2.

LITERATURE REVIEW

2.1 Amino acids

In 1956, a research program started at Kyowa Hakko Kogyo Co., Ltd.,

Tokyo that was aimed at obtaining a microorganism that could accumulate

glutamic acid extracellular. Among many isolates we found a colony that might

be fit for the purpose, named this isolate Micrococcus glutamicus No.534.

Further study revealed that this microorganism could accumulate glutamic

acid at a limiting concentration of biotin present in the medium. This suggested

that biotin must play a key role in the physiology of the cells and their

glutamate forming capability. By microscopic observation of cultures at various

stages, we found that the cell form can change considerably. For this reason,

and due to further taxonomical studies, we renamed the bacterium

Corynebacterium glutamicum. From mutational work on this organism, together

with discoveries regarding key regulatory features, it was found that many

amino acids, such as lysine, arginine, ornithine, threonine, etc., could be

accumulated. Most of these amino acids are now produced commercially

[Ikeda.K, 2002].

Amino acids produced by such a process are all in their natural (L) form

and this gives microbial production a big advantage over chemical synthesis.

Thus, a new industry called amino acid fermentation was born. The

commercial production of amino acids up to the discovery of C. glutamicum had

relied on the decomposition of natural protein and the isolation of its

constituent amino acids. The new process, on the contrary, was a biosynthetic
process using carbohydrate and ammonium ions. Therefore our process can

contribute to the amino acid supply and also helps to increase the absolute

amount of protein in the world. Since the world population continues to

increase year by year, so will the demand for amino acids and protein. After

World War II, two new fermentation industries were born in Japan. These are

the amino acid and nucleotide fermentation industries [Ikeda.K, 2002].

2.2 Role of monosodium glutamate

Amino acid production was born in Japan, and to do so we have to go

back to the year 1908. At that time Prof. Kikunae Ikeda at the University of

Tokyo found that monosodium glutamate (MSG) had a potent taste enhancing

power [Ikeda et.al., 1908]. He found this phenomenon through a careful

examination of the decomposition products of konbu, a type of seaweed.

During these studies he found a small crystal. This was glutamic acid, which

he discovered had a sour taste. Then he added NaOH to a glutamic acid

solution and tasted again. Surprisingly, it had changed into a beautiful taste.

That was the aim of his studies, since he was searching for the potent essence

of a flavor or taste enhancer. By the addition of only a few milligrams of MSG to

various foods, their taste was noticeably improved [Kinoshita et.al., 1987]. His

real intention was to improve nutrition and increase the short life expectancy of

the Japanese at that time. He finally got the idea that even if the same food was

eaten, its value might be increased if the taste is enhanced. In this sense, an

improvement in taste might contribute to relieving malnutrition. Therefore,

professor Ikeda began to search for the essence of good taste. Konbu had been
traditionally used in Japanese food as a taste enhancer, so he believed it

should contain the essence of flavor. This led to the discovery of MSG, whose

commercial production was essential to make use of its taste-enhancing

properties for the daily food of the Japanese [Kinoshita et.al., 1987].

Mr.Saburosuke Suzuki was the man who supported Prof. Ikeda’s desire.

Wheat gluten was chosen as the raw material to obtain MSG. But this task was

very difficult. Concentrated HCl must be used for decomposition of gluten, but

no anticorrosive vessels were available in those days. So clay pots were used,

but they were fragile and their use was very dangerous. Moreover, the gas from

HCl caused serious damage to the health of the residents living near the

factory. He had to face an onslaught of accusations and complaints.

Consequently, he had to move his factory to a remote location. His struggle to

produce MSG continued for ten years, before he finally became confident of

commercial success. Once MSG appeared in the market, its miracle power

overwhelmed the food market and it became an essential food additive. Mr.

Suzuki’s company is now known as Ajinomoto Co., Inc [Ikeda .K, 1908].

After World War II, Dr. Benzaburo Kato set up Kyowa Hakko Co., Inc. in

1945. Because of the shortage of food, the Japanese suffered great hunger.

Everywhere malnourished patients were seen. Dr. Kato was deeply worried by

this situation and thought of an idea for relieving the miserable situation by

supplying plenty of protein as food. To implement his idea, he asked me to

establish a commercial process that could supply food protein by a

fermentation process.

