Citric acid production

CITRIC ACID PRODUCTION , METHODS OF ITS PRODUCTION, PURIFICATION OF CITRIC ACID AND ITS USES.

1. C ITRIC A CID P RODUCTION

2. INTRODUCTION Citric acid or 2-hydroxy-1,2,3- propane tri carboxylic acid (C 6 H 8 O 7 .H 2 O) The name is derived from the Latin word citrus which refers to tree of genus Citrus including lemon trees. Citric acid is the most important commercial product, which is found in almost all plant & animal tissues. Citric acid is a weak organic acid found in citrus fruits( lemon ). It is a good natural preservative and is also used to add an acidic (sour) taste to foods and soft drinks.

3. Citric acid in its pure form is readily soluble in water and colorless. Citric acid can be derived from natural sources ( e.g ; lemon, lime and orange) or synthetic source (chemical reaction and microbial fermentation). At room temperature, citric acid is a white crystalline powder Lemons and limes have particularly high concentrations of the acid; it can constitute as much as 8% of the dry weight of these fruits (about 47 g/L in the juices).

4. H ISTORY The discovery of citric acid has been credited to the 8th century Muslim alchemist Jabir Ibn Hayyan (Geber). Citric acid was first isolated in 1784 by the Swedish chemist Carl Wilhelm Scheele, who crystallized it from lemon juice.  Industrial-scale citric acid production began in 1890 based on the Italian citrus fruit industry. In 1893, C. Wehmer discovered  Penicillium  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.

5. In 1917, the American food chemist James Currie discovered certain strains of the mold  Aspergillus niger  could be efficient citric acid producers. The pharmaceutical company Pfizer began industrial-level production using this technique two years later.

6. M ICROBIAL S TRAIN Large number of micro-organisms including bacteria, fungi and yeasts have been employed to produce citric acid. The main advantages of using this micro-organisms are: Its easy of handling Its ability to ferment a variety of cheap raw materials High yields

7. Various strains of genera fungi, yeast and bacteria were reported i.e., Penicillium luterum , Penicillium restrictum , Penicillium citrinum , Trichoderma viride , etc. Only mutants of Aspergillus (Aspergillus niger ) and yeasts genus Candida have almost exclusively been utilized

8. S UBSTRATE The basic substrates for citric acid production in plants using the surface method of fermentation is cane molasses Plants using submerged fermentation can not only cane molasses, but a substrate of higher purity i.e ; hydrolysed starch, technical and pure glucose, refined or raw sugar, purified and condensed beet or cane juice. Substrate commonly used beet molasses, Sucrose, Starch, Alkanes, Oils and Fats.

9. B IOCHEMISTRY Citric acid is excreted from cells in response to unfavorable intracellular condition caused by increased levels of TCA. A crucial prerequisite for overflow of citric acid from A.niger cells is therefore increased level of Kerbs Cycle intermediates caused by anaplerotic reactions.

10. F ermentation Fermentation is the technique of biological conversion of complex substrates into simple compounds by various microorganisms such as bacteria and fungi. In this metabolic breakdown, they also release several additional compounds apart from the usual products of fermentation such as carbon dioxide and alcohol. These additional compounds are known as secondary metabolites.

11. T ypes of c itric a cid f ermentation Industrial citric acid production can be carried out in three different ways: Surface fermentation Submerged fermentation Solid state fermentation

12. S urface f ermentation First individual process of citric acid production was liquid surface culture. It is also known as stationary batch fermentation process which completed in 8-12 days. It is carried out in fermentation chambers with a number of trays arranged in shelves. Fungal mycelium develops on the surface of the medium on these trays.

13. Contd… After sterilizing the fermentation medium contained in the trays, it is inoculated with spore suspension and incubated at 28-30℃ for 24 hours. Spores germination started at pH 6-6.5 which can be visualized with naked eye as continuous mycelium on the surface. Heat generated during process controlled by proper aeration.

14. S teps f or c itric a cid p roduction Molasses substrate acidified with phosphoric acid to a pH 6-6.5 and heated at temperature 110℃ for 15-45 minutes. Potassium hexa-cyanoferrate is added to the hot substrate to precipitate trace metals. Inoculate media by adding dry conidia mixed with sterile air and spread as an aerosol over the trays. Kept temperature constant at 30℃ by means of air current.

15. Contd.. With in 24 hours after inoculation, germinating spores start forming a 2-3 cm cover blanket of mycelium floating on the surface of substrate. At this stage add ammonium ions as a result pH drops to 2. Fully developed mycelium floats as a thick white layer on the nutrient solution. Recovery of mycelium to extract citric acid.

