What is Citric Acid And How is It Made?

Citric acid (C₆H₈O₇) is a six-carbon tricarboxylic acid which is also known as  2-hydroxy-1,2,3 – propane tricarboxylic acid. 

It was first isolated from lemon juice by a chemist called Karl Scheels in 1874 in England. The acid has a melting point of 153°C and decomposes at higher temperatures. The acid has a molecular weight of 210.14 g/mol. It also possesses three different pK_a values, at pH 3.1, 4.7 and 6.4, owing to the presence of three functional groups of carboxylic acid in its structure.

The acid is commonly used in the pharmaceutical, food and beverage industry as an acidulant. Commercially it is one of the most important organic acids to be manufactured because of its great versatility and widespread use. About 70% of total production is used by the food industry whilst 12 % is used by the pharmaceutical industry. The main applications are:

• Beverages: adds tartness and helps develop and complement fruit and berry flavours. It provides an antimicrobial action and is commonly used as a preservative. Regularly used for pH adjustment to provide uniform acidity.
• Confectionary: lends tartness. Reduces sucrose inversion. helps with colour darkening in hard boiled sweets. Performs like an acidulant.
• The acid is often used in manufacturing metabolites for the pharmaceutical industry and consumer healthcare. It is also a valuable ingredient in the synthesis of other acids.

In biochemistry, citric acid is a major substrate of energy metabolism.

Manufacture Of Citric Acid

Most production occurs by fermentation of fungus. Two types of fungal fermentation are used: liquid surface culture and submerged fermentation with the latter being the more popular of the two. Fermentation conditions were established in the 1930s and 40s with the testing various fermentation media. The main microorganism used in fermentation is the fungus Aspergillus niger because it accumulates citric acid if the metabolic conditions are right (Papagianni, 2007; Show et al., 2015). Candida species which are yeasts have also been tried and tested.

In recent years the yeast Yarrowia lipolytica has been exploited because it lends itself well to particular types of fermentation systems such as the chemostat style of process and it can use glycerol as a substrate (Papanikolaou et al., 2002). It is also feasible to use low-grade substrates for citric acid production and a number have been tested over many years.

The Fermentation Medium

The quality and yield of citric acid depends entirely on the carbon and carbohydrate source which is for the vast majority of general fermentation, sugar. The type of sugar affects the metabolic activity of the microorganism used and seems to be more influential here than in other types of fermentation where nitrogen and nutrients might be more influential. A case in point is xanthan production for example. Based on Vandenberghe’s study (1999), sucrose is better than fructose, glucose and galactose. 

If there is a suitable source of sugar then citric acid can be fermented. Molasses from the sugar industry also serves as a readily-available substrate if sucrose is unavailable. It is a waste product of the sugar processing industry and has for many years been relatively cheap to obtain in a crude form. It has a range of minerals and nutrients which can be additionally supplemented with if growth is to be optimised (Clark, 1962).

As with many fermentations using waste sources, the presence of heavy metals is highly problematic. Ironically, trace elements such as zinc, iron, copper and manganese are essential and it has been a well researched area in citric acid production by A. niger. (Tomlinson et al., 1950) However, subsequent studies show that careful management of trace minerals is needed when heavy metals are present in excess. Molasses is treated with ferrocyanide (commonly calcium ferrocyanide or potassium ferrocyanide) before citric acid production by fermentation to remove heavy metal ions, particularly iron (Fe³⁺), which can negatively affect the fermentation process (Clark et al., 1965). 

Molasses often contains iron and other trace metals as impurities. These metals:

• Can inhibit the growth of the citric acid-producing microorganism, typically Aspergillus niger.

• Stimulate unwanted metabolic pathways, such as the production of oxalic acid, gluconic acid, or other byproducts instead of citric acid.

• Disrupt enzyme systems involved in the citric acid biosynthetic pathway.

Ferrocyanide forms insoluble metal ferrocyanide complexes (e.g., Prussian blue with iron), which can then be removed by filtration or sedimentation.

By removing iron and other inhibitory metals:

• The tricarboxylic acid (TCA) cycle in Aspergillus niger functions more efficiently in the direction that favours citric acid accumulation.

• There is less oxidative damage to cells caused by iron-catalyzed reactions like the Fenton reaction (which produces reactive oxygen species).

• The fermentation environment is more controlled and favourable for citric acid biosynthesis.

Iron and copper in molasses can catalyze the growth of contaminant organisms or spoilage pathways. Ferrocyanide helps minimize this risk by removing those catalytic metals.

One study found that the addition of as little as 2 ppb of manganese to ferricyanide-treated beet molasses reduced citric acid production by 10% as well as causing undesirable changes in the morphology of the mould (Clark et al., 1966). Normally, A. niger forms pellet-like forms but heavy metal poisoning changes it to the filamentous form. .

Fermentation Media

Other fermentation media include apple pomace with a small addition of methanol can be used.  Olive- mill wastewater (OMW) is a liquid byproduct of the olive oil industry and it is extremely difficult to treat because of its high phenolic content (Ammar et al., 2005). Orange process waste is also a suitable source of sugar for this type of fermentation (Aravantinos-Zafiris et al., 1994).

An alternative method is  to use solid-state fermentation (Vandenberghe et al., 2000) which also employs Aspergillus species to produce the acid. Virtually all the substrate for this type of fermentation is derived from agricultural waste because it can supply most of the nutrients needed for microbial growth. Dhillon et al., (2011) list these substrates as sugarcane bagasse, fruit pomace, wheat, rice, maize and grain brans, wheat and rice straw, coconut coir pith, newspaper, fruit wastes, tea and coffee wastes, cassava waste, and distiller grains amongst others. Whether all these have been tested with citric acid fermentation using SSF remains to be seen but they have been tried elsewhere. There are a considerable number of other substrates available from waste material which will undoubtedly surface in research papers and patents (AW Sansome-Smith, 2019).

