Citric acid production

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

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

2. Introduction Citric acid (C 6 H 8 O 7 ) 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

4. Strains For Citric Acid Production Many strains excrete traces of citric acid as a metabolite of primary metabolism V arious 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 O nly 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 µ gl 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

10. Solid State Fermentation

11. Submerged Fermentation  

12. Factors Affecting Citric Acid Production Factor affecting citric acid fermentation are the type and i. T he concentration of carbon source, ii. Nitrogen and phosphate limitation, iii. pH (pH>5) iv. Aeration v. Trace Elements vi Lower Alcohols

13. Product Recovery F irst step - Separation of biomass from fermentation broth separated mycelia retain about 15 % of the citric acid formed during fermentation Surface Process F ermentation fluid drain H ot water introduction to wash out the remaining citric acid from the mycelial mats F iltration 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/m 3 ) 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 D rying  of  citric  acid monohydrate – rotary drying equipments , fluidised bed dryers Disadvantages large amount of lime required F ormation 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- E lution 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 C itric acid separation by liquid membranes, the tertiary amines can also be used 4. Microporous hollow fibres   P ermeator 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 C itric acid recovery of up to 99 %   

18. 5. Electrodialysis E nables separation of salts from a solution and their simultaneous conversion into the corresponding acids and bases using electrical potential and mono- or bipolar membranes I ntegrating 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 E lectrodialytic 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 p h adjustment Carbonation

20. Food Industry Uses Jellies and jams Gelling agent, tartness and flavour Soft drinks and syrups Acidulant , N atural 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

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.