Lactic Acid Fermentation: Types, Pathways, and Production

Yogurt and sourdough are both made by lactic acid bacteria, yet they taste completely different: yogurt is cleanly sour, while sourdough is tangy, a little fizzy, and complex. Sauerkraut bubbles as it ferments; a vat of yogurt does not. The reason is that "lactic acid bacteria" are not all the same. One group makes almost nothing but lactic acid. The other makes lactic acid plus carbon dioxide and alcohol. Knowing which group is at work tells you what the food will taste like, whether it will fizz, and how it is made. That single split is the key to understanding lactic acid fermentation.

Lactic acid (2-hydroxy propanoic acid) is a three-carbon organic acid obtained by carbohydrate fermentation due to microorganisms (Lactic acid bacteria) or chemical synthesis. Its structure is CH₃-CHOH-COOH: a methyl group at one end, a carboxyl (acid) group at the other, and a hydroxyl group on the central carbon. Its molecular formula is C₃H₆O₃. Because the central carbon is a chiral center, lactic acid exists as two optical isomers: L(+) lactic acid and D(-) lactic acid.

Figure: Optical isomers of Lactic acid (Okafor, 2007)

Lactic acid was discovered first in 1780 by Swedish chemist Scheele and commercially produced in 1881 by Charles E. Avery in Littleton, Massachusetts, USA. Commercially, it is produced by fermentation methods or chemical synthesis. Before the advent of refrigeration or the modern canning method, lactic acid fermentation was used to preserve dairy products, vegetables, and meat for a more extended period.

What is lactic acid fermentation?

Lactic acid fermentation is the process in which certain bacteria (and some fungi) break down sugars and produce lactic acid as the main product. It happens without oxygen (anaerobically). The bacteria that do this are called lactic acid bacteria (LAB), and they include the genera Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, and Pediococcus.

The process matters for two reasons. First, it preserves food: as lactic acid builds up, the pH drops, and the acidic conditions stop spoilage and disease-causing microbes from growing. This is how yogurt, cheese, sauerkraut, kimchi, and many pickles are made and kept. Second, purified lactic acid is a valuable industrial chemical used in food, cosmetics, pharmaceuticals, and biodegradable plastics.

Before refrigeration and canning existed, lactic acid fermentation was one of the main ways people preserved dairy, vegetables, and meat through the winter. It is one of the oldest food-preservation methods, and it is covered alongside the others in the article on food preservation methods.

Two types: homofermentative and heterofermentative

Lactic acid bacteria are divided into two groups based on what they produce from sugar. This distinction is the heart of the topic.

Homofermentative bacteria convert sugar almost entirely into lactic acid, with little or nothing else. "Homo" means one: essentially one product. They use the Embden-Meyerhof-Parnas (EMP) pathway, the same glycolysis pathway used in most cells, and yield about 2 molecules of lactic acid and 2 ATP per glucose. Examples include Lactobacillus delbrueckii, Lactococcus lactis, and Streptococcus thermophilus. These make cleanly sour products like yogurt and many cheeses.

Heterofermentative bacteria convert sugar into a mixture: lactic acid plus ethanol (or acetic acid) plus carbon dioxide. "Hetero" means different: several products. They use the phosphoketolase pathway (a branch of the pentose phosphate pathway) and yield only about 1 lactic acid and 1 ATP per glucose, along with the CO₂ and ethanol. Examples include Leuconostoc mesenteroides and Lactobacillus brevis. The CO₂ they release is why sauerkraut and sourdough bubble and develop a more complex, tangy flavor.

How to tell them apart

The quickest bench clue: if the fermentation produces gas, it is heterofermentative. A homofermentative culture makes acid without bubbling. For the laboratory test that shows this, see the article on the carbohydrate fermentation test.

Industrial production of lactic acid

Beyond its role in food, lactic acid is manufactured on a large scale as an industrial chemical. Roughly 90% of the world's lactic acid is made by fermentation rather than chemical synthesis, because fermentation can produce a single optical form (either L(+) or D(-)) whereas chemical synthesis produces a mixture. The rest of this article covers how that industrial fermentation and purification is done.

