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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.
1.3.2.2 Lactic Acid
Lactic acid (C3H6O3) history is dated long back in 1780 when it was first discovered in sour milk by Swedish chemist, Scheele (Figure 1.3). However, in 1847 lactic acid was discovered as a final product of fermentation, and its commercial production from microorganisms is new. It is colorless to light yellow in color available in solid or liquid form. It is widely found in nature among human beings, animals, plants, and microorganisms, in two isomeric forms, i.e. L (+) and D (−) isomers, and as a racemic mixture (DL‐lactic acid). Originally, lactic acid was used as a preservative but now has a wide range of applications in food industry as a flavor enhancer in juices, jams, syrup, etc. Recently, polylactide (formed by condensation of lactic acid) a biodegradable thermoplastic that requires pure lactic acid is used for food packaging.
Microbial production of lactic acid utilizes two types of bacteria heterofermentative and homofermentative bacteria. As the name suggests heterofermentative bacteria produces other by products apart from lactic acid, while homofermentative bacteria solely produce lactic acid. A part of lactic acid group bacteria (LAB), Lactococcus and Lactobacillus are the most important producer of lactic acid. Twenty‐two different Lactobacillus species are identified utilizing different substrate. Lactobacillus delbruekii requires glucose as a carbon source, while Lactobacillus pentosus grow on sulfite wastewater (Breed et al., 1957). Lactobacillus xylans is homofermentative utilizing xylose. Other genera of LAB include Streptococcus, Pediococcus, and Leuconostoc. Majority of species of the genus Streptococcus are pathogenic to humans like Streptococcus pyogenes, Streptococcus pneumoniae, etc. Out of these, Streptococcus thermophilus, a homofermentative facultative anaerobic is nonpathogenic and used to produce curd rich with Gamma‐amino‐butyric acid (GABA) (Linares et al., 2016). Leuconostoc mesenteroides synthesizes D‐Lactic acid in pure form. Wild‐type L. mesenteroides is grown on lactic acid and strains resistant to lactic acid were isolated giving a production of 76.8 g/l as twice as wild‐type strain (Ju et al., 2016).
Lactic acid production by LAB is mostly affected by demand to keep pH of the culture acidic which implies that the strain should be acid tolerant. The complete genome sequencing of Lactobacillus lactis opens the opportunity for genetic manipulation to increase lactic acid production (Bolotin et al., 2001). After a year of full‐genome sequencing Patnaik et al. (2002) identified new lactobacillus species through genome shuffling, which can produce three times the lactic acid compared to the wild type at pH 4.0.
Similarly, mutant strain of Lactobacillus delbrueckii was also obtained by genome shuffling giving 40 g/l lactic acid under low pH conditions (John et al., 2008). Apart from pH, high temperature, salts, lactate, and alcohol‐tolerant Lactococcus lactis strain expressing E. coli chaperone DnaK proteins were made (Sugimoto et al., 2010). This shows multiple resistance effect of DnaK protein. Acid‐resistant strain of Lactobacillus casei under acid stress shows an overexpression of RecO protein as compared to wild‐type strain (Wu et al., 2012). This indicates the role of RecO protein in acid stress. Therefore, RecO protein from L. casei is engineered in L. lactis under the effect of nisin inducible expression system (Wu et al., 2013). The engineered strain grows well in stress condition, and there was an increase in lactate dehydrogenase (LDH) enzyme and hence lactic acid.
LAB cannot utilize starch as a carbon source due to unavailability of an enzyme α‐amylase. α‐Amylase hydrolyzes complex sugars like starch to release simple sugars like glucose which can be easily used as carbon source by microorganisms. Keeping this in mind high‐yielding lactic acid strain Lactococcus lactis IL 1403 was engineered with α‐amylase from Streptococcus bovis. The strain generated can easily be grown on starch and giving a yield of 1.57 g/l/h (Okano et al., 2007). Production of pure lactic acid in a particular isomeric form is of interest. Lactobacillus plantarum expressing S. bovis α‐amylase and LDH deficient produces pure D‐lactic acid from corn starch (Okano et al., 2009). As the strain is LDH deficient therefore cannot produce L‐lactic acid and can be grown on starch as expresses α‐amylase. The strain was able to produce D‐lactic acid with the optical purity of 99.6%.
In addition to bacteria, filamentous fungi Rhizopus oryzae also accumulates lactic acid when grown on mineral medium and starch or xylose (Koutinas et al., 2007).
1.3.2.3 Succinic Acid
Succinic acid (C4H6O4) is the intermediate metabolite of krebs or TCA cycle. In earlier times, it was formed by distillation of amber and therefore called as amber acid or spirit of amber (Song and Lee, 2006). After that it was produced from butane through maleic anhydride. It is used as a flavoring agent in food industry. It has also a range of applications in dyes, perfumes, and in the manufacturing of clothing, paint, fibers, etc. Succinic acid is widely used as precursor for chemicals such as 1,4‐butanediol, γ‐butyrolactone, N‐methyl‐2‐pyrrolidone, tetrahydrofurane, and 2‐pyrrolidone (Pateraki et al., 2016). These chemicals are further derived into biodegradable polyesters such as polybutyrate succinate, polyamides, pesticides, and green solvents (Sun et al., 2015). This leads to the increased demand of succinic acid which was 50 000 metric tons in 2016 to double by 2025 (Chinthapalli et al., 2018).
Like citric acid, succinic acid is also synthesized in almost all plants, animals, and microorganisms. Various microorganisms like E. coli, Actinobacillus succinogenes, Mannheimia succiniciproducens, etc. produce succinic acid. Among them M. succiniciproducens gives the highest yield of 1.64 mol/mol glucose and productivity of 6.02 g/l/h (Lee et al., 2016). M. succiniciproducens was first isolated from the bovine rumen of Korean cow. Availability of whole genome sequence of M. succiniciproducens makes it easier candidate for genetic engineering (Lee et al., 2005). Glucose‐6‐phosphate 1‐dehydrogenase (zwf) gene is upregulated when succinic acid production is increased. Overexpression of zwf gene in M. succiniciproducens increases succinic acid synthesis (Kim et al., 2017).
Escherichia coli produce succinic acid in a very scarce amount, but is the model organism for genetic manipulation due to its fast growth and availability of genetic toolboxes. One of the strains NZN111 is generated by knocking out pyruvate formate lyase and LDH (Singh et al., 2009). This leads to the inhibition of formic and lactic acid, increasing the succinic acid production. But the strain generated was not able to thrive anaerobically producing lactic acid. This was solved by further overexpressing the gene for malate dehydrogenase in the same strain producing 31.9 g/l of succinic acid (Wang et al., 2009). Spontaneous chromosomal mutation of glucose phosphotransferase generates strain AFP111, which can anaerobically grow on glucose giving productivity of 0.87 g/l/h (Chatterjee et al., 2001). Prolonged anaerobic conditions can hamper cell growth giving low production rates. To produce succinate under aerobic conditions, five genes were inactivated in E. coli, namely, succinate dehydrogenase, pyruvate oxidase, acetate kinase phosphotransacetylase, aceBAK operon repressor, and glucose phosphotransferase (Lin et al., 2005). Apart from inactivation of these genes phosphoenolpyruvate carboxylase gene was overexpressed giving productivity of 1.08 ± 0.06 g/l/h of succinic acid.
Species of yeast like Y. lipolytica and Saccharomyces cerevisiae are also prospective host for succinic acid production. A robust Y. lipolytica