Citric acid production

microbial production of citric acid, koji process, surface and submerged fermentation, recovery , purification and applications of citric acid

1. Citric Acid Production

2. INTRODUCTION Citric acid (2-hydroxy-1,2,3- propane tri carboxylic acid) is the most important commercial product, which is found in almost all plant & animal tissues. Citric acid is the most important organic acid produced in tonnage and is extensively used in food and pharmaceutical industries. Citric acid is a weak organic acid found in citrus fruits(lemon). It is good ,natural preservative and is also used to add an acidic taste to food and soft drinks. More than million tonnes are produced every year by fermentation.

3. HISTORY Citric acid was first isolated in 1784 by the Swedish chemist carl Wilhelm Scheele, who crystallize it from lemon juice. Industrial scale citric acid production began in 1890 based on the Italian citrus fruit industry. In 1893, C. Wehmer discovered penicillin mold could produce citric acid from sugar. However, microbial production of citric acid did not become industrially important until world war I disrupted Italian citrus exports.

4. Applications of Citric Acid: 1. Citric acid, due to its pleasant taste and palatability, is used as a flavoring agent in foods and beverages e.g., jams, jellies, candies, desserts, frozen fruits, soft drinks, wine. Besides brightening the colour , citric acid acts as an antioxidant and preserves the flavors of foods 2. It is used in the chemical industry as an antifoam agent, and for the treatment of textiles. In metal industry, pure metals are complexed with citrate and produced as metal citrates. 3. In pharmaceutical industry, as trisodium citrate, it is used as a blood preservative. Citric acid is also used for preservation of ointments and cosmetic preparations. As iron citrate, it serve as a good source of iron.

5. Applications of Citric Acid: 4. Citric acid can be utilized as an agent for stabilization of fats, oils or ascorbic acid. It forms a complex with metal ions (iron, copper) and prevents metal catalysed reactions. Citric acid is also used as a stabilizer of emulsions in the preparation of cheese. 5. In detergent/cleaning industry, citric acid has slowly replaced polyphosphates.

6. Microbial Strains for Citric Acid Production: Microbial Strains for Citric Acid Production: Many microorganisms can produce citric acid. The fungus aspergillus niger is most commonly used for industrial production of citric acid. The other organisms (although less important) include A . Clavatus , A. Wentii , penicillium luteum, Candida catenula , C. Guilliermondii and corynebacterium sp. Only mutants of A. Niger and yeast genus Candida have almost exclusively been utilized.

7. Advantages of using this micro-organism are: Its ease of handling. Its ability to ferment a variety of cheap raw materials. It provides high yield

8. Biosynthesis of Citric Acid: Citric acid is a primary metabolic product (of primary metabolism) formed in the tricarboxylic acid (Krebs) cycle. Glucose is the predominant carbon source for citric acid production. The biosynthetic pathway for citric acid production involves glycolysis wherein glucose is converted to two molecules of pyruvate . Pyruvate in turn forms acetyl CoA and oxaloacetate which condense to finally give citrate.

9. Carbohydrate source A wide range of raw materials can be used for the supply of carbohydrates. These include molasses (sugar cane or sugar beet), starch (from potatoes), date syrup, cotton wastes, banana extract, sweet potato pulp, and brewery waste and pineapple waste water. A high yield of citric acid production occurs if the sugars that are rapidly metabolised are used e.g. sucrose, glucose, maltose. At present, cane molasses and beet molasses are commonly used.

10. The concentration of carbohydrate significantly influences citric acid production. Ideally, the sugar concentration should be 12-25%. At a concentration less than 5% sucrose, citric acid formation is negligible.

11. Trace metals: Certain trace elements (Fe, Cu, Zn, Mn, Mg, Co) are essential for the growth of A. Niger. Some of the trace metals particularly Mn 2+ , Fe 3+  and Zn 2+  increase the yield of citric acid. These ions promote glycolysis and reduce respiration; both these processes promote citric acid production. pH: pH of the medium is kept around 5 when molasses is used and at pH 3 when sucrose used. Advantage with low pH is that the risk of contamination is very minimal, since many organisms cannot grow at this pH.

12. Nitrogen source: Ammonium salts, nitrates and urea are the nitrogen sources used in the media for citric acid production. All the three compounds are equally good sources, as long as they do not adversely affect the pH of the medium.

13. Koji Process It also refers to as “Solid-state fermentation”. The koji process was first introduced in Japan. It is related to the use of agro -industrial residues for citric acid production. In Koji process, raw materials like apple pomace, sugar cane, beet molasses etc. can be used. These raw materials are utilized by the Aspergillus niger . The pH and moisture content of the raw material is adjusted to 4-5 and 70% respectively. Then the raw material is cooled at 30-60 degrees Celsius and after that, inoculate A. niger . After inoculation, the medium is transferred into large trays of 3-5cm depth and incubated at 25-30 degrees Celsius for 3-7 days. At last, the citric acid is extracted from the fermentation tank. The starch content of the raw material is degraded into citric acid by the amylase enzyme of the Aspergillus niger . The koji process does not require the substrate pre-treatment because the trace elements do not affect the production of citric acid.