Raw Material

Raw materials used for the production of lactic acid should have the following desirable properties:

1. Cheap and readily available.
2. Low level of contaminants.
3. Should produce little or no by-products.
4. Little or no-pre treatment is required before the fermentation.
5. The cost of product purification should be low if refined materials are used.

The most commonly used raw materials for lactic acid production are whey, molasses, starchy and cellulosic materials. Among these, starchy and cellulosic materials are mainly used as they are cheap, abundant, and renewable.

Some starchy materials used are sweet sorghum, rice, corn, cassava, wheat, rye, and barley. These materials consist of mainly α(1,4)- and α(1,6)-linked glucose, so before the fermentation, they are hydrolyzed to fermentable sugars.

Cellulosic materials such as corncob, waste paper, and agricultural residues like alfalfa fiber, corn stover, wheat straw, and wheat bran have been used for lactic acid production. Industrial waste products like molasses and whey are used as raw materials for lactic acid production. Molasses (a waste product of the sugar manufacturing process) contains a large amount of sucrose, and whey (a by-product of the dairy industry), contains lactose, protein, fat, and mineral salts.

So, for utilization of whey lactose, it is added with an additional nitrogen source, such as yeast extract, to produce lactic acid. Along with these raw materials, supplements such as yeast extract and corn steep liquor are also added.

Bacteria

The choice of microorganisms depends on the raw materials that must be fermented. The organism has desirable characteristics such as:

• Being able to ferment cheap raw materials rapidly.
• Requiring minimum nitrogenous substance.
• Producing low amounts of cell mass with negligible by-products.
• Providing a high yield of lactic acid under low pH and high temperatures is used for lactic acid fermentation.

An organism’s choice depends on the type of raw materials to be fermented. The most used organisms are lactic acid bacteria. Recently, the mold Rhizopus has also been used for lactic acid production, because it can ferment starch directly and makes purification simpler.

For industrial lactic acid production, homofermentative bacteria are preferred, because they convert sugar almost entirely into lactic acid with few by-products, which makes purification easier and the yield higher. The workhorse is Lactobacillus delbrueckii.

Different species are chosen to match the raw material's sugar. Amylase-producing L. amylophilus and L. amylovorus can ferment starchy materials directly. L. delbrueckii ferments sucrose; L. helveticus ferments lactose and galactose; L. lactis ferments glucose, sucrose, and galactose; and L. brevis and L. pentosus are used with pentose sugars from materials such as wheat straw.

Fermentation

Lactic acid is corrosive, so wood or stainless steel fermenters with capacities between 25,000 and 110,000 liters are used. In order to avoid contamination, sterilization is done by steaming before introducing the broth.

Figure: Lactic acid fermentation

The most frequently used method for lactic acid production is batch, fed-batch, repeated batch, and continuous fermentations. Mainly two bioreactor systems are used for lactic acid production: constant cell recycle fermentation process and fed-batch fermentation.

During the fermentation process, as a carbon source, raw materials are added along with 5% starter culture, mostly Lactobacillus, and calcium carbonate or lime is added to neutralize the lactic acid as it forms, keeping the pH from dropping too low (typically held around 5 to 6). The fermentation runs warm, around 45 to 50°C for thermophilic strains.

For the production of lactic acid, various fermentation approaches are used:

Batch fermentation

During batch fermentation, carbon source, nitrogen source, and other components are added before starting the fermentation. Mainly two types of batch fermentation occur such as:

• Solid State Fermentation (SSF): Fermentation occurs in a single reaction vessel and has a rapid processing time. Raw materials such as wheat bran, rice bran, barley, fruit pulps, and sugarcane bagasse are used as carbon sources with no or negligible amount of water.
• Separate hydrolysis and fermentation process: During this fermentation, raw materials are pre-treated, discarding unnecessary compounds such as lignin in the case of lignocellulosic biomass. These raw materials are then subjected to enzymatic saccharification, and the hydrolysate formed is subjected to fermentation.