16. S ubmerged f ermentation Submerged fermentation is a process involving the development of microorganisms in a liquid broth. The broth medium inside the nutrient substratum is in liquid form and organism can grow throughout the broth medium. Process carried out in bioreactor, completed in 5-12 days. Organism after 1-2 days of inoculation, grows as a pallet of 0.5 cm in diameter and suspend freely in the medium.

17. Contd… So organism with huge contact surface area can take up the oxygen and nutrient. Air flow introduced into vessel at high speed, agitation equipment mixes and breaks the bubbles. Carbon source present in the medium decomposed anaerobically by microorganisms.

18. S teps f or c itric a cid p roduction Beet molasses, nutritive salts such as ammonium nitrate added. pH of substrate maintained at 5.5-5.9. Process run in one or two stages by using hydrophilic spores suspension. Development of hyphae and aggregation generally requires a period of 9-25 hours at 32℃. Mycelia aggregation can be detected after 24 hours of inoculation. After 6-8 days citric acid purified.

19. S olid- s tate f ermentation In this process, microbes are grown in low water containing insoluble material which act as a physical support and source of nutrients as well. Solid substrate is moistened with 70% moisture pH 4.5 to 6.0 and temperature between 28-30℃ required. Process completed within 4-5 days.

20. S teps f or c itric a cid p roduction Solid substrate is soaked with water up to 65-70% water content. After the removal of excess water mass undergoes a steaming process. Sterile starch paste is inoculated by spreading Aspergilus niger conidia in the form of aerosol on the substrate surface. pH maintained at 5-5.5 and temperature at 28-30℃. Growth can be enhanced alpha-amylase. Citric acid obtained at the end.

21. S eparation of c itric a cid Solvant extraction This process can be applied when the fermented must contain low amount of impurities. Citric acid can be recovered from the extract by distilling off the solvant . The aqous solution purified citric acid is crystallized by concentration.

22. S eparation P rocess

23. P urification of c itric a cid Purification is a simple way of getting pure citric acid followed by two simple techniques. 1. Precipitation 2. Filteration Precipitation is the most commonly used technique. It is performed by the addition of calcium oxide hydrate (milk of lime) to form the slightly soluble tri-calcium citrate tetrahydrate . The precipitated tri-calcium citrate is removed by filtration and washed several times with water.

24. It is then treated with sulphuric acid forming calcium sulfate, which is filtered off. Mother liquor containing citric acid is treated with active carbon and passed through cation and anion exchangers. Several anion-exchange resins are commercially available. Precipitation by metal ions:- Metal salts with lower solubilities can formed by enzymes and proteins.

25. Nucleic acids which are present in microbial cells must be removed because they reduced the resolution of separation. Precipitation by organic solvants Used industrially because it is inexpensive and simple. By adding an organic solvant to an aqous fermentation broth the dielectric constant will decrease and causing the solubility to decrease.

26. F iltration Filters use some porous material along with applied pressure to push smaller particles through the filter. Separating elements of the solution based on size. Batch Filtration:- Usually performed under the constant pressure that moves the liquor through the filter. Filter cake will build up as filtration proceeds and resistance to broth flow will increase.

27. Can be used to remove the cells but does not work well for animal cell debris. Ultrafiltration Utilize a membrane to separate particles that are much larger than the solvant used. Successful removal occurs in the particle size range of 10 solvant molecular diameters to 0.5.

28. Rotary Drum Filtration Rotary vaccum filters can be used to remove proteins , enzymes , mycelia cells. Solution is vaccumed upward where it croses a filter septum removed by positive displacement pump. Filter cake is removed after each rotation to give fresh surface for filteration .

29. F actors e ffecting on p roduction of c itric a cid It was shown that the factors mainly affecting the citric fermentation are : The type and concentration of carbon source nitrogen phosphate limitation pH Aeration Oligo elements concentration Time of fermentation

30. 1. N utrient Aspergillus niger grows well in media containing carbohydrates nitrogen (as ammonium or nitrate ions) phosphate, low amounts of potassium magnesium sulfate, and trace metals such as iron, manganese, zinc, and copper. The nitial sugar concentration plays an important role. The highest citric acid conccentrations were observed in cultures grown at high initial sugar concentrations (15−20% w/v)

31. Further increase of sugar concentration resulted in a decrease of acid concentration by 15%. This is due to osmotic effects In addition to carbohydrates, nitrogen phosphate concentrations have a strong influence on citric acid production. nitrogen or phosphate concentration less than 0.2% (w/v) in the medium appears to be adequate.

32. 2- Inhibitors and Stimulants The most important stimulants used for i mproving citric acid yield by A. niger are methanol E thanol These chemicals have been found to retard growth , delay sporulation and increase citric acid production. Addition of ethanol resulted in a twofold increase in CS activity, and a 75% decrease in ACH activity. Other stimulants such as fats and oils significantly increased the production of citric acid.