14. Surface Culture Process It also refers to as “ liquid surface fermentation ”. Surface culture fermentation was the first method introduced for citric acid production in  1919 . In liquid surface fermentation, the culture medium (molasses and salts) (5-6 pH) is added to the aluminium shallow trays up to 5-20 cm deep. The process is carried out in the fermentation chamber which provides uniform air circulation, maintains relative temperature and humidity. First, the spores of  A.niger  is blown onto the surface of the culture medium for about  5-6 days  and then dry air is passed. Now, the pH of the culture medium is adjusted between  1.5-2   pH. After 24 hours, the spores start to germinate and the growth of  white mycelium  is observed on the surface of the culture medium. After the utilization of sugar content by the mould , the remaining liquid is separated from the mycelial mat. In the surface culture process, a small amount of citric acid is produced as the primary metabolites by the  A. niger .

15. Submerged Culture Process It also refers as “Submerged culture fermentation”. About 80% of the citric acid production is carried out through submerged fermentation method. Submerged fermentation makes the use of black Aspergillus i.e. A. japonicus. It is performed in a bioreactor made of stainless steel compiled with proper aeration, cooling system, impellers etc. For carbon source, substrate like beet molasses, corn starch etc. are used as a substrate. For the nitrogen source, ammonia is used. The substrate used in this method requires pre-treatment like the addition of nutrients, sterilization etc. The A. japonicus is inoculated into the culture medium and maintained at 30degrees Celsius. Submerged fermentation is mostly carried out in a batch bioreactor in which 1500kg of citric acid and 500kg of biomass can be produced from the 2500kg glucose and 860kg of oxygen.

16. Recovery of Citric Acid The product formed after fermentation is the fermented liquor looks hazy due to the presence of antifoaming agents, mycelia etc. Therefore to separate these things, a slurry of calcium hydroxide i.e. Ca (OH)2 to form a precipitate of calcium citrate. The precipitate of calcium citrate is filtered and washed. After filtration, a filtrate forms which is treated with the sulphuric acid for the precipitation of calcium as “Calcium sulphate” (CaSO4). Calcium sulphate is then treated with the activated carbon by which it gets demineralized after passing it consecutively from the ion exchange bed. The solution obtained from this is subjected to the circulating crystallizers. The crystals formed as a result of crystallization are then removed by centrifugation. After the completion of these steps, the remaining solvent is dried, sieved and then packed. The remaining mother liquor is again recovered by the same process.

17. Uses The production of citric acid is necessary for its versatile uses in the different fields that are given below: Food industries: Citric acid is used for the production of jams, jellies, candies, frozen fruits etc. In certain foods, citric acid is used as an “Artificial flavouring agent”. Beverage industries: Citric acid is used for the production of soft-drinks and distilled beverages like wine. Hospitals: Citric acid is used as “Effervescent agent” at the time of blood transfusion. Cosmetic industries: Citric acid is used in cosmetic products like astringent lotions, hair gels etc.

18. Factors Affecting the Production There are some factors which can directly or indirectly affect the production process or the fermentation of citric acid.

19. Carbon Source Concentration: For most of the industrial production, carbon sources like glucose and sucrose are recommended as they provide a good source of carbon for the growth of the biomass. Galactose acts as an alternative source of glucose and sucrose, but it allows low microbial growth and thus not favours the cumulation of citric acid. Other carbon sources like cellulose, starch etc. allows limited growth and may slow down the growth rate of the microorganisms, which will lead to minimal production. The optimal sugar concentration ranges between 10-14% and the sugar concentration below 2.5% will not produce citric acid.

20. Nitrogen Source Concentration: Ammonium salts like urea, ammonium sulphate etc. results in a decrease in pH, which is crucial for citric acid production. The nitrogen concentration must be in between 0.1-0.4 N/L. High nitrogen source will increase the microbial growth which will consume more sugar and in turn decrease the yield of citric acid.

21. Phosphorous Source Concentration: For the best production and growth of fungi, potassium dihydrogen phosphate is considered best for the phosphorous source. The concentration of phosphorous must be in between 0.5-5.0 g/l for the maximum yield. If phosphorous present in excess, it leads to the production of sugar acids which will decrease the carbon dioxide fixation and thus, stimulate the fungal growth.

22. Presence of Trace Elements: Divalent metals such as iron, zinc, manganese, copper etc. produce at the time of fermentation. If KH2PO4 is added to the zinc, it favours the production but the trace elements like manganese, iron, and high concentration of zinc leads to a reduction in yield.

23. Lower Alcohols Concentration: Lower alcohols like ethanol, methanol etc. enhance the citric acid fermentation. The concentration of lower alcohol must be in between 1-3%. Lower alcohols not only stimulate the citric acid production but also promotes the sporulation of microorganisms.

24. Miscellaneous Compounds: Calcium fluoride, sodium fluoride etc. are the miscellaneous compounds which accelerate the citric acid production whereas potassium ferrocyanide decreases the yield.

25. pH Concentration: The pH of the culture medium is directly related to the growth of microorganisms and their metabolic activities. A pH changes to acidic by the microbial activity, like for Aspergillus, the pH drops to 3.0. The pH below 2.0 is optimal for the production of citric acid and a pH of 2.2 is best for the growth of mould .

26. Aeration System: Aeration increases the dissolved oxygen concentration in the culture medium and thus increases the yield of citric acid. The aeration system also reduces the fermentation time. The concentration of oxygen must be above 25% saturation.