ABOUT L GLUTAMIC ACID FERMENTATION

2. Introduction
 The history of the first amino acid production dates back to
1908 when Dr. K. Ikeda, a chemist in Japan, isolated
glutamic acid from kelp, a marine alga, after acid hydrolysis
and fractionation.
 He also discovered that glutamic acid, after neutralization
with caustic soda, developed an entirely new, delicious
taste.
 Glutamic acid is a non-essential amino acid.
 This was the birth of the use of monosodium glutamate
(MSG) as a flavour-enhancing compound.
 Though chemical methods are available, it is manufactured
predominantly by microbial fermentation.
 Chemical synthesis yields a racemic mixture (D and L
forms)

4. Why Produced on a Large Scale?
 Food Production:
 As flavour enhancer, to improve flavour.
 As nutritional supplement.
 Beverage
 As flavour enhancer: in soft drink and wine.
 Cosmetics
 As Hair restorer: in treatment of Hair Loss.
 As Wrinkle: in preventing aging.
 Agriculture/Animal Feed
 As nutritional supplement: in feed additive to enhance nutrition.
 Other Industries
 As intermediate: in manufacturing of various organic chemicals.
 Glutamate is medically used as a neurotransmitter.

5. Production strains
 Many microbes are capable to synthesize glutamic acid
including bacteria, yeast, actinomycetes etc.
 Corynebacterium glutamicum (Micrococcus glutamicum) was
first isolated in in 1957 and used by Kyowa Hakko because of
high extraction of glutamic acid.
 It is gram positive, non-sporulating, non-motile bacterium,
requires biotin as a growth factor (imp for α-ketoglutarate
dehydrogenase activity) .
 Mutants are found to over produce glutamic acid.
 Lysozyme sensitive mutants are able to convert 40% carbon
source into glutamic acid in presence of 100µg/l biotin.
 Other genera are Brevibacterium, Microbacterium and
Arthrobacter.

6. Biosynthesis
 The glucose breaks down into C3 and C2 fragments by
organism through EMP and PP pathway; enters in to TCA
cycle.
 The main precursor for glutamic acid is α-ketoglutarate. It
forms in TCA cycle via citrate and isocitrate.
 Reductive amination of α-ketoglutarate with NH4+ ions
leads to formation of glutamic acid with the help of
reduced NADP dependent α-ketoglutarate
dehydrogenase.
 NADPH2 is then generated by reductive amination of α-
ketoglutarate .

9.  The stoichiometry of glutamic acid formation
from glucose and acetate are as follows.
 C6H12O6 + NH3 + 1.5O2 C5H9O4N + CO2 +
3H2O2
 3C2H4O2 + NH3 + 1.5O2  C5H9O4N + CO2 +
3H2O2
 One mole of glutamic acid is produced from 1
mole of glucose or 3 moles of acetate.
 Actual conversion rate is 50-70%

10. Effect of permeability on glutamic acid
production
 Production and excretion of glutamic acid depends
on cell permeability. Increase in permeability can
be attained in many ways through:
Biotin deficiency
Oleic acid deficiency in oleic acid auxotrophs
The addition of saturated fatty acid derivatives
The addition of penicillin
Glycerol deficiency in glycerol auxotrophs

11.  All glutamic acid producers require biotin as a
growth factor , an essential coenzyme in fatty acid
synthesis.
 Biotin conc. greater than 5µg/ml increase oleic
acid synthesis result into high phospholipid
content in cm, leads to great decrease in glutamic
excretion.
 Glutamic acid accumulates in cell (25-35 5µg/mg
dry weight) result in to feedback inhibition.
 On the other hand deficiency of biotin causes
membrane damage through reduction in
phospholipid synthesis leads to glutamic acid
excretion.
 To solve the problem oleic acid (Saturated fatty
acid) auxotrophs are generated.

