citric acid production by microbial fermentation followed by product recovery processes.

1. CITRIC ACID PRODUCTION
Presented by:
N.Nachal (18FET210)
Nishank Waghmare (18FET211)

2. 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

3. 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.

4. 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

5. 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

6. 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.

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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 %

15. • 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

16. 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

17. 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 %

18. 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.

19. 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

20. 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

21. REFERENCES
Yuguo, Z., Zhao, W., & Xiaolong, C. (1999). Citric acid production from the mash of dried sweet potato
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.