CITRIC ACID PRODUCTION

Presented by:

N.Nachal (18FET210)

Nishank Waghmare (18FET211)

Introduction

• Citric acid (C6H8O7) is a weak organic tricarboxylic acid found in

citrus fruits

• Citrus fruits (lemons, oranges, tomatoes, beets etc.) are those fruits

which contains sufficient amount citric acid and they are classified

as acid fruits

• Citric acid is produced by three method fermentation, chemical

synthesis and extraction from citrus fruits

History of Citric Acid Production

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

Zahorsky in 1913 patented a new strain -Aspergillus niger

Currie 1917 opened the way for industrial citric acid fermentation using a new micro-organism

In 1960’s practice of submerged fermentation gained popularity.

Strains For Citric Acid Production

• Many strains excrete traces of citric acid as a metabolite of primary

metabolism

• Various strains of genera fungi, yeast and bacteria were reported such as:

Penicillium luterum, Penicillium purpurogenum, Penicillium restrictum,

Penicillium janthinellum, Penicillium citrinum, Paecilomyces divaricatum,

Mucor piriformis, Trichoderma viride, Sacharomycopsis lipolitica,

Arthrobacter paraffineus, Corynebacterium sp. et al

• Only mutants of Aspergillus (Aspergillus niger) and yeasts genus Candida

have almost exclusively been utilized

Biochemistry

• Citric acid is excreted from the cells in response to

unfavourable intracellular condition caused by increased levels

of tricarboxylic acids (TCA)

• A crucial prerequisite for overflow of citric acid from A. niger

cells is therefore increased level of Krebs cycle intermediates

caused by anaplerotic reactions

Influence of the Trace Metals

• In citric acid technology absence of iron and manganese in the fermentation

substrate plays the most crucial role

• Iron ions in higher concentration than 1.5 mg/l strongly affect cellular

morphology, by promoting unproductive filamentous mycelial growth form

• 1 µgl of manganese could completely ruined the production yield of and

caused organism’s morphology to switch from microbial pellets, known as

citric acid productive form, to unproductive filamentous growth.

Substrates

• The basic substrate for citric acid fermentation in plants using the surface

method of fermentation is beet or cane molasses

• Plants using submerged fermentation can use not only beet or cane molasses,

but a substrate of higher purity such as hydrolysed starch, technical and pure

glucose, refined or raw sugar, purified and condensed beet or cane juice

• Substrates commonly used- Beet molasses, Cane molasses, Sucrose , Syrups,

Starch, Hydrol, Alkanes, Oils and fats

Production Processes

• Surface or submerged fermentation technique dominated over traditional

method of preparing citric acid by extraction from various juices

• Promising results were obtained in fed-batch process and by continuous

fermentation

• Citric acid fermentation using immobilized A. niger cells on various kinds

of carriers as glass, polyurethane foams, entrapment in calcium alginate

beds, polyacrylamide gels, agar, agarose , cellulose carriers, metal screens

and polyester felts

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 temperature 110 ºC 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 cooled medium, or as a dry conidia mixed with sterile air and 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 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 stops after 8 - 14 days.

Recovery of mycelium to extract citric acid

Solid State Fermentation

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 aerosol or as a liquid conidia suspension on the substrate surface

The pH of the substrate is about 5 to 5.5, and incubation temperature 28 to 30 ºC. Growth can be accelerated by adding α-amylase, although the fungus can hydrolyze starch with its own α-

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. On other cases, mechanical passes are also used to obtain more citric acid from the

cells

Submerged Fermentation

Beet molasses substrate (12 - 15 %. reducing sugar content ), Nutritive salts, such as ammonium nitrate or potassium dihydrogen phosphate are added. pH of 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 . Amounts of spores are 5 to 25 x 106 per litre of

substrate

The development of the hyphae and the aggregation generally requires a period from 9 to 25 hours at temperature of 32 ºC

Mycelia aggregation and spherical pellets, the productive form can be detected after 24 hrs of inoculation.