Amino acids are the building blocks that constitute proteins. Which are

very important to living systems for their survival Amino acids comprise about
16% N2 that distinguish them carbohydrates and fats in the body

[Lichtenberger, 1996]. Amino acids are the building blocks used to make

proteins and peptides .With the exploitation of new uses and the growing

markets of amino acids, production of amino acid technology has made large

progress during the latter half of the 20th century. Fermentation technology

has played a crucial role in the progress made and currently the fermented

amino acids represent important products of biotechnology in both volume and

value. This area is highly competitive in the world market and process

economics are of primary importance. For cost effective production, many

technologies have been developed to establish high productive fermentation

and recovery process [Herman et.al., 2003].

As the building blocks of life, amino acids have played an important role

in both human and animal nutrition and health maintenance [Balch et.al.,

1990]. Proteins are probably the most important class of biochemical

molecules, although of course lipids and carbohydrates are also essential for

life. On account of its functionality and the special features arising from

chirality, this class of compounds is biochemically extremely important and of

great interest for the chemical industry [Leuchtenberger, 1996]. Of the twenty

standard protein amino acids, the nine are essential the rest of the amino acids

are non essential including L-Glutamic acid.

2.3 Amino acid production

The production of amino acids is a big industrial factor in both the

chemical and biotechnological industries. There has been always hard

competition between these two fields to produce amino acids in a cheap and

energy reducing mode. Amino acids have many special properties which make
them very valuable, as for example their contribution to nutrition, the taste,

the chemical features and their importance in physiological activities. The

proteinogenic amino acids are the building blocks of proteins, they are

important intermediates on the pathway from the genetic to the protein level.

The varied use of amino acids is as supplements to human and animal

food, medical infusions, cosmetics and intermediates in the chemical industry.

According to data from 1995 the whole market is estimated at 3 billion US $ in

1995. Divided in 38% for food, 54% for feed and 8% for other applications

[Leuchtenberger et.al., 1996]. Figure 2.1 shows an overview of all methods to

gain amino acids in industry.

Chemical synthesis Protein hydrolysis (extraction)

R C COOH

NH2

Enzymatic synthesis Fermentation, cultivation

(Microbial overall producers)

Fig. 2.1: Principle possibilities to produce L-amino acids or D/L-

amino acids in the case of chemical synthesis (modified

according to Leuchtenberger et al. 1988).

It is possible to synthesize all amino acids in the traditional chemical way

but for many of them it would be much more profitable to produce with
different methods. The advantage of the enzymatic synthesis and the direct

fermentation is the modern enantio selective production of either the L-

enantiometric or D-enantiomeric form. There are examples for each of the

production possibilities mentioned in Figure 2.1. Glycine is the only non-chiral

amino acid, therefore the chemical process is without competition because

there is no racemic product mixture to purify. L-asparagine, L-arginine, L-

histidine and L-cysteine for example are produced by extraction from protein

hydrolysates, L-tryptophan and L-aspartic acid are obtained using enzymes or

immobilized cells.

The barrier for multi enzyme systems is reached when the effectiveness

of the microbial cell as enzyme membrane reactor is much higher in spite of

side reactions and by-products. On this account the direct fermentation is the

preferable process in commercial aspects for L-lysine and L-Glutamic acid [Kole

M et.al, 1986, Kinoshita et.al, 1961 and Kiefer et.al., 2004].

A major problem is the strong regulated biosynthesis in wild type

microorganisms. The produced amino acid itself restricts the formation of

necessary enzymes (feedback repression) and/or reduces the activity of key

enzymes for the metabolic building pathway (feedback inhibition) [Trotschel

et.al., 2005]. In a suitable strain the control mechanisms have to be

deactivated. In addition, side reactions and the degradation of end and

intermediate products have to be blocked.

2.4 Discovery of L-Glutamic acid

Glutamic acid was first discovered by Ritthausen in 1866.Some of the

seed proteins, especially the prolamines, yielded 20-45 percent of glutamic acid
on hydrolysis. The man who first noted in 1908 the commercial importance of

Glutamic acid was Kikunae Ikeda. He discovered that monosodium glutamate

(MSG sodium salt of glutamic

acid strongly enriched the flavor. Glutamate was obtained by decomposing

plant proteins such as soybean and wheat.

