fermentation technolgy

1. GLUTAMIC ACID FERMENTATION

2. INTRODUCTION
• Glutamic acid was discovered by Ritthausen in 1866.
• In 1908 professor Ikeda found that the material enhancing the taste of
sea weed (tangle) soup came from a sodium salt of glutamic acid
(monosodium glutamate).
• Since then monosodium glutamate has been widely used in both the food
industry and by the general public as a flavor-enhancing agent.
• The demand of glutamic acid has increased rapidly. Before 1956 glutamic acid
was mainly obtained by the hydrolysis of plant proteins . gluten

3. • In 1956 Japanese investigators reported the possibility ofproducing glutamic acid
by rnicrobiological Means, and began on an industrial scale the production of this
amino acid

5. 333.8 °CBoiling point

6. APPLICATIONS
• Gluatamic acid is Important in brain metabolism hence various analogues of
glutamic acid are used in treating various neuropathic diseases.
• Since 1908, monosodium glutamate has been used as a flavor enhancing agent
both by the food industry and the general public.
• Poly glutamic acid (PGA) is a naturally occurring anionic polymer that is
biodegradable, edible, and non-toxic towards human and environment .
• It is a good candidate for various industrial applications including thickener,
bitterness reliving agent , Cryoprotectant.
• Curable biological adhesive
• heavy metals absorbers .

7. • Cosmetics
- L-Glutamic Acid is widely used as Hair restorer in Cosmetics.
- As Hair restorer: in treatment of Hair Loss.
- As Wrinkle: in preventing aging

9. Microorganisms
• Corynebacteriumspp. (C. glutamicum; C. lilum)
Brevibacterium spp. (B. divericartum: B. alanicum)
Microbacterium spp. (M. flavumvar. glutamicum)
Arthrobacter spp. (A. globiformis; A. aminofaciens)

10. PRODUCTION OF GLUTAMIC ACID BY
WILD TYPE BACTERIA
• (i) Organisms: Wild type strains of the organisms of the four genera
mentioned above are now used for the production of glutamic acid.
• The preferred organism is however Corynebacterium glutamicum. The
properties common to the glutamic acid bacteria are:
• (a) they are all Gram-positive and non-motile;
•(b) they require biotin to grow;

11. • (c) they lack or have very low amounts of the enzyme alpha-ketoglutarate,
which is formed by removal of CO2 from isocitrate formed in TCA cycle
(citric acid cylce).
• Since alpha -ketoglutarate is not dehydrogenated it is available to form
glutarate by reacting with ammonia.
• (ii) Conditions of the fermentation: The composition of a medium which
has been used for the production of glutamic acid is as follows (%):
glucose, 10; corn steep liquor 0.25; enzymatic casein hydrolysate 0.25;
K2HPO(Dipotassium phosphate )4 0.1, Mg. SO4, 7 H2 O, 0.25; urea, 0.5.
• It should be noted that besides glucose, hydrocarbons have served as
carbon sources for glutamic acid production.
• The optimal temperature is 30° to 35° and a high degree of aeration is
necessary.

12. • (iii)Biochemical basis for glutamic acid production: Studies by several
workers have clarified the basis for glutamic acid production as
summarized below.
• (a) Glutamic acid production is greatest when biotin is limiting; that is,
when it is sub optimal. When biotin is optimal, growth is luxuriant and lactic
acid, not glutamic acid, is excreted.
• The optimal level of biotin is 0.5 mg per gm of dry cells; with higher
amounts glutamic acid production falls.
• (b) The isocitrate-succinate part of the TCA cycle is needed for growth. It
is only after the growth phase that glutamic acid production becomes
optimal.
• (c) An increase in the permeability of the cell is necessary so as to permit
the outward diffusion of glutamic acid, essential for high glutamic acid

13. • This increased permeability to the acid can be achieved in the following ways:
• (i) ensuring biotin deficiency in the medium
• (ii) treatment with fatty acid derivatives,
• (iii) ensuring oleic acid deficiency in mutants requiring oleic acid (C16 - C18).
• (iv)addition of penicillin during growth of glutamic acid bacteria, Cells treated in
one of the first three ways above have cell membranes in which the saturated to
unsaturated fatty acid ratio is abnormal, therefore the permeability barrier is
destroyed and glutamic acid accumulates in the medium.
• The major factor in glutamic acid production by wild type organism is thus
altered permeability.
• Treatment with penicillin prevents cell-wall formation. Cell wall inhibiting
antibiotics such as penicillin and cephalosporin have enabled the use of
molasses .which are rich in biotin for glutamic acid production.