Another method of obtaining the acid is by extraction from fruit juices. A high acid fruit juice is treated with calcium oxide to form calcium citrate. The citrate salt is insoluble and precipitates out of solution. The citrate salt is recovered by filtration. Sulphuric acid is added to the precipitate to form calcium sulphate (gypsum) and the citric acid is recovered in solution. Some citric acid is commercially produced from waste citrus fruits produced in South and Central America.

Aeration Of Fermentation

Citric acid fermentations need to be fully aerated through out the course of the fermentation. That means there must be an adequate supply of air if not oxygen. Compared to other types of fermentation, loss of oxygen provision has a detrimental effect on the growth and metabolism of fungus like A. niger which then impacts yields. Approaches to improve oxygen delivery have been tried by using what are known as oxygen vectors such as n-dodecane (Jianlong, 2000). 

The key measure for oxygen in solution is the kLa (volumetric oxygen transfer coefficient). Adding an oxygen-vector can almost double the kLa for oxygen. A 5% v/v addition of n-dodecane improved both citric acid yield, and biomass production without causing any damage to the fungus. It’s an idea that should be more universally applied to other types of fermentation such as xanthan production for example where the rapid rise in viscosity reduces kLa.

References

Ammar, E., Nasri, M., Medhioub, K., (2005) Isolation of phenol degrading Enterobacteria from the waste water of olive oil extraction process. W. J. Microbiol. Biotechnol. 21, pp. 253–259

Angumeenal, A. R., & Venkappayya, D. (2013). An overview of citric acid production. LWT-Food Science and Technology, 50(2), pp. 367-370.

Aravantinos‐Zafiris, G., Tzia, C., Oreopoulou, V., & Thomopoulos, C. D. (1994). Fermentation of orange processing wastes for citric acid production. Journal of the Science of Food and Agriculture, 65(1), pp. 117-120.

Clark, D. S. (1962). Submerged citric acid fermentation of sugar beet molasses. Effect of ferrocyanide control. Industrial & Engineering Chemistry Product Research and Development, 1(1), pp. 59-62.

Clark, D. S., Ito, K., & Tymchuk, P. (1965). Effect of potassium ferrocyanide on the chemical composition of molasses mash used in the citric acid fermentation. Biotechnology and Bioengineering, 7(2), pp. 269-278

Clark, D. S., Ito, K., & Horitsu, H. (1966). Effect of manganese and other heavy metals on submerged citric acid fermentation of molasses. Biotechnology and Bioengineering, 8(4), pp. 465-471

Clark, D. S., & Lentz, C. P. (1963). Submerged citric acid fermentation of beet molasses in tank‐type fermenters. Biotechnology and Bioengineering, 5(3), pp. 193-199.

Hang, Y.D. & Woodams, E.E.  (1984) Apple pomace: A potentials substrate for citric acid production by Aspergillus niger. Biotechnol. Lett. 6: 763 (Article).  

Jianlong, W. (1998). Improvement of citric acid production by Aspergillus niger with addition of phytate to beet molasses.
Bioresource Technology, 65, pp. 243–245

Max, B., Salgado, J.M., Rodríguez, N., Cortés, S., Converti, A., Domínguez, J.M. (2010) Biotechnological production of citric acid. Brazilian J. Microbiol. 41(4), pp. 862–875

Najafpour, G.D. (2007) Production Of Citric Acid. In: Biochemical Engineering And Biotechnology.

Papagianni, M. (2007) Advances in citric acid fermentation by Aspergillus niger: Biochemical aspects, membrane transport and modeling. Biotechnol. Adv., 25(3) pp. 244-263 (Article)

Papanikolaou, S., Muniglia, L., Chevalot, I., Aggelis, G., & Marc, I. (2002). Yarrowia lipolytica as a potential producer of citric acid from raw glycerol. Journal of Applied Microbiology, 92(4), pp. 737-744

Prado, F.C., Vandenberghe, L.P.S., Woiciechowski, A.L., Rodrígues-León, J.A., Soccol, C.R. (2005) Citric acid production by solid-state fermentation on a semi-pilot scale using different percentages of treated cassava bagasse. Brazilian J. Chem. Eng. 22(4), pp. 547–555 

Sansome-Smith, A.W. (2019)  “Commercial production of citric acid from waste materials using waste materials: Impact on quality and price” Private communication to citric acid suppliers. 

Show, P. L., Oladele, K. O., Siew, Q. Y., Aziz Zakry, F. A., Lan, J. C. W., & Ling, T. C. (2015). Overview of citric acid production from Aspergillus niger. Frontiers in Life Science, 8(3), pp. 271-283 (Article)

Soccol, C.R., Vandenberghe, L.P., Rodrigues, C., Pandey, A. (2006) New perspectives for citric acid production and application. Food Technol. Biotechnol. 44(2), pp. 141–149

Tomlinson, N., Campbell, J. J. R., & Trussell, P. C. (1950). The influence of zinc, iron, copper, and manganese on the production of citric acid by Aspergillus niger. Journal of Bacteriology, 59(2), pp. 217-227.

Vandenberghe, L. P. S., Soccol, C. R., Pandey, A., & Lebeault, J. M. (1999). Review – microbial production of citric acid. Brazilian Archives of Biology and Technology, 42, 263e276

Vandenberghe, L. P., Soccol, C. R., Pandey, A., & Lebeault, J. M. (2000). Solid-state fermentation for the synthesis of citric acid by Aspergillus niger. Bioresource Technology, 74(2), pp. 175-178 (Article).