12.  Oleic acid addition repress acetyl CoA carboxykase
(biotin containing enzyme). Here addition of oleic acid
leads to synthesis of cm with lower phospholipid
content.
 Such strains can also excrete glutamic acid in presence of
biotin.
 The addition of penicillin in the growth phase promotes
excretion of glutamic acid in presence of biotin.
 It is added to the fermentation medium containing large
amount of biotin after 8-10 hours of inoculation at conc.
of 5-300 unit/ml.
 Use of penicillin or saturated fatty acids makes it
possible to use cheaper raw materials like sugar cane or
sugar beet molasses, which otherwise cannot be utilized
due to high biotin content.

13. Conditions to manufacture/Factors
affecting fermentation
Carbon sources:
 Wide variety of sources can be used.
Glucose and fructose are frequently used.
As an alternative unrefined sources like
molasses or at a lesser extent starch
hydrolysates are used.
 Europe: beet molasses
 Japan: Acetate
 Process using methanol, ethanol,
acetaldehyde or alkanes have also been
developed, but cost effectiveness largely
depends on price of petroleum.

14. Nitrogen sources:
 In addition to ammonium salts, ammonia can be used.
 Addition of ammonia provides pH control as well.
 In the acidic pH range with excess ammonia glutamine
is produced instead of glutamic acid.
 Most glutamic acid producers possess urease activity
so urea can be used.
Growth factors:
 Biotin (in media with 10% glucose: 5µg/l)
 With low glucose conc., its demand decreases
 For acetate: 0.2-1.0µg/l
 L-cysteine and thiamine may be necessary

15. O2 supply:
 It should neither be too low nor too high.
 Under oxygen deficiency excretion of lactate and
succinate occurs.
 High oxygen in presence of ammonium ion deficiency
causes growth inhibition and production of α-
ketoglutarate.
 In both case glutamic acid production get lowered.
 Optimal yield is obtained at Kd value of 3.5X10-6 mole
O2/atm.min.ml.

16. Production Processes
 The manufacturing process of glutamic
acid by fermentation
 comprises :-
 a. fermentation,
 b. crude isolation,
 c. purification processes.

17.  A typical fermentation from glucose with
Brevibacterium divaricatum runs as follows (1975):
 At the beginning of the fermentation, 0.65ml/l of oleic
acid is added. The pH is set at 8.5 with ammonia and is
automatically maintained at 7.8 during the course of
the fermentation.
 After beginning growth of the culture (14hr), the
temperature is increased from 32-33 ˚C to 38 ˚C.

18.  After metabolism of the glucose down to a level of
0.5-2%, glucose feeding is done until the
fermentation is completed; 160g/L are fade on the
average.
 Aeration is controlled so that the CO2 content in
the exhaust gas does not exceed 4.5 vol%.
 The glutamic acid content is analyzed hourly. As a
rule, the fermentation is stopped after 30-35 hours
with a glutamic acid yield of about 100g/L.
 If molasses from starch sachharification is
substituted for glucose, the glutamic acid yield is
94g/L after 36 hours.

19.  The glutamic acid yields with different carbon sources
are listed in below table.

22. Separation and purification
 After the fermentation process, specific method is require
to separate and purify the amino acid produced from its
contaminant products, which include:
 Centrifugation.
 Filtration.
 Crystallisation.
 Ion exchange.
 Electrodialysis.
 Solvent extraction.
 Decolorisation.
 Evaporation.

23. Separation and purification of
Glutamic acid
 After fermentation, the cells may be filtered using a rotary
vacuum filter.
 The glutamic acid crystal is added to the sodium hydroxide
solution and converted into monosodium glutamate (MSG).
 MSG is more soluble in water, less likely absorb moisture and
has strong umami (MSG taste) taste.
 The MSG is cleaned by using active carbon, which has many
micro holes on their surface.
 The clean MSG solution is concentrated by heating and the
monosodium glutamate crystal is formed.
 The crystal produce are dried with a hot air in a closed system.
 Then, the crystal is packed in the packaging and ready to be
sold.