Citric Acid Production
- Citric acid is the most important commercial product which is found in almost all plant and animal tissues.
- The molecular formula of citric acid is C6H8O7, 2-hydroxy-1,2,3-propane tricarboxylic acid.
- It is widely used organic acid in the field of food (60%) and pharmaceuticals (10%).
- In 1784 W.SCHEELE’s first time isolated from lemon juice as calcium citrate, which treated with sulphuric acid gave citric acid in the liquid phase.
Microorganism used for Citric Acid Production
- A large number of micro-organisms have been employed to produce citric acid. These include bacteria, fungi, and yeasts. But A.Niger and saccharomycopsis sp. are employed for commercial production because it has several advantages.
Advantages of using this micro-organism are:
- Its ease of handling.
- Its ability to ferment a variety of cheap raw materials.
- It provides high yields.
MICRO-ORGANISMS USED FOR CITRIC ACIC PRODUCTION
- Candida tropicalis
- Hansenula anamola
- Yarrowia lipolytica
- Bacillus licheniformis
- Arthrobacter paraffinens
- Corynebacterium species
- Aspergillus nagger
- A. carbonarius
Raw materials that are used for citric acid production are divided into two groups:
- Substances with low ash content
e.g. cane or beet sugar, dextrose syrups, and crystallized dextrose
- Materials which have high ash content.
e.g. cane and beet molasses, crude unfiltered starch hydro-lysates.
CITRIC ACID CYCLE
Steps of Citric Acid Cycle
- The citric acid cycle is also called the tricarboxylic acid cycle (TCA).
The steps include in the citric acid cycle are:
- Formation of Citrate
- Formation of Isocitrate via cis-Aconitate
- Oxidation of Isocitrate to a-Ketoglutarate and CO2
- Oxidation of a-Ketoglutarate to Succinyl-CoA and CO2
- Oxidation of Succinate to Fumarate
- Hydration of Fumarate to Malate
- Oxidation of Malate to Oxaloacetate
Accumulation of citric acid
- Giving rise such a mutant organism that can carry an incomplete citric acid cycle for its accumulation
By inhibiting enzymes:
- By altering environmental conditions ( pH, Temperature)
- Treat medium with ferrocyanide or ion exchange versions so that the enzymes involved in the TCA cycle inhibit except citrate synthase.
Citric acid production techniques
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 heated at T 110c for 15 to 45 min.
- Potassium hexacyanoferrate is added to the hot substrate, to precipitate or complex trace metals (Fe, Mn, Zn) and to act in excess as a metabolic inhibitor restricting growth and promoting acid production.
- Inoculation is performed in two ways, as a suspension of conidia added to the cooling medium, or as a dry conidium mixed with sterile air and spread as an aerosol over the trays.
- The temperature is kept constant at 30 degrees during the fermentation by means of air current
- Within 24 hours after inoculation, the germinating spores start forming a 2-3 cm cover blanket of mycelium floating on the surface of the substrate. As a result of the uptake of ammonium ions, the pH of the substrate falls to 2.0.
- The fully developed mycelium floats as a thick white layer on the nutrient solution. The fermentation process stop after 8-14 days.
- Recovery of mycelium to extract citric acid.
- These all steps followed during the surface fermentation to produce citric acid
- The solid substrate is soaked with water up to 65-70% of water content. After the removal of excess water, the mass undergoes a steaming process.
- Sterile starch paste is 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 T 28-30C.growth can be accelerated by adding Alpha-amylase. although the fungus can hydrolyze starch with its own alpha-amylase. During the citric acid production pH dropped 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. In other cases, mechanical passes are also used to obtain more citric acid from the cells.
- These all steps followed during the solid-state fermentation to produce citric acid
- Beet molasses substrate (12-15%, reducing sugar content) nutritive salts, such as ammonium nitrate or potassium dihydrogen phosphate are added, pH of the substrate is maintained at 5.5 to 5.9.
- The process can usually run in one or two stages, using hydrophilic spores suspensions or germinated conidia from the propagator stage.
- The development of the hyphae and the aggregation generally requires a period from 9 to 25 hours at T 32c
- Mycelia aggregation and spherical pellets, the productive form can be detected after 24 hours of inoculation.
- The change of pH in this phase is 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 purified from mycelium.
- These all steps followed during the submerged fermentation to produce citric acid
- The biomass is separated by filtration. Then the liquid broth is transferred to the recovery process.
Purification is a simple way of getting pure citric acid followed by two simple techniques.
- 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. 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.
- After purification, It can be produced in two forms
- Contains one water molecule for every citric acid molecule
- Require repeated crystallization until the water content is approx. 7.5- 8.
- Processed to remove all water from the end product
- Prepared by dehydrating the monohydrate citric acid product at a temperature above 36.6°C.
FACTORS AFFECTING CITRIC ACID PRODUCTION
- Carbon source
- Nitrogen Source
- Phosphorus Source
- Trace Element
- the pH of fermentation medium
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.