Citric Acid Fermentation

 Citric acid -widely used organic acid in the field of food (60%) and pharmaceuticals (10%). 

It plays a pivotal role in food and beverage industries and pharmaceutical, chemical cosmetic, and other industries for applications such as acidulation, antioxidant, flavor enhancement, preservation, and plasticization and as a synergistic agent.

In 1784, W. Scheele, first time isolated from lemon juice as calcium citrate, which when treated with sulphuric acid gave citric acid in the liquid phase.

 Citric acid fermentation is one of the primitive fermentations but still very much in use.

Microorganisms used for Citric Acid Production

A large number of micro-organisms employed to produce citric acid- bacteria, fungi, and yeasts.

Bacteria:

·         Bacillus licheniformis

·         Arthrobacter species

·         Corynebacterium species

Fungi:

·         Aspergillus niger

·         Aspergillus awamori

·         Aspergillus carbonarius

  Yeasts

·         Candida tropicalis

·         Hansenula anomola

·         Yarrowia lipolytica

Aspergillus niger is most commonly used for citric acid production. Among the yeast species, Yarrowia lipolytica is known as a potential producer of citric acid

 

Aspergillus niger employed for commercial production (first by James Curie)

 

Ø  ease of handling

Ø  ability to ferment a variety of cheap raw materials

Ø  high yields

 Raw materials

·          cane or beet sugar, dextrose syrups, crystallized dextrose, cane and beet molasses, crude starch hydrolysates.

Metabolism

 

· The citric acid cycle production- tricarboxylic acid cycle (TCA).

       

Accumulation of citric acid

In media for commercial production citric acid can be accumulated by, 

1) By using mutants:

·         Use a mutant organism with an incomplete citric acid cycle for its accumulation

2) By inhibiting enzymes in TCA cycle:

·         Done by altering environmental conditions ( pH, Temperature)

·         Treating the medium with ferrocyanide or ion exchange versions so that the enzymes involved in the TCA cycle are inhibited except citrate synthase.

 

The industrial citric acid production can be carried in three different ways:

·         Surface fermentation

·         Submerged fermentation

·         Solid-state fermentation

 

SURFACE FERMENTATION PROCESS

·         Molasses substrate (15-20% of sucrose, added nutrients) acidified with, phosphoric acid to a pH 6.0-6.5 and sterilized.

·         Potassium hexacyanoferrate is added to the hot substrate, to precipitate or complex trace metals (Fe, Mn, Zn) and in excess, it acts  as a metabolic inhibitor restricting growth and promoting acid production.

·         Inoculation - suspension of Aspergillus nigerconidia added to the cooling medium, or as a dry conidium spread as an aerosol over the trays.

·         The temperature is kept constant at 30 ^oC during the fermentation by means of air current

·         Within 24 hours after inoculation, the germinating spores  form a 2-3 cm cover blanket of mycelium floating on the surface of the substrate. The fully developed mycelium floats as a thick white layer on the nutrient solution

·         The fermentation process continues till 8-14 days. The pH of the substrate falls to 2.0.

·         Recovery of mycelium carried out to extract citric acid.

 

 SOLID STATE FERMENTATION

·         The solid substrate is soaked with water up to 65-70% of moisture content and sterilized

·         Inoculated by spreading Aspergillus niger conidia in the form of an aerosol or as a liquid conidia suspension on the substrate surface

·         The pH of the substrate is about 5-5.5 and incubation at 28-30^oC.

·         Growth can be accelerated by adding α-amylase (the fungus can also hydrolyze starch with its own α -amylase).

·         During the citric acid production pH drop to values below 2.

·         The solid-state surface process takes 5 to 8 days at the end of which the entire is extracted with hot water.

 

SUBMERGED FERMENTATION

·         Molasses/sucrose substrate (12-15%, reducing sugar content) supplemented with  ammonium nitrate or potassium dihydrogen phosphate are added

·         pH of the substrate is maintained at 5.5 to 5.9.

·         Inoculated with spore suspensions or germinated conidia.

·         pH change from 5.5 to 3.5 for beet molasses substrate and 2.2 for the sucrose substrate.

·         Fermentation lasts up to 6-8 days and later citric acid is recovered

 

Recovery

The biomass is separated by filtration. Then the liquid broth is transferred to the recovery process.

·         Precipitation is the first step-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.

·         It is then treated with sulphuric acid forming calcium sulfate, which is filtered off.

·         Treatment with activated carbon and passing through cation and anion exchangers for further purification

·         After purification, it can be produced as Monohydrate or Anhydrous.

Mono hydrate- Contains one water molecule for every citric acid molecule. Require repeated crystallization until the water content is approx. 7.5- 8.

Anhydrous- Processed to remove all water from the end product. Prepared by dehydrating the monohydrate citric acid product at a temperature above 36.6°C.

 Critical factors in fermentation

·         Carbon source

·         Nitrogen Source

·         Phosphorus Source

·         Trace Elements- copper, manganese, magnesium, iron, zinc, molybdenum required n ppm range

·         the pH of fermentation medium-starts from 5, falls to ~3, maintained at low pH, for decreased risk of contamination and better yield

·         Aeration

 Uses of Citric Acid

·         It is used in the detergent industry as a phosphate substitute.

·         Used as a preservative and flavoring agent.

·         Emulsifying agent in ice cream.

·         Also used as an antioxidant.