Fed-Batch Fermentation

During fed-batch fermentation, all the raw materials, such as carbon sources, nitrogen sources, and other components, are added at regular intervals during the fermentation process without the removal of the fermentation broth. It is helpful to maintain low substrate concentration by supplying nutrients to the fermentation culture, reducing substrate inhibition.

Continuous Fermentation

During continuous fermentation, fresh medium is added to the fermenter while withdrawing already existing broth at the same rate, maintaining the concentration of substrates and products.

During this fermentation method, the tank is filled with sweet whey at a temperature of 45°C. Mash is then inoculated with Lactobacilli culture, which is agitated and maintained at 45°C. When the pH of the mash drops to 5 (usually after 12 hours), lime is added to strengthen the pH between 5-5.8. At the end of 24 hours and after that, every 12 hours, the lactose content of whey is measured. When lactose is less than 1%, whey is introduced into the fermentation tank from the whey storage tank, and fermentation broth is extracted simultaneously from the fermenter, maintaining the continuous cycle.

After the fermentation is complete, recovery of lactic acid is made. Fermentation of lactic acid is an easy process, but the recovery of acid is difficult.

Separation and Purification of Lactic Acid

Lactic acid separation starts after the completion of the fermentation process and proceeds to the filtration process to avoid microorganism contamination that may enter the ion exchange column.

Several methods are present for the separation of lactic acid from the fermentation broth, which is called the downstream process, and a few are discussed below:

Neutralization and Precipitation of Salt with Acid

The most conventional method for the separation step in LA production by fermentation is precipitation. The step involved is the addition of excess calcium carbonate or calcium hydroxide to the acid produced for neutralization, and pH is maintained around 5 to 6 to make a calcium salt of acid called calcium lactate.

The fermentation broth is treated with sulphuric acid to precipitate the calcium sulfate or gypsum, which is filtered. The filtrate containing free organic acid is evaporated to obtain pure Lactic acid. The steps of chemical reactions involved are shown below:

Fermentation: Under anaerobic conditions, glucose is broken down to form lactic acid.

Sugar →  Lactic acid

C₆H₁₂O₆ → 2 CH₃CHOHCOOH

Neutralization Step: During fermentation, calcium hydroxide neutralizes lactic acid.

Lactic acid + calcium hydroxide → Calcium lactate + Water

2 CH₃CHOHCOOH + Ca(OH)₂ → Ca(CH₃CHOHCOO)₂ + 2 H₂O

Acidification Step: Fermentation broth is treated with sulphuric acid to precipitate calcium sulfate or gypsum, which is filtered.

Calcium lactate+ sulphuric acid →Lactic Acid+ calcium sulfate (gypsum)

Ca(CH₃CHOHCOO)₂ + H₂SO₄ → 2 CH₃CHOHCOOH + CaSO₄

Figure: Lactic acid recovery by precipitation

Solvent Extraction or Liquid-liquid Extraction

Solvent extraction or liquid-liquid extraction is the process in which one or more solutes are separated based on their relative solubilities in two immiscible liquids, usually water (polar) and an organic solvent (non-polar). The most crucial factor that contributes to the extraction are:

• Distribution coefficients (ratio of the LA concentration in the solvent phase to the aqueous phase).
• Easy separation of the liquid phase.
• Selectivity of the extractant.
• Choice of solvent extraction.

The characteristics of the solvent include chemically stable, regenerable, selectivity, low corrosivity, low toxicity, and low viscosity, and diluents are used if extractants have high viscosity.

Some of the extractants used are Tri-n-octylamine-Tripropylamine (2:8 w/w), Tri-n-butyl phosphate (TBP), Tri-n-octylamine (1 M), and so on.

Figure: Lactic acid recovery by solvent extraction method

Separation with Membranes

In the membrane separation processes, the solutes are transferred by a semi-permeable barrier that separates the two phases, which restricts the transport of components from one stage to another. Microfiltration, Ultrafiltration, and electrodialysis technology are used for lactic acid separation, which does not produce a salt residue.

The membrane separation process is highly selective so that it can ensure high levels of purification and separation. It can also be integrated with conventional fermenters, allowing simultaneous production and purification.