14. FERMENTOR PRODUCTION OF AMINO ACID
•
21.6.1 Fermentor Procedure
Starting from shake flasks the inoculum culture is grown in shake flasks and transferred
to the first seed tank (1,000–2,000 liters) in size.
• After suitable growth the inoculum is tansferred to the second seed tank (10,000–
20,000 liters),
• which serves as inoculum for the production tank (50,000–500,000 liters).
• The fermentation is usually batch or fed-batch .
• In batch cultivation all the nutrients are added at once at the beginning of the
fermentation, except for ammonia.
• which is added intermittently to help adjust the pH, and fermentation continues until
sugar is exhausted.

15. • In a fed-batch process, the fermentor is only partially filled with
medium and additional nutrients added either intermittently or continuously
until an optimum yield is obtained
• . The fed-batch appears preferable for the following reasons:
(a) Most amino acid production requires high sugar concentrations of up to 10%.
If all were added immediately, acid would be quickly produced which will inhibit
the growth of the microorganisms and hence reduce yield
• (b) Where auxotrophic mutants are used, excess supply of nutrients leads to
reduced production due to overgrowth of cells or feed back regulation by the
nutrient.
(c) During the lag phase of growth, the oxgen demand of the organism may
exceed that of the organism leading to reduced growth

16. 21.6.2 Raw Materials
• The main raw materials used are cane or beet molasses and starch hydrolysates
from corn or cassava as glucose.
• In the US, the preferred carbon source is corn syrup from corn, whereas in
Europe and South America it is beet molasses.
• As nitrogen source, inorganic sources such as ammonia or ammonium sulfate is
generally used.
• Phosphates, vitamins and other necessary supplements are usually provided with
corn steep liquour.

17. FERMENTATION
The usual culture medium for glutamic acid fermentationcontains a carbon source
such as glucose, the acid hydrolysate ofstarch, molasses, or a mixture of these
substances.
A nitrogen source such as urea, and other chemicals is present.
The prepared culture medium is sterilized in a fermentor by steam.
When the temperature of the medium cools down to 30°C, the micro-organism
is added to the fermentor in a proper inoculum size.
The micro-organism is incubated for thirty-six to forty-eight hours during which
time the Ph(7-7.8), temperature(30-35) , and aeration rate are carefully controlled.

18. 21.6.4 Down Stream Processing
• After fermentation, the cells may be filtered using a rotary vacuum filter.
Sometimes filtration can be improved by using filter aids.
• These filter aids, usually, kiesselghur which are based on diatomaceous earth,
improve the porosity of a resulting filter cake leading to a faster flow rate.
• Before filtration a thin layer is used as a precoat of the filter (normally standard
filters).
The extraction method of the amino acid from the filtrate, depends on the level
of purity desired in the product.
• However two methods are generally used: the chromatographic
(ion exchange) method or the concentration-crystallization method.

19. Crystallization
• Crystallization is often used as a method to recover the amino acid. Due to the
amphoteric character (contains both acidic and basic groups) of amino acids,
their solubility is greatly influenced by the pH of the solution and usually show
minima at the isoelectric point (zero net charge).
• Since temperature also influences the solubility of amino acids
and their salts, lowering the temperature can be used in advance as a means of
obtaining the required product.
• Precipitation of amino acids with salts, like ammonium and calcium salts, and
with metals like zinc are also commonly used.

20. Ion exchange
• resins have been widely used for the extraction and purification of amino
acids from the fermentation broth.
• The adsorption of amino acids by ion exchange resinsis strongly affected by the
pH of the solution and by the presence of contaminant ions.
• There are two types of ion exchange resins; cation exchange resins and anion
exchange resins.
• Cation exchange resins bind positively charged amino acids (this is in the
situation where the pH of the solution is lower then the isoelectric point (IEP) of
the amino acid),

21. • whereas anion exchange resins bind negatively charged amino acids (pH of the
solution is higher than IEP).
• Extraction of the bound amino acid(s) is done by introducing a solution
containing the counterion of the resin.
•
• Anion exchange resins are generally lower in their exchange capacity and
durability than cation exchange resins and are seldom used for industrial
separation.
• In general, ion exchange as a tool for separation is only used when other steps
fail, because of its tedious operation, small capacity and high costs

23. According to the findings of the scientific panel of the PFA, Chinese salt
contains Monosodium glutamate (MSG) which can cause headaches,
fatigue, palpitations, nausea and vomiting, sweating, flushing and numbness
of the face, more so among people who are sensitive to it.
It can also cause hypertension and is extremely hazardous for pregnant
women, the panel found.
According to some reports, regular use of this ingredient can cause long-
term issues like high blood pressure, autism, hormonal imbalance, epilepsy,
food allergies, asthma, reduction in bile formation, cancer and possible
sterility in females.