In1955, Kinoshita aimed to find an appropriate organism, which could

convert non proteinaceous raw material to find appropriate organism, which

could convert non proteinaceous raw material into an amino acid and excrete it

out of cells abundantly. A screening programme for such microorganisms was

started in1955, headed by Dr.Shigezo Udaka He found the novel bacterium

(Corynebacyerium glutamicum), which can accurate 10.3 g/l glutamic acid in

the medium. This left no doubt that glutamic acid produced was the result of

direct bacterial fermentation process [Kinoshita .S, 1987].

The discovery of a potent glutamic acid producing bacterium by

Kinoshita et al [Kinoshita et.al., 1957] was the start of the subsequent

development of amino acid production by regulation of biosynthetic

metabolism. At that time, a two step L-Glutamic acid production process was

used. The glutamic acid producing bacterium first discovered was reported as

Micrococcus glutamicus in 1958, and other glutamic acid producing coryne

form species, all potent strains, were subsequently isolated by many others.

C.glutamicum, C.lilium, C.herculis, Brevibacterium flavum, B.lactofermentum,

B.divaricatum, B.ammoniagenes, B.thiogenetalis, Mycobacterium

ammoniaphilum is among the potent glutamic acid producing strains. Some

more of the glutamic acid secreting bacteria can be easily found in nature as

Escherichia coli, Bacillus megaterium, Bacillus circulans, Bacillus cereus and

Sarcina lutea.All of these organisms can produce glutamic acid from

carbohydrates [Abe et.al., 1972]. Representative strains producing glutamic

acid by direct fermentation were classified into four genera; Corynebacterium,

Nicrobacterium, Arthrobacter and Brevibacterium.

Kinoshita et al. [Kinoshita et.al., 1957] introduced the first fermentation

process for industrial production of amino acids by microorganisms. Bacterial

cultures were used for glutamic acid production due to increasing demand for

monosodium glutamate as a flavoring agent. He discovered a bacterium

Micrococcus glutamicus through, which he introduced a new approach for the

screening of microorganisms capable of accumulating glutamic acid in the

medium.

Fig.2.2: L-Glutamic Acid Chemical Structure (Chemical Formula:

C5H9NO4 & M.WT: 147.13)

2.5 .Corynebacterium glutamicum

2.5.1 Strain information

In 1957 Kinoshita et al. isolated a bacterial strain which was able to

overproduce L-Glutamic acid in minimal media with glucose as carbon source

and release the product in the extracellular environment. The isolated soil

bacterium was named Corynebacterium glutamicum. In taxonomic terms it

belongs to the family of Corynebacteriaceae. Its cell wall formation is very

characteristic (gram positive), especially the existence of mycolic acids which

surround the entire cell as a structured layer [Eggling et.al., 2001]. The wild

type strains are mostly able to grow aerobically on basic minimal media

containing a carbon source like glucose, phosphate, sulphate, ammonia and in

addition biotin due to the fact that this bacterial species is completely biotin

deficient. Furthermore Corynebacterium glutamicum is immobile and non

sporulating. Since the isolation in 1957 high amounts of L-Glutamic acid have

been produced with new developed or advanced strains of this species [Eggling

et.al., 2001].

Figure 2.3 shows the cell wall of Corynebacteriaceae has a special

structure which is different from other gram positive bacteria the peptidoglycan

layer is connected to the hetero polysaccharide arabinogalactan. The external

mycolic acid layer is linked again with the arabinogalactan [Eggling et.al,

2003].

Fig. 2.3: Formation of the Corynebacterium glutamicum cell wall

(right) in comparison to gram-positive (left) and gram-

negative bacteria (middle) (Eggeling et al. 2003).

 Because of many experiences the scientists gained over the last decades

about this organism and its metabolic fluxes in context of amino acid

production, Corynebacterium glutamicum has become the most important

bacterial strain for amino acid overproduction. It has been observed that the

regulatory system is much simpler than that of Escherichia coli [Tosaka et.al.,

1986]. There is information of the production of L-Glutamic acid [Kinoshita

et.al., 1961], L-lysine [Eggling et.al., 1999] and L-valine [Blombach et.al., 2007]

using strains of Corynebacterium glutamicum .

