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Citric acid (2‐hydroxy‐propane‐1,2,3‐tricarboxylic acid) is colorless, odorless, in its pure form, and is water soluble. It is widely distributed in plants and animals and is omnipresent in nature as the intermediate compound in krebs cycle or tricarboxylic acid cycle. It is used as flavoring agent in food industry (Soccol et al., 2006). It has a major role in pharmaceutical industry as a blood preservative, in chemical industry as antifoam agent, and in soap industry as stabilization of fats and oils (Max et al., 2010). Its estimated production capacity is approximately 2 million tons per year (Steiger et al., 2017). Citric acid synthesis pathway along with other organic acids synthesis is shown in Figure 1.4.

It was first discovered in lemon (citrus fruit) and now about 99% of citric acid is produced from microbial fermentation. Wehemer (1893) discovered the production of citric acid by Penicillium glaucum, but could not scale up the process (Vandenberghe et al., 1999). In 1917, strains of Aspergillus niger were found to synthesize citric acid and soon became the method of choice for industrial production of citric acid (Chen and Nielsen, 2016). Highest producing strains of Aspergillus can accumulate 200 g/l citric acid under optimum conditions (Steiger et al., 2017). For improved production rate, metabolic and genetic engineering of Aspergillus strains are done which can tolerate low pH levels and can secrete excess citric acid in the medium (Ruijter et al., 1997; Steiger et al., 2019). Carbon source utilization plays an important role in industrial production of citric acid. Despite giving higher yields glucose cannot be used industrially as it is not cost efficient. Therefore, liquified corn starch, glycogen, etc. are used. Using liquified corn starch leads to the production of isomaltose with the action of enzyme glucosidase and cannot be used for citric acid production. There is another enzyme glucoamylase that releases glucose from starch and is beneficial. So, Wang et al., (2016) deleted the glucosidase encoding gene and overexpressed glucoamylase gene in A. niger. This leads to the decrease in residual sugar to about 88.2% and increase in citric acid production to 16.9%, reaching up to 185.7 g/l.

From Figure 1.4, it can be seen that oxaloacetate and Acetyl‐CoA act as a precursor for citric acid production. Oxaloacetate is generated by malate dehydrogenase and fumarase while Acetyl‐CoA by pyruvate dehydrogenase, acetyl‐CoA synthetase, and ATP‐citrate lyase (ACL1, ACL2). ATP‐citrate lyase, as the name suggests consumes citrate to synthesize Acetyl‐CoA. There was an increase in citric acid production when ACL1 was deleted from A. niger (Meijer et al., 2009). But when both the subunits ACL1 and 2 were deleted, there was a decrease in levels of citric acid (Chen et al., 2014). Overexpression of fumarase gave increased citrate levels, while malate dehydrogenase only expedites levels initially. Role of citrate exporter (CexA) is also very important. Citric acid produced inside the cell and not able to get released in the medium could lead to its inhibition. CexA is the main transporter of citric acid in A. niger. Overexpression of this transporter gene leads to increased production of citric acid (Steiger et al., 2019).

Figure 1.4 Biosynthetic pathway for production of organic acids.

Apart from A. niger, a well‐established producer of citric acid, several strains of bacteria and yeast are also used for citric acid production. Some other producers of citric acid are yeasts which include Candida oleophils, Candida guilliermondi, Saccaromicopsis lipolytica, Hansenula anamola, Candida parapsilosis, Candida tropicalis, Candida citroformans, and Yarrowia lipolytica. Among bacterial species, i.e. B. licheniformis, Arthrobacter paraffinens, and Corynebacterium sp., were also used previously for citric acid production by using many raw materials as a substrate with the percentage yield of 27–88% per sugar consumed by the microbial strains.