Figure: Lactic acid recovery by membrane

Uses of Lactic Acid

Lactic acid has many uses, some of which are listed below:

1. It is used in the chemical industry as a pH regulator, neutralizer, cleaning agent, and green solvent.

2. Used in cosmetics products such as moisturizers, anti-acne agents, anti-tartar agents, skin-lightening agents, and skin-rejuvenating agents.

3. Used in the food industry, such as preservatives, flavors, acidulants, and improving microbial quality.

4. Used in the pharmaceutical industry, such as in dialysis solutions, tableting, surgical sutures, controlled drug delivery systems, prostheses, and mineral preparations.

How to Remember

Homo = one, Hetero = many.
Homofermentative makes one product (lactic acid). Heterofermentative makes many (lactic acid + ethanol + CO₂). The Greek prefix is the whole answer.

Gas is the giveaway.
If the fermentation bubbles, it is heterofermentative (the bubbles are CO₂). No gas means homofermentative. This is also exactly how the lab carbohydrate fermentation test tells them apart: gas in the Durham tube.

Two pathways, two yields.
Homofermentative uses ordinary glycolysis (EMP) and gets 2 ATP. Heterofermentative uses the phosphoketolase pathway and gets only 1 ATP (it "spends" carbon making CO₂ and ethanol instead of capturing all of it). More products, less energy.

Yogurt is clean, sauerkraut is complex.
Homofermentative yogurt and cheese taste cleanly sour. Heterofermentative sauerkraut and sourdough are tangy and gassy. The flavor tells you which group did the work.

Key exam facts in one table

Where Students Get Confused

What is the difference between homofermentative and heterofermentative bacteria?
Homofermentative bacteria make almost only lactic acid from sugar. Heterofermentative bacteria make lactic acid plus ethanol (or acetic acid) and carbon dioxide. The simplest test is gas: heterofermentative fermentation produces CO₂ and bubbles, homofermentative does not.

Why do sauerkraut and sourdough bubble but yogurt does not?
Because sauerkraut and sourdough are made by heterofermentative bacteria that release carbon dioxide gas, while yogurt is made by homofermentative bacteria that produce lactic acid without gas.

Is lactic acid fermentation the same as the lactic acid that builds up in muscles?
The chemistry is related but the context differs. In muscle, cells briefly convert pyruvate to lactate when oxygen is short, using the same reduction step. Lactic acid fermentation in food is that process carried out by bacteria as their normal way of life, producing enough acid to preserve and flavor food.

Why is homofermentative fermentation preferred for making industrial lactic acid?
Because it produces lactic acid as nearly the only product. That means fewer by-products to remove, easier purification, and a higher yield of pure acid. Heterofermentative bacteria would contaminate the product with ethanol and other compounds.

Do homo- and heterofermentative bacteria use the same pathway?
No. Homofermentative bacteria use the Embden-Meyerhof-Parnas pathway (ordinary glycolysis). Heterofermentative bacteria use the phosphoketolase pathway, which is why they produce extra products and less ATP.

References

1. Okafor N. Modern Industrial Microbiology and Biotechnology. Enfield (NH): Science Publishers; 2007.
2. Wee YJ, Kim JN, Ryu HW. Biotechnological production of lactic acid and its recent applications. Food Technol Biotechnol. 2006;44(2):163–172.
3. Komesu A, Maciel MRW, Filho RM. Separation and purification technologies for lactic acid: a brief review. BioResources. 2017;12(3):6885–6901. https://doi.org/10.15376/biores.12.3.6885-6901
4. Din NAS, Lim SJ, Maskat MY, Mutalib SA, Zaini NAM. Lactic acid separation and recovery from fermentation broth by ion-exchange resin: a review. Bioresour Bioprocess. 2021;8(1). https://doi.org/10.1186/s40643-021-00384-4
5. Narayanan N, Roychoudhury PK, Srivastava A. L(+) lactic acid fermentation and its product polymerization. Electron J Biotechnol. 2004;7(2):167–178.
6. Jay JM, Loessner MJ, Golden DA. Modern Food Microbiology. 7th ed. New York: Springer; 2005.