2.5.2 L-Glutamic acid production with Corynebacterium glutamicum

The process to gain L-Glutamic acid with Corynebacterium glutamicum

by direct fermentation [Kinoshita et.al 1961] is very well investigated. Key

factors for the cultivation process in order to reach high amounts of L-Glutamic

acid are the optimal concentration of biotin to influence and support cell

growth and the secretion of the product in the extracellular environment

[Clement et.al., 1986]. Another important factor to prevent side reactions and

by-products is the oxygen supply. Under partially anaerobic conditions

other/additional products like lactic acid could be obtained [Ikeda et.al.,

2002].Enzymes given in Fig.2.4 are listed below

1. Phosphoenolpyruvate carboxylase.
2. Pyruvate kinase.
3. Pyruvate carboxylase.
4. Pyruvate dehydrogenase.
5. Citrate synthetase.
6. Aconitase.
7. Isocitrate dehydrogenase.
8. L-glutamate dehydrogenase (GDH).
9. α-ketoglutarate dehydrogenase (KDH).
10. Isocitrate lyase.
11. Malate synthethase.
Fig. 2.4: Regulation of L-Glutamic acid biosynthesis in

Corynebacterium glutamicum (Leuchten-berger 1996);

straight lines represent feedback inhibition, dashed

lines represent feedback repression.

The most important factor for L-Glutamate overproduction is the activity

of the enzymes GDH and KDH (Figure 2.4). In overproducers the conversion

velocity of α-ketoglutarate to L-Glutamic acid with GDH is 150 times higher

than the side reaction of the substrate with KDH which leads back to the citric

acid cycle [Shiio et.al, 1980]. In Figure 2.4 the versatile regulation mechanisms
in biological pathways for L-Glutamic acid (feedback inhibition and repression)

are shown, the problems in modifying these metabolic fluxes in desired

directions are quite obvious due to the complex city and various connections in

these metabolic cycles.

2.6 Bio chemical amino acid degradation

Apart from sugars, amino acids are common substrates for microbial

cell growth. Sources of amino acids, like peptones, tryptones and casamino

acids are used in laboratory media. The degradation of individual amino acids

depends on the availability of other energy sources and on the C/N ratio.

The first step in biochemical amino acid degradation is, in general, the

removal of the α-amino acid group. The reaction products are 2-oxo acids.

There are different biochemical ways to perform this reaction. They are, as

follows:

- The oxidative deamination

- The transamination

- The β-elimination

After the α-amino group removal further degradation steps take place

according to the back bones of the 2-oxo acids (aliphatic, heterocyclic and

aromatic compounds).

The biochemical degradation products of amino acids are normally

intermediates of the citric acid cycle or precursors of these intermediates. They

are converted to carbon dioxide and water or will be applied in the

gluconeogenesis. The degradation of L-Glutamic acid and other amino acids

which are important in this thesis is shown in Figure 2.4. The amino acids can

be divided in two groups of catabolic degradation, the glucogenic and the

ketogenic amino acids. The ketogenic amino acids are converted to acetylCoA

or acetoacetate. Afterwards they are transformed to fatty acids or ketone

bodies. Lysine, leucine and threonine belong to this group of amino acids

which can be degraded only this way. The degradation products of these three

amino acids cannot be used for gluconeogenesis. Tryptophane, tyrosine, iso-

leucine and phenylalanine are amino acids which can be metabolized on both

biochemical ways for degradation, the ketogenic and the glucogenic one. The

other amino acid groups, shown in Figure 2.4, are degraded through the

glucogenic ways. This means that their degradation products are available for

gluconeogenesis. Glucose precursors like pyruvate, α-ketoglutarate and

succinylCoA are synthesized on these biochemical pathways. Methionine is

converted to succinyl-CoA, glutamic acid to α-ketoglutarate and alanine as well

as glycine is degraded through transamination to pyruvate and following

acetyl-CoA. The biochemical degradation is an important explanation approach

to discuss increased and decreased amino acid concentrations under nutrient

deficient condition phases in shake flask and bioreactor cultivations. Under

those conditions the microorganisms try to find possibilities to maintain the

metabolism by degradation or conversion of available substances, like e.g. the

conversion of amino acids.