The change of pH in this phase is from 5.5 to 3.5, for beet molasses substrate, and to 2.2 for the sucrose substrate

Fermentation last upto 6-8 days and later citric acid is purified from mycelium

Factors Affecting Citric Acid Production

Factor affecting citric acid fermentation are the type

and

i. The concentration of carbon source,

ii. Nitrogen and phosphate limitation,

iii. pH (pH>5)

iv. Aeration

v. Trace Elements

vi Lower Alcohols

Product Recovery

• First step - Separation of biomass from fermentation broth

• separated mycelia retain about 15 % of the citric acid formed during fermentation

Surface Process

Fermentation fluid drain

Hot water introduction to wash out the remaining citric acid from the mycelial mats

Filtration cake (not more than 0.2 per cent of citric acid), is dried to yield a protein-rich feed

Submerged process

• Heating (70 ºC) for 15 min – protein coagulation

• Oxalic acid removal by adding calcium hydroxide (2.7-2.9 pH,70-75°C) Calcium

oxalate precipitation follwed by centrifugation

Recovery techniques

1. Precipitaion

• Precipitation of the insoluble tri-calcium citrate by the addition of an equivalent

amount of lime to the citric acid solution

• To obtain large crystals of high purity, milk of lime containing calcium oxide (180-

250 kg/m3) is added gradually at a temperature of 90°C and pH – 7

• The minimum loss of citric acid due to solubility of calcium citrate is 4-5 %

• Calcium citrate is then filtered off and subsequently treated with concentrated sulphuric

acid (60-70 per cent) to obtain citric acid

• The filtrate (25-30% citric acid) is treated with activated carbon to remove residual

impurities or may be purified in ion-exchange columns

• The purified solution is then concentrated in vacuum evaporators at temperature below

40°C (to avoid caramelization) - crystallized

• Drying of citric acid monohydrate – rotary drying equipments, fluidised bed dryers

Disadvantages

• large amount of lime required

• Formation of large amounts of liquid and solid wastes

2. Solvent extraction

process can be applied when the fermented musts contain a low amount of impurities

• Trioctylamine - amine-citric acid complex

• aliphatic alcohols, ketones, ethers

• organophosphorus compounds - tri-n-butylphosphate and alkylsulphoxides

citric acid can then be recovered from the extract either by distilling off the solvent

the aqueous solution purified citric acid is subsequently crystallized by concentration

3. Ion exchange

The efficiency of the ion-exchange separation process may be greatly enhanced by applying a

simulated moving bed counter-current flow system

Disadvantage-

Elution of citric acid from the adsorption bed may require a large amount of desorbent

3. Liquid membranes

Liquid membranes containing mobile carriers consist of an inert, mobile ion-exchange agent

Citric acid separation by liquid membranes, the tertiary amines can also be used

4. Microporous hollow fibres

Permeator consists of two sets of identical hydrophobic microporous hollow fibres

One set carries the feed solution of citric acid and the other the strip solution

The organic liquid membrane is contained in the shell side between these two sets of hollow

fibres

Citric acid recovery of up to 99 %

5. Electrodialysis

Enables separation of salts from a solution and their simultaneous conversion into the

corresponding acids and bases using electrical potential and mono- or bipolar membranes

Integrating bipolar membranes with anionic and cati-onic exchange membranes -

electrodialytic separation of salt ions and their conversion into base and acid

Pretreatment steps :

Filtration of the broth, removal of ionogenic substances (especially Ca++ and Mg++ ions) and

neutralization by means of sodium hydroxide

Electrodialytic step - the sodium citrate solution is converted into base and citric acid, which

is simultaneously concentrated and for the most part purified.

Applications of Citric Acid

• Citric acid is accepted as GRAS (generally recognized as safe)

- approved by the Joint FAO/WHO Expert Committee on Food Additives

• Citric acid monohydrate is widely used as

Preservative

Flavour enhancer

Sequestrant

Emulsifying agent

ph adjustment

Carbonation

Food Industry Uses

Jellies and jams Gelling agent, tartness and flavour

Soft drinks and syrups Acidulant , Natural fruit flavour, tartness

Frozen fruits Inactivation of oxidative enzymes, protects ascorbic acid by inactivating trace metals

Animal fats and oils Shows Synergism with other antioxidants, Sequestrant

Dairy products emulsifier, acidifying agent in many cheese products Antioxidant

Cosmetics pH adjustment, antioxidant, buffering agent

Wines and ciders Prevents turbidity, prevents browning in some white wines adjusts pH, inhibits oxidation.

Fruits and vegetable juices Stabilizer

Pharmaceuticals Antioxidant, anticoagulant, acidulants

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with its dregs by Aspergillus niger in an external-loop airlift bioreactor. Process Biochemistry, 35(3-4),

237-242.

Berovic, M., & Legisa, M. (2007). Citric acid production. Biotechnology annual review, 13, 303-343.

Swain, M. R., Ray, R. C., & Patra, J. K. (2011). Citric acid: microbial production and applications in food

and pharmaceutical industries. Citric Acid: Synthesis, Properties and Applications, Edition, 1, 97-118.

Gupta, S., & Sharma, C. B. (2002). Biochemical studies of citric acid production and accumulation by

Aspergillus niger mutants. World Journal of Microbiology and Biotechnology, 18(5), 379-383.