33. Inhibitors Addition of some inhibitors such as calcium fluoride, sodium fluoride, potassium fluoride, hydrogen peroxide, naphthaquinone , methylene blue, sodium azide , and sodium arsenate to the different media increased (by 30–40%) citric acid concentration.

34. Inoculum To prepare inoculum A. niger is grown in standard media for molds. It is usually cultivated on potato dextrose agar (PDA) slants In petri dishes at 28–30°C for 3–5 days. The spores obtained are suspended in sterile water .

35. When mycelia pellets are used, they are grown in submerged fermentation for 2–3 days in medium that has the same composition as the production medium. The production medium is then inoculated at a concentration of 5–10% (v/v). In solid-state fermentation, the medium is inoculated with 5% (v/w) of the inoculum containing 108 spores/mL to give a concentration of 0.5*107 spores/g wet substrate.

36. F ermentation T ime The optimum time for the maximum production of citric acid depends on the strain used the chemical composition of the medium the fermentation system the conditions under which fermentation takes place. In the surface culture, fermentation time is usually completed in 10–20 days, In the submerged culture incubation time is much shorter (5–10 days).

37. In solid-state fermentation the fermentation time depends strongly on the amount of inoculum used the moisture content of the substrate, the initial pH the temperature particle size of medium

38. pH When A. niger is used For the production of citric acid, the initial pH is dependent on the medium employed. In synthetic media The initial pH of the medium is usually adjusted to 2.5–3.5, In the case of molasses The initial pH must be neutral or slightly acidic in order for germination and growth of the microorganism to occur. The pH of the medium is adjusted with HCl, H2SO4, or NaOH.

39. The pH of the substrate decreases during fermentation (from 7.0 to 2.0) due to the production of citric acid and other acids generated in the TCA cycle. When yeasts are used For the production of citric acid, the pH is often adjusted to 6.0–7.0 by addition of lime, calcium carbonate, or sodium hydroxide.

40. A eration and A gitation Aspergillus niger is an aerobic microorganism. Aerating and ag agitating in t he fermentation broth normally satisfies the oxygen demand of a fermentation process. Agitation is important for adequate mixing mass transfer heat transfer. a lso maintains homogeneous chemical and physical conditions in the culture by continuous mixing.

41. The concentration of dissolved oxygen increased with the increase of speed of agitation. It fell rapidly during the first 2 days of fermentation after which it increased more slowly due to the rapid increase of biomass concentration (6.0–9.0 g/L) observed at the same time.

42. U ses of c itric a cid Food Additive Citric acid is used in various food products. It is used in soft drinks and beverages. Citric acid can also be used in various candies. Sometimes white powder is present on the candies, that white powder is citric acid. Prevent crystallization of sucrose, produce dark colour in hard candies, inversion of sucrose.

43. Dairy products Used as emulsifier in ice creams and processed cheese. Used as acidifying agent in many cheese products and as an antioxidant. Fruits and vegetable juices Acts as stabilizer in commercially prepared juices of fruits and vegetables.

44. Cleaning Agent Citric acid can also be used as cleaning agent. Citric acid can be used in detergents and soaps. Cleaners that are utilized in kitchen contain the citric acid.

45. Cosmetics Citric acid is used in cosmetics. It can also be used as home masks because it can remove the dead skin. Skin tone can be improved by the citric acid. Wrinkles and acne scars can be reduced by the citric acid. Citric acid can also be used in hand wash and shampoos. Water Softener Citric acid can be used in the detergents as water softener because it is organic acid. Citric acid is a weak acid so it is a strong softener for water.

46. Industrial uses Citric acid is used in the manufacturing of detergent and also in the tanning of leather. It can also be used to preserve the blood. Gelatin desserts Adjusts pH to the desired levels and helps in setting of gelatin desserts.

47. Jellies and jams Gelling agent, provides the desired degree of tartness, tang and flavor. Frozen fruits Lowers pH to inactivate oxidative enzymes. Protects ascorbic acid by inactivating trace metals. Wines and ciders Prevents turbidity of wines. Adjusts pH, inhibits oxidation.

48. Pharmaceuticals Used in tablets in combination with bicarbonates. Antioxidant in vitamin preparations. Used as anticoagulant. Beverages Provides flavor and tartness. Increases the effectiveness of antimicrobial preservatives.

49. Metal cleaning Removes metal oxides from surface of ferrous and nonferrous metals. Others Used in copper platting, metal cleaning, printing inks, photographic reagents, polymers and waste treatment. To cure kidney disorders Sodium citrate, acetic acid is used to prevent kidney stones.

50. S ide e ffects Taking excess of citric acetate in combination with sodium citrate may lead to kidney failure. Taking citric acid with empty stomach may lead to stomach or intestinal side-effects. It may also lead to muscle twisting or cramps. It can also cause weight gain, swelling, fast heart rate.