Organic Acid Fermentation

Citric and Fumaric Acid

CITRIC ACID

• Citric acid is a weak organic acid.• It is a natural • In biochemistry, the conjugate base of citric

acid, citrate, is important as an intermediate in the citric acid cycle.

• 1.5 million tonnes of citric acid is produced every year by fermentation.

CITRIC ACID

• Citric acid accumulates during the controlled fermentative growth of particular species of Penicillin and Aspergilius.

• A.niger, A.clavatus,P. leutum, P. citrinum, M. piriformis.

• But only A. niger is used for commercial production: can accumulate high amounts of citric acid while growing on a carbohydrate medium.

Parameters affecting bioprocess

Fungi: important to choose a stable fungal strain for producing .

• A. niger- commercially used strain.1.efficient strains( high yield)2.Posses uniform biochemical properties3.Produce small amounts of oxalic acid.4.Easily cultivable

A. niger

• Great variation in terms of morphology and physiology in strains.

• Carefully selected for positive characteristics:

1. Citric Acid yield2. Amount of sporulation3. Strain stability.

• Selection of favourable fermentation medium- most critical factor in obtaining high-level accumulation of citric acid by A. niger.

• Nutrient deficiency in terms of trace metals or phosphate is required.

• This may vary with the strain used.• Medium should ne slightly deficient in phosphates or

one or more of the metals Mn, Fe,Zn and Cu.

Surface-culture process

• Culture is inoculated across the surface of the production media.

• Stationary Fermentation• Old practice but still practiced.

Parameters affecting bioprocess

Inoculation Preparation: spores of A. niger inoculated in shallow pans at 25oC for 4-14 days.

Source of Carbon: Sucrose is considered as the best source for citric acid production

• Sucrose> 15% should not be used as excess remains unconverted.

Parameters affecting bioprocess

• Substitution with fructose or glucose: yield decreased

• Beet Molasses: used extensively-requires pretreatment.

• Ferrocyanide or Ferricyanide is added to molasses before sterilization

• The metals of beet molasses: iron exchange resins

Parameters affecting bioprocess

Inorganic Salts: Nitrogen, Potassium, Sulphur

and Magnesium salts are needed for the fermentation.

• Essential to add salts in minimum quantity- higher conc. Affects fermentation adversely.

Parameters affecting bioprocess

• Eg: more than 2.5gms of NH4No3 – decreases the yield of citric acid.

• The type and quantity of ions required also depends on the fungal strain being used.

• eg: A.niger 62: requires 0.1mg/l of iron while A.niger 59 requires 10mg/l of iron for optimum production.

Parameters affecting bioprocess pH: adjusted to 3.4-3.5 using HCl• certain strains: high yield at pH1.6-2.2

• Low pH values in A.niger citric acid fermentation have following advantages:

1. Formation of Citric acid favored2. Formation of oxalic acid suppressed

3. Chances of contamination reduces

Parameters affecting bioprocess

• Aeration: proper aeration is required to maintain continuous citric acid production.

• Time: 7-10 days• Yields: 60-80 gms/ 100 gms of sugar

incorporated.

RECOVERY

• Mycelium filtered• Filtrate treated with Calcium hydroxide producing

calcium citrate• Calcium citrate filtered and washed• Treated with equivalent amount of sulphuric acid

releasing citric acid- leaving precipitates of calcium sulphate.

• Decolorize using activated charcoal• Evaporation and crystallization

Submerged Culture

• In this process the fungus is grown dispersed in liquid medium.

• Phosphate limitation plays a significant role in the production of citric acid by A.niger.

• Fungal spores are used to seed inoculum media designed- to develop cell mass not citric acid production.

• The fungus is then transferred to media designed citric acid production.

• This method also utilizes pre-treated beetroot molasses as carbon sources.

Submerged Culture using Yeast

• Candida guilliermondii grown submerged on media containing glucose or black strap molasses- used for citric acid production.

• Duration of fermentation lesser than A. niger with a yield of 110g/l

• Candida lipolytica – using paraffin as carbon source- citric acid production.

APPLICATIONS

• Used as an acidulant in food and pharmaceutical industry

• As a chelating agent• In the production of carbonated beverages

FUMARIC ACID• Manufacture of resins.

• Can also be converted Maleic acid by heating in acid solution: used in manufacture of alkyd resins, unsaturated polyester coating compounds and plasticizers.

• Most of the microbes produce fumaric acid as an intermediate of TCA cycle.

• But only Mucorales and Rhizopus accumulate fumaric acid.

• Rhizopus nigricans used for commercial production.

• Strain selection is very important because:1.Some strains of Rhizopus don’t produce

fumaric acid.2.While other produce mixture of fumaric and

lactic acid.3.Also certain species of Rhizopus inculding R.

nigricans lack invertase activity- cannot utilize sucrose directly- molasses can be used as carbon source.

Fermentation Process• Highly aerobic- deficiency of oxygen leads to accumulation

of ethanol.• Temperature: 28 to 30OC• Surface or Submerged fermentation.• Medium contains: Hexoses, salts and ammonia or urea.• Ratio of carbon to nitrogen compounds in the medium is

important: controls the yields.• Conc. Of trace elements is critical: Zn should be limiting-

excess leads to formation of other organic acids.

RECOVERY• Fumaric acid is poorly soluble in water.• Saturation occurs at approx. 0.7g/100ml.• Fumaric acid crystallizes in medium to form a gel- thickens

the media and coating of mycelium:: slows or stops fermentation.

• R. nigricans: cannon withstand high acidity itself.• To neutralize and prevent crystallization- sodium or

potassium carbonate is added- to maintain pH between 5-6.• Calcium carbonate cant be used- calcium fumarate also

crystallizes forming gel.• Fumaric acid is harvested by acidifying so that the acid

crystalizes.