Citric Acid Production- Microbes, Methods, Steps, Factors and application

Citric acid derives its name from the Latin word citrus, a tree whose fruit is like the lemon. Citric acid is a natural acid found in citrus fruits like lemons, limes, oranges, and grapefruits. It is a primary metabolic product formed in the tricarboxylic acid (or Krebs) cycle.

It is a common component of various fruits and vegetables but is most abundant in citrus fruits. Chemically, citric acid is a tricarboxylic acid with the chemical formula C6H8O7.

Citric acid, a key player in various industries, is widely used in food and beverages, pharmaceuticals, and even household products.

Properties of Citric acid

History of Citric acid production

Micro organism used in Citric acid production

The primary microorganism employed in citric acid production is Aspergillus niger, a fungus known for its efficiency in citric acid synthesis. This microorganism can use diverse carbon sources and flourish in a range of environmental conditions making it an ideal candidate for large-scale industrial processes. 

                     Table : List  of microorganism found to accumulate/produce citric acid

Microbial synthesis of Citric Acid

• Citric acid serves as a key product in primary metabolism and is generated within the tricarboxylic acid (Krebs) cycle. The primary carbon source for the production of citric acid is glucose.
• The biosynthetic pathway for citric acid involves glycolysis, where glucose undergoes conversion into two pyruvate molecules.
• Pyruvate transforms into acetyl CoA. Pyruvate carboxylase converts pyruvate to oxaloacetate is also a key enzvme in citric acid production. Acetyl CoA and oxaloacetate, eventually condense to produce citrate. During the synthesis citric acid there are tenfold increase in the Citrate synthase.
• The overproduction of citric acid necessitates a specific combination of unique nutritional conditions, including an excess of carbon source, hydrogen ions, and dissolved oxygen, along with suboptimal concentrations of specific trace metals and phosphate.
• These factors work together to synergistically impact the fermentation performance. A shortage of manganese or limitations in phosphate and nitrogen can inhibit the anabolism of A. niger.
• This inhibition results in the breakdown of proteins, leading to an elevated concentration of ammonium ions. Interestingly, this increase can counteract the inhibitory effects exerted by citric acid on phosphofructokinase, acting as a positive end-effector.
• Elevated levels of NH4+ and glucose also suppress the synthesis of α-ketoglutarate dehydrogenase, hindering the catabolism of citric acid through the Krebs cycle, ultimately causing its accumulation.
• Consequently, the accumulation of citric acid is attributed, in part, to the rapid influx of substances and a decrease in outflow velocity, as illustrated in Figure below

Production process

There are two process through which citric acid can be produced industrially.

• 1. Surface process
• 2. Submerged process

Yield

Yield of citric acid : the calculation indicates that from 100 grams of sucrose, it is theoretically possible to generate 112 grams of anhydrous citric acid or 123 grams of citric acid-1 hydrate. However, the actual yield of citric acid is reduced compared to the calculated values due to the oxidation of sugar to CO2 during the trophophase.

Factors Affecting Citric Acid Fermentation

Various factor affect the production of citric acid which are –

Carbon Source –

The sugars that are quickly assimilated by the microorganism allow high final yield of citric acid.

Various sources of carbohydrates can be utilized as raw materials. These sources include molasses (derived from sugar cane or sugar beet), starch (obtained from potatoes), date syrup, cotton wastes, banana extract, sweet potato pulp, brewery waste, and pineapple wastewater.  In general, sucrose is preferable to glucose as A. niger has an extracellular mycelium-bound invertase that is active at low pH.

The carbon source concentration is also important in citric acid production. In batch processes, the ultimate citric acid yield rises with the initial sugar concentration.

Optimal productivities are typically attained within the range of 14–22% sugar. This is because excessively high concentrations of the carbon source tend to inhibit α-ketoglutarate dehydrogenase.

Conversely, low glucose levels result in reduced mycelium size and altered morphology.

Nitrogen and phosphate limitations

Complex media like molasses are often rich in nitrogen, However, in laboratory, media are supplemented with ammonium salts (such as ammonium nitrate and sulfate) to enhance fermentation.

Addition of nitrogen sources, especially ammonium salts, leads to a decrease in pH, favoring the fermentation process.

Alternative Nitrogen Sources: Urea and yeast/malt extract have been successfully employed as alternative nitrogen sources in citric acid production.

Phosphate favors enhanced biomass growth.

pH of culture medium

The pH of the medium plays a critical role in two key stages of the citric acid production process.

Spore Germination:

pH > 5 is essential for the germination of spores, the starting point of all fermentations.

Spore germination involves the absorption of ammonia, releasing protons and consequently lowering the pH.

Production Phase:

During the production phase, maintaining a low pH (pH ≤ 2) is of utmost importance.

Low pH values mitigate the risk of contamination by other microorganisms.

In addition, the low pH inhibits the production of undesirable organic acids, such as gluconic and oxalic acid

Aeration

Fluctuations in aeration rates can negatively impact performance. Excessive aeration may lead to a low partial pressure of dissolved CO2 in the broth. Conversely, elevated levels of CO2 in the gas phase can be detrimental to the final concentrations of citrate and biomass

Trace elements

a balanced concentration of trace metals (Zn, Mn, Fe, Cu ) are required for growth of biomass and production of citric acid. e.g.

• Zinc – 0.3 ppm
• Iron – 1.3 ppm
• Manganese – 3 mg/l

Manganese are important in cell wall synthesis, sporulation and production of secondary metabolites. therefore care must be taken while preparing medium.

Application of citric acid

1. Food and Beverages: Citric acid is commonly used in the food and beverage industry to enhance the flavor of products. It is a key ingredient in soft drinks, candies, jams, and jellies. Additionally, it is used in the preparation of citric acid solutions for home canning and preserving.
2. Pharmaceuticals: In the pharmaceutical industry, citric acid is used as a buffering agent to control the acidity of certain medications. It is also employed in the formulation of effervescent tablets and syrups.
3. Cleaning Products: Due to its acidic and chelating properties, citric acid is found in many household cleaning products. It is effective in removing mineral deposits, especially in descaling agents for coffee makers and dishwashers.
4. Cosmetics: Citric acid is used in cosmetics and personal care products for its pH-adjusting and antioxidant properties. It is often present in skincare products, shampoos, and lotions.5.  Industrial Processes: Citric acid is utilized in various industrial processes, such as the textile industry for dyeing and finishing, and in the metal industry for cleaning and treating surfaces.

References

• Max B, Salgado JM, Rodríguez N, Cortés S, Converti A, Domínguez JM. Biotechnological production of citric acid. Braz J Microbiol. 2010 Oct;41(4):862-75. doi: 10.1590/S1517-83822010000400005. Epub 2010 Dec 1. PMID: 24031566; PMCID: PMC3769771.
• Luciana P. S. Vandenberghe, Carlos R. Soccol, Ashok Pandey. Jean-Michel Lebeault. Microbial production of citric acid.  Braz. arch. biol. technol. 42 (3) • 1999. https://doi.org/10.1590/S1516-89131999000300001
• National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 311, Citric Acid. Retrieved February 1, 2024 from https://pubchem.ncbi.nlm.nih.gov/compound/Citric-Acid.