CITRIC ACID PRODUCTION

Introduction
Today over 99% of the worlds output of citric acid is produced microbially. Approximately70% of citric acid produced is used in the food and beverage industry for various purposes, 12% in pharmaceuticals and about 18% for other industrial uses. Citric acid is a weak organic acid, and it is a natural preservative and is also used to add an acidic, or sour, taste to foods and soft drinks. In biochemistry, it is important as an intermediate in the citric acid cycle and therefore occurs in the metabolism of virtually all living things.

Citric acid is a tricarboxilic acid having the formula shown below and is an intermediate in Krebs cycle:

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History

Citric acid was first isolated in 1784 by the Swedish chemist Carl Wilhelm Steele, who crystallized it from lemon juice. It is a 6-carbon containing tricarboxylic acid. Industrial-scale citric acid production began in 1890 based on the Italian citrus fruit industry. In 1893, C. Wehmer discovered that 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. In 1917, the American food chemist James Currie discovered that certain strains of the mold Aspergillus niger could be efficient citric acid producers, and Pfizer began industrial-level production using this technique two years later, followed by Citrique Belge in 1929.

Uses of citric acid:

1. As an acidulant in food (e.g. Jams, preserved fruits, fruit drinks, etc.) and pharmaceutical industries. 2. As chelating and sequestering agent (e.g. in the tanning of animal skin) 3. In the production of carbonated beverages. 4. Citrate and citrate esters as plasticizers.

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Strains for citric acid production :

It is very important to select suitable strain for citric acid production. Many strains excrete traces of Citric acid as a metabolic of primary metabolism. Ex. o Aspergillus niger, o A. wentii, o A. clavatus, o Penicillium luteum, o P. citrinum, o Mucor piriformis, o Paecilomyces divaricatum, o Citromyces pfefferianus, o Candida guilliermondii, o Saccharomycopsis lipolytica, o Tricoderma viride, o Arthrobacter paraffineus, and o Corynebacterium sp. o However, Only mutants of Aspergillus niger are used for commercial production. o Compared to Penicillium strains, the Aspergilli produce more citric acid per unit time. A. niger strains among these fungal strains are used for the following reasons: 1. They are efficient (i.e. high-yielding) strains. 2. The possess fairly uniform biochemical properties. 3. The produce a negligible or small amount of oxalic acid, provided fermentation conditions (i.e.pH and salts) have been adjusted to favour the formation of Citric acid. 4. They can easily be cultivated.

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INDUSTRIAL PROCESS
There are various fermentation processes used in manufacture of citric acid.
A. Surface culture process B. Submerged culture process A.SURFACE CULTURE PROCESS

1. Preparation of inoculum
The spores of Aspergillus niger strain required to inoculate shallow pans are produced by growing the fungus from a stock culture on a suitable solid sporulation medium at 25 C for 4 to 14 days. In the composition formula of inoculum medium, the presence of trace amounts of manganese salts, unless balanced against proper amounts of zinc or iron salts, may lower yields of citric acid in the actual fermentation receiving these spores. Suspension of spores is obtained by suspending the grown spores in a suitable diluent, such as water containing a wetting agent. The spores of inoculum are added to the production medium so as to keep them floating on the surface, since this is a surfaceculture process. This may be accomplished by a suitable special inoculating device.

2. Source of carbon

Sucrose, according to laboratory studies, is the best source of carbon among various tested organic substances, particularly sugars, in producing high yields of Citric acid.

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 It was also reported that sucrose concentration more than 15% should not be used, since excess amount of sugar remained unconverted. When part of sucrose was substituted by fructose or glucose, it resulted in lower yield. Beet molasses is extensively used as a carbon substrate in the fungal production of Citric acid on a commercial basis.

3. Inorganic salts

Apart from the carbon, hydrogen and oxygen supplied by the added carbohydrate, the trace metals, namely nitrogen, potassium, phosphorus, sulphur, and magnesium are needed in the fermentation medium used for Citric acid production. According to Currie (1917), the fermentation medium with the following chemical composition is the most favorable medium in the manufacture of Citric acid: No. 1 2 3 4 5

Component
Sucrose NH4NO3 MgSo4.7H2O KH2PO4 HCl to pH

Gms./Liter
125-150 2.0-2.5 0.20-0.25 0.75-1.0 3.4-3.5

Doelger and Prescott reported the following medium to be most satisfactory No. 1 2 3 4 5

Component
Sucrose NH4NO3 MgSo4.7H2O KH2PO4 pH

Gms./Liter
140 2.23 0.23 1.0 2.20 to 1.60

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 Many scientists have studied the effect various metals on the production of citric acid. Therefore, the proper amount of various metals should be added to the medium.

4. pH

pH should be adjusted to 3.4 to 3.5 with HCl Generally, calcium carbonate is not added to the production medium to bring about the neutralization of Citric acid produced because its presence favours contamination. Moreover, its absence favours higher yields of Citric acid and a shorter fermentation time.

Low pH values in Aspergillus niger-Citric acid fermentation are desirable for the following reasons: (a) Sterilization of medium is more readily effected. (b) Formation of citric acid is favoured. (c) Formation of oxalic acid is suppressed. (d) The danger of contamination is minimized.