Fig. 2.5: Biochemical amino acid degradation to intermediates of
the citric acid cycle. The glycogenic degradation is
colored in green, the ketogenic one in red / violet (Voet
et al. 2002).
2.7. Production of L-Glutamic acid by fermentation

The demand for utilization of L-Glutamic acid as a human dietary

supplement, flavour enhancer, chemical in biochemical processing,

pharmaceutical and cosmetic industries is fastly growing in recent times. It is

largely imported amino acid in India and the total worldwide production of L-
Glutamic acid is more than 1.5 million tons per year through fermentation.

Besides the existing methods of industrial fermentations, many efforts are

being pursued to improve the glutamic acid production especially under the

stand point of cheaper available substrates to help go down the production

costs. So better and cheaper methods of L-Glutamic acid production has

always been an issue which the scientific community have pursued with zeal.

A Survey of literature revealed the various methods of production of L-

Glutamic acid. In the year 1866, L-Glutamic acid was discovered by Karl

Heinrich Leopold Ritthausen. Later in the year 1907, Kikunae Ikeda identified

L-Glutamic acid crystals in konbu broth which gives a undeniable flavor in

many foods especially in seaweed and he termed this flavor Umami [Ikeda,

2002].Industrially the L-Glutamic acid was produced from vegetable proteins

like wheat proteins, soya bean proteins upon acid hydrolysis with HCl was first

reported by Ajinomoto Co., Inc., Tokyo, Japan.

Later a glutamic acid producing bacterium Micrococcus glutamicus or

Corynebacterium glutamicum was discovered by Kinoshita et.al. In 1957 which

produces 30 g/L of L-Glutamic acid in glucose medium [Kinoshita et.al 1957].

In the same year Donald A. Kita and Jackson Heights N.Y produced L-

Glutamic acid by using different strains of Cephalosporium through

fermentation of substrates like distiller’s solubles, cornsteep liquor, wheat

gluten etc. and the yield varies from 1 to 31 g/L.

In 1959, Kwei-chao chao and J.W Foster reported that, the Bacillus

strain 14B22 Utilized 3% glucose medium and liberated 12.5 mg/ml of

Glutamic acid.
 L-Glutamic acid can be produced by the fermentation of saccharide

material using five different strains of Brevibacterium consisting of atleast one

member of this group desthio biotin, biotin disulfoxide and biocytin and the

yield exceeds 4.0 g/dl.This was first reported by shinichi motozaki et.al. in

1963.

A Direct method of production of L-Glutamic acid by fermenting a

suitable nutrient media with a biotin requiring microorganism Micrococcus

glutamicus M-560 was developed by Thomas Philips in 1963. The yield was 40

g/L when grown in 3L molasses medium containing 37.5 parts per billion of

biotin and 4000 U/L of penicillin.

Shiio.et.al. in 1964 used four different strains of glutamic acid

producing microorganisms for L-Glutamic acid production from the substrates

like sodium acetate, potassium acetate or acetic acid. Brevibacterium flavum

ATCC No.13826 produced 15 g/L, Brevibacterium roseum ATCC No. 13825

produced 14.8 g/L, Brevibacteruim lactofermentus ATCC No. 13869 produced

14.3 g/L and Corynebacterium acetoacidophilum ATCC No. 13870 produced 7.3

g/L of glutamic acid.

The effect of penicillin on L-Glutamic acid by Corynebacterium

hydrocarboclastus M104 which assimilates glucose (G-medium) and/or

hydrocarbons (H-medium) containing kerosene, n-dodecane, n-Tetradecane

and n-hexadecane was first reported by Shinichirootsuka et.al. in 1964. The

yield of L-glutamic acid was 6.3 g/L in the G-medium and 2.1 g/L, 1.9 g/L,

4.0 g/L and 2.8 g/L in the H-medium containing these hydrocarbons.

It was reported by Joji Takahashi et.al. in 1965 that L-Glutamic acid

production by corynebacterium can be induced by the effect of different

natural nutrients.The yield was 5 g/L in the medium containing 3% n-

paraffins, 0.01% corn steep liquor mineral salts etc.

A screening test was performed by Wee Chong Tan and Bernard-

Malin.in 1964, to isolate a glutamic acid producing microorganism from soil by

using a Selective medium containing glucose, urea etc. when it is subjected to

U.V and X-ray treatment it liberated 10mg/ml of broth.