce fewer acids and many neutral products, thus carrying out acetone-butanol fermentation.  This fermentation had great importance during World War 1 due to the need for acetone for the production of munitions.

Requirements for butyric acid fermentation

  1. Stirred tank reactor
  2. Substrate eg, rice straw
  3. Sodium hydroxide for delignification and swelling of rice straw
  4. cellulolytic butyrate-producing mixed culture
  5. fermentation medium: pretreated rice straw, tryptone, yeast, NaCl, caco3, D-cysteine hydrochloride, chloroform.

Procedure of Butyric Acid Fermentation

1. Experimental set-up

  • A stirred tank reactor is set up by wrapping the heating wire to control the temperature and attached with a wet gas meter to measure biogas.
  •  Later acidified water is filled into a water seal and wet gas meter to avoid the dissolution of carbon dioxide present in biogas. 
  • pH is controlled using a NaHCo3 buffer.
Procedure of Butyric Acid Fermentation
Figure: Production of butyric acid from acid hydrolysate of corn husk in fermentation by Clostridium tyrobutyricum: kinetics and process economic analysis. Image Source: Zhiping Xiao et al. 2018.

2. Sodium hydroxide treatment to the substrate

  • Substrate such as rice straw is treated with 1% sodium hydroxide solution for delignification as well as swelling of biomass to increase digestibility, for 72h at 50 degrees Celsius.
  • The solid residue is later filtered and washed with buffer.

3. Preparation of inoculum

  • Cattle manure, pig manure compost, cornfield soil, and rotten wood is used to make cellulolytic butyrate-producing a mixture that contains cellulolytic and xylanolytic bacteria, butyrate-producing bacteria, and other acidogenic bacteria.
  • For preparation, this mixture is transferred to a seed medium containing pretreated substrate, tryptone, yeast extract, NaCl, CaCO3, D-Cysteine hydrochloride, and a filter paper strip as an indicator of pH change. 
  • Poured with nitrogen gas for 10 min to create anaerobic conditions. 
  • Now, autoclaved at 115 degrees Celsius for 20 mins. 
  • Following inoculation of stored culture, the bottle is incubated.

4. Butyric acid fermentation

  • Fermentation medium is prepared the same as seed medium used to prepare inoculum. With the extra addition of chloroform as a methanogenic bacteria indicator.
  • The reactor of fermentation and medium do not pour with nitrogen gas and autoclaving
  • Inoculation of 1 L of inoculum into 20L of the fermentation medium is suggested.
  • Batch fermentation: no additional medium is added after the initial change, aside from NaHCo3 buffer.
  • Semi-continuous fermentation: 3L fermentation broth is discarded and 3 L fresh fermentation medium is added on the fourth day and every day thereafter

Products of batch and semi-continuous fermentation

  • The degradation rate of substrate is usually higher in batch fermentation than in semi-continuous, because of longer residence time.
  • Although, reacter volumetric productivity is greater in semi-continuous
  • The aging of carboxylic acid-fermenting strains and Na+ can be minimized due to the inhibition of end-products in semi-continuous fermentation.
  • Efficiency, productivity, and nonproductive time are enhanced in a semi-continuous process.
  • Thus, semi-continuous is a better process for butyric acid fermentation than batch fermentation.

Uses of butyric acid

  • Inhibit the growth of other bacteria: It was observed that butyric acid produced from the fermentation by the bacterium Staphylococcusepidermidis inhibits the growth of Staphylococcus aureus. This was observed in the patient suffering from atopic dermatitis. Butyric acid act like a histone deacetylase inhibitor by inducing the histone lysine in human keratinocytes.
  • Anti-inflammatory effect: Butyric acid has an anti-inflammatory effect by suppressing certain proteins that produce inflammation. It helps in controlling the inflammatory immune response by regulating the T cells which results in inflammation. 
  • Digestion and gut health: Butyric acid supplementation improves the symptoms of ulcerative colitis and Crohn’s disease .it also helps to reduce the gut permeability and “leaky gut” which is a major problem in autoimmune disorders such as colitis.
  • Role in colon cancer: Butyric acid is the major short-chain fatty acid produced in the large bowel and acts as an important regulator in colorectal cancer. butyric acid induces the signaling pathway which activates apoptosis of the cancerous cells in the colon.


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  3. Borycka-Kiciak K, Banasiewicz T, Rydzewska G. Butyric acid – a well-known molecule revisited. Prz Gastroenterol. 2017;12(2):83-89. doi: 10.5114/pg.2017.68342. Epub 2017 Jun 13. PMID: 28702095; PMCID: PMC5497138.
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  6. National Center for Biotechnology Information. “PubChem Compound Summary for CID 264, Butyric acid” PubChem, https://pubchem.ncbi.nlm.nih.gov/compound/Butyric-acid. Accessed 13 May 2022.
  7. Xiao, Z., Cheng, C., Bao, T. et al. Production of butyric acid from acid hydrolysate of corn husk in fermentation by Clostridium tyrobutyricum: kinetics and process economic analysis. Biotechnol Biofuels 11, 164 (2018). https://doi.org/10.1186/s13068-018-1165-1

About Author

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Khushi Jain

Khushi did her bachelor's in microbiology from Ramlal Anand College, Delhi University, and completed her master's in microbiology from the Central University of Punjab, Bathinda. She has a hands-on training experience in genomic DNA manipulation techniques. She is also the co-author of a research paper related to amoebiasis and its prevalence in India in a scientific journal. She is the main author of a research paper on malaria prevalence in India in pregnant women which is in the process of publication. Her area of interest is genetics, recombinant DNA technology, and microbiology.

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