5. Ratio of surface area to volume

This is an important bioparameter in this process. Rate of bioconversion of sugar to acid depends on this ratio. The lower the ratio of the volume to surface area, the higher is the yields of Citric acid Usually, the production medium is placed in Shallow Pan in such a way that a shallow layer of medium with a depth of 1.0 to 2.5 cm is formed.

6. Aeration

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It is necessary to the surface of the seeded medium. From laboratory studies it has been found that, the rate of aeration, either higher or lower than the optimum rate gives lower yields of Citric acid.

7. Time

With the shallow-pan method (i.e. Surface Culture Process) the period required for the progress and termination of the fermentation process in the range of 7 to 10 days. It has been reported that yield of Citric acid may be in the range of 60 to 80 gm. (or higher) of anhydrous Citric acid per 100 gm. of incorporated sugar. Wells, Moyer and May obtained a maximum yield of 90.7 per cent of Citric acid from glucose on the basis of sugar consumed. The recovery operation for citric acid from harvested fermentation broth is difficult for the following important reasons. 1. The presence of Unconverted sugar 2. The presence of other acid fermentation products(e.g. oxalic acid) 3. The presence of Trace elements as impurities. Usually, recovery of Citric acid is practiced as under: 1. The fermentation liquor is drained off to separate the mycelium. In addition, any intracellular Citric acid present in the mycelium is obtained by pressing the mycelial mat. 2. The recovered fermentation liquor, as in step (1), is treated with milk of lime forming the precipitate of calcium citrate. 3 The precipitate of calcium citrate obtained from hot neutral aqueous solution, as in step (2), is filtered & washed. 4 Calcium citrate is treated with an equivalent of sulphuric acid to liberate Citric acid, leaving behind the precipitate to calcium sulphate. 5 The precipitated solution, as in step (4), is filtered & washed. 6 An impure solution of Citric acid is subjected to decolorization by the activated carbon. Then, it is also demineralized. 7 Finally, the pure Citric acid solution is evaporated and is Crystallized from the solution.

8. Yields

9. Recovery of Citric acid

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 The alternative method is the counter current extraction method. Organic solvents (100 parts of tri-n-butyl phosphate & 5 to 30 parts of n-butyl acetate) are employed for the purpose of extracting Citric acid. Then Citric acid is extracted from the organic phase with lime water. Lastly, the the precipitate of calcium citrate is trated with sulphuric acid as previously discussed.

B. SUBMERGED CULTURE PROCESS Aspergillus niger

In this process the fungus is grown dispersed through a liquid production medium. o Usually fermentation is carried out in fermentation vessel consisting a sterilizable tank of the capacity of thousands of gallons. The fermentation tank is equipped with a mechanical agitator and a sparger. Relation between phosphate and citric acid production was established in submerged production. o Relation between Aspergillus morphology and citric acid production was also established. o Specific quantities of Ferrocynide lead to formation of an optimum mycelial form. o Production was preferred from the Beet molasses. Production of inoculam. o Spores are produced under controlled aseptic condition. o Spores are harvested in the inoculation medium which is designed to develop cellular mass and to control morphology rather than to produce citric acid.
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o Then transferred to production medium under aseptic condition. o In contrast to inoculation medium, production medium contains constituents so as to favour citric acid production. o High aeration rates are necessary. o Samples are regularly withdrawn aseptically to determine the citric acid and sugar content. o In addition to this, pH, dissolved oxygen, and solid content are determined.

YEAST
The belief that industrial production of Citric acid could be possible only by Aspergillus niger, was disproved in about 1969 to 1970. A challenging patent was issued in 1970 by demonstrating the production of citric acid by the species of yeast, Candida lipolytica. Fermentation time is shorter than fermentation with fumgi. Strain improvement yields 110 gms/ltr. Hydrocarbon can be used as raw material.

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Uses of citric acid:

Food additive Water softening Others o Citric acid is Citric acid is used in biotechnology and the pharmaceutical industry to passivate high-purity process piping (in lieu of using nitric acid). o commonly employed in wine production as a substitute or improver where fruits containing little or no natural acidity are used. It is mostly used for inexpensive wines due to its low cost of production. o Citric acid can be used in shampoo to wash out wax and coloring from the hair. It is notably used in the product "Sun-in" for bleaching, but is generally not recommended due to the amount of damage it causes. o Citric acid is also used as a stop bath as part of the process for developing photographic film. The developer is normally alkaline, so a mild acid will neutralize it, increasing the effectiveness of the stop bath when compared to plain water. o Citric acid is used as one of the active ingredients in the production of anti-viral tissues. o Citric acid can be used in food coloring to balance the pH level of the normally basic dye.
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Citric acid may be used as the main ripening agent in the first steps of making mozzarella cheese

o Citric acid is used as a good alternative to nitric acid in the process of stainless steel o Citric acid can be used as a delay to prompt natural cement. It can delay the very rapid setting time substantially. o Citric acid is one of several acids that is used by home brewers to modify brewing water for making beer.

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