BIOTECHNOLOGY OF CITRIC ACID PRODUCTION

CONCLUDED BY- Musharraf Ali

M.Tech(BCE)-IIT(BHU)

1- INTRODUCTION-

• Citric acid is ( 2-hydroxy-1,2,3 propane tricarboxylic acid)-

• Molecular weight: 192g.

• It is naturally found in fruits such as lemon, orange, pineapple, plum, and pear

• Citric acid was first commercially obtained from lemon juice, crystallized in 1784 by Carl Wilhelm Scheele, a Swedish chemist.

• In 1893 Wehmer investigated citric acid production from sucrose with strains of Mucor and Penicillium.

• In 1917 Currie described the first production of citric acid with Aspergillus niger.

• In 1952, America Miles Company (Miles Laboratories, Inc., Elkhart, IN) used the submerged fermentation process in a pilot program on a plant scale to produce citric acid.

• After 1952, many countries used glucose or beet and cane molasses as substrates to produce citric acid.

. Two methods are commonly used for the production

of citric acid by A. niger: surface fermentation and submerged culture fermentation.

• The current annual world production of citric acid among 35 countries is ~600,000 tons.

• USA is still a net importer of citric acid, while Western Europe is a net exporter.

• Citric acid is used in the food and beverage industries (70%), in pharmaceuticals (12%), and in other industries and applications (18%).

2- MICROORGANISMS USED FOR THE PRODUCTION OF CITRIC ACID:-

3- BIOSYNTHESIS OF CITRIC ACID

4- FACTORS AFFECTING CITRIC ACID PRODUCTION

1- Nutrient- Aspergillus niger grows well in media containing carbohydrates(sucrose,

glucose, fructose, maltose, mannose, and starch), 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 concentrations were observed in cultures grown at high initial sugar concentrations (15−20% w/v).Further increase of sugar concentration (i.e., 250 g/L)) resulted in a decrease of acid concentration by 15%. The decreased concentration of acid encountered with the highest concentration treatment was probably due to osmotic effects.

In addition to carbohydrates, nitrogen and the phosphate concentrations have a strong influence on citric acid production. Generally, a nitrogen or phosphate concentration less than 0.2% (w/v) in the medium appears to be adequate.

2- Inhibitors and Stimulants

The most important stimulants used forimproving citric acid yield by A. niger are methanol & ethanol.These chemicals have been found to retard growth, delay sporulation, & increase (by 30–50%) 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.

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

3- Inoculum:

To prepare inoculum, A. niger is grown in standard media for molds. It is usually cultivated on potato dextrose agar (PDA) slants or in petri dishes at 28–30°C for 3–5 days. The spores obtained are suspended in sterile water containing 0.1% Tween 80.

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).

When spores are used as inoculum, the substrate is inoculated with 0.5–1.0% (v/v) of the inoculum to give a final concentration of ~106 spores/mL.

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.

4 Fermentation Time

The optimum time for the maximum production of citric acid depends on the strain used, the chemical composition of the medium, the fermentation system, and generally, the conditions under which fermentation takes place.

In the surface culture, fermentation time is usually completed in 10–20 days, while in the submerged culture incubation time is much shorter (5–10 days). 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, and the particle size of the medium.

5- Temperature

Asperillus niger and other fungi used in the production of citric acid from synthetic media or molasses in submerged fermentation have an optimum temperature between 25 & 30'C.

Also, no significant differences were noted in biomass yield, specific biomass production rate, and specific sugar uptake rate among cultures grown at 25 and 30°C. Increasing the fermentation temperature from 25 to 40°C significantly affected specific citric acid production rate (0.157 to 0.111 g citric acid/g biomass dry weight/d) and biomass dry weight (20.5 to 30g/L).

Perlman and Sih reported that a two stage process, where A. niger was incubated at 28–30°C for 2–3 days and then for a week at 20°C, resulted in higher yields than when the temperature was maintained at 28–30°C.

6- 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, while 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. 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.

7- Aeration and Agitation

Aspergillus niger is an aerobic microorganism and therefore requires oxygen. Aerating and agitating the fermentation broth normally satisfies the oxygen demand of a fermentation

process.Agitation is important for adequate mixing, mass transfer, and heat transfer. It not only assists mass transfer between the different phases present in the culture, but also

maintains homogeneous chemical and physical conditions in the culture by continuous mixing.

The concentration of dissolved oxygen increased with the increase of speed of agitation. In the cultures agitated at 300, 400, 500, and 600 rpm the concentration of dissolved oxygen from the second to the 12th day remained at ~22%, 30%, 40%, and 50% of the initial saturation level, respectively. 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.

5- SUBSTRATES USED FOR THE PRODUCTION OF CITRIC ACID-

6-Simplified Flow chart for citric acid production

7- RECOVERY OF CITRIC ACID

• The fermentation broth obtained either from surface or submerged fermentation is filtered to remove mycelia or cells and other suspended impurities.

• Citric acid is precipitated from the filtrate as calcium citrate by the addition of lime slurry at 95°C for 1 h or 50°C for 20min.

• The precipitate is washed to remove soluble impurities and treated with sulfuric acid to precipitate calcium sulfate and regenerate the citric acid.

• The solution is then filtered to remove CaSO4. The liquid is decolorized with charcoal and ion exchangers.

• The purified solution is concentrated by evaporation and run into low temperature crystallizers.

• Finally, the crystals are removed by centrifugation. Citric acid is marketed as an anhydrous crystalline chemical, as a monohydrate or as a crystalline sodium salt.