Production of Organic Acids - Citric and Fumaric Acids

1. Organic Acid Fermentation
Citric and Fumaric Acid

2. CITRIC ACID
• Citric acid is a weak organic acid.
• It is a natural
• In biochemistry, the conjugate base of citric
acid, citrate, is important as an intermediate
in the citric acid cycle.
• 1.5 million tonnes of citric acid is produced
every year by fermentation.

3. CITRIC ACID
• Citric acid accumulates during the controlled
fermentative growth of particular species of
Penicillin and Aspergilius.
• A.niger, A.clavatus,P. leutum, P. citrinum, M.
piriformis.
• But only A. niger is used for commercial production:
can accumulate high amounts of citric acid while
growing on a carbohydrate medium.

4. Parameters affecting bioprocess
Fungi: important to choose a stable fungal
strain for producing .
• A. niger- commercially used strain.
1.efficient strains( high yield)
2.Posses uniform biochemical properties
3.Produce small amounts of oxalic acid.
4.Easily cultivable

5. A. niger
• Great variation in terms of morphology and
physiology in strains.
• Carefully selected for positive characteristics:
1. Citric Acid yield
2. Amount of sporulation
3. Strain stability.

6. • Selection of favourable fermentation medium- most
critical factor in obtaining high-level accumulation of
citric acid by A. niger.
• Nutrient deficiency in terms of trace metals or
phosphate is required.
• This may vary with the strain used.
• Medium should ne slightly deficient in phosphates or
one or more of the metals Mn, Fe,Zn and Cu.

7. Surface-culture process
• Culture is inoculated across the surface of the
production media.
• Stationary Fermentation
• Old practice but still practiced.

8. Parameters affecting bioprocess
 Inoculation Preparation: spores of A. niger
inoculated in shallow pans at 25o
C for 4-14
days.
 Source of Carbon: Sucrose is considered as
the best source for citric acid production
• Sucrose> 15% should not be used as excess
remains unconverted.

9. Parameters affecting bioprocess
• Substitution with fructose or glucose: yield
decreased
• Beet Molasses: used extensively-requires
pretreatment.
• Ferrocyanide or Ferricyanide is added to
molasses before sterilization
• The metals of beet molasses: iron exchange
resins

10. Parameters affecting bioprocess
 Inorganic Salts: Nitrogen, Potassium, Sulphur
and Magnesium salts are needed for the
fermentation.
• Essential to add salts in minimum quantity-
higher conc. Affects fermentation adversely.

11. Parameters affecting bioprocess
• Eg: more than 2.5gms of NH4No3 – decreases
the yield of citric acid.
• The type and quantity of ions required also
depends on the fungal strain being used.
• eg: A.niger 62: requires 0.1mg/l of iron while
A.niger 59 requires 10mg/l of iron for
optimum production.

12. Parameters affecting bioprocess
 pH: adjusted to 3.4-3.5 using HCl
• certain strains: high yield at pH1.6-2.2
• Low pH values in A.niger citric acid fermentation have
following advantages:
1. Formation of Citric acid favored
2. Formation of oxalic acid suppressed
3. Chances of contamination reduces

13. Parameters affecting bioprocess
• Aeration: proper aeration is required to
maintain continuous citric acid production.
• Time: 7-10 days
• Yields: 60-80 gms/ 100 gms of sugar
incorporated.

14. RECOVERY
• Mycelium filtered
• Filtrate treated with Calcium hydroxide producing
calcium citrate
• Calcium citrate filtered and washed
• Treated with equivalent amount of sulphuric acid
releasing citric acid- leaving precipitates of calcium
sulphate.
• Decolorize using activated charcoal
• Evaporation and crystallization

15. Submerged Culture
• In this process the fungus is grown dispersed in liquid
medium.
• Phosphate limitation plays a significant role in the
production of citric acid by A.niger.
• Fungal spores are used to seed inoculum media designed-
to develop cell mass not citric acid production.
• The fungus is then transferred to media designed citric
acid production.
• This method also utilizes pre-treated beetroot molasses as
carbon sources.

16. Submerged Culture using Yeast
• Candida guilliermondii grown submerged on
media containing glucose or black strap
molasses- used for citric acid production.
• Duration of fermentation lesser than A. niger
with a yield of 110g/l
• Candida lipolytica – using paraffin as carbon
source- citric acid production.

17. APPLICATIONS
• Used as an acidulant in food and
pharmaceutical industry
• As a chelating agent
• In the production of carbonated beverages

18. FUMARIC ACID
• Manufacture of resins.
• Can also be converted Maleic acid by heating in acid
solution: used in manufacture of alkyd resins, unsaturated
polyester coating compounds and plasticizers.
• Most of the microbes produce fumaric acid as an
intermediate of TCA cycle.
• But only Mucorales and Rhizopus accumulate fumaric
acid.
• Rhizopus nigricans used for commercial production.

19. • Strain selection is very important because:
1.Some strains of Rhizopus don’t produce
fumaric acid.
2.While other produce mixture of fumaric and
lactic acid.
3.Also certain species of Rhizopus inculding R.
nigricans lack invertase activity- cannot utilize
sucrose directly- molasses can be used as
carbon source.

20. Fermentation Process
• Highly aerobic- deficiency of oxygen leads to accumulation
of ethanol.
• Temperature: 28 to 30O
C
• Surface or Submerged fermentation.
• Medium contains: Hexoses, salts and ammonia or urea.
• Ratio of carbon to nitrogen compounds in the medium is
important: controls the yields.
• Conc. Of trace elements is critical: Zn should be limiting-
excess leads to formation of other organic acids.

21. RECOVERY
• Fumaric acid is poorly soluble in water.
• Saturation occurs at approx. 0.7g/100ml.
• Fumaric acid crystallizes in medium to form a gel- thickens
the media and coating of mycelium:: slows or stops
fermentation.
• R. nigricans: cannon withstand high acidity itself.
• To neutralize and prevent crystallization- sodium or
potassium carbonate is added- to maintain pH between 5-6.
• Calcium carbonate cant be used- calcium fumarate also
crystallizes forming gel.
• Fumaric acid is harvested by acidifying so that the acid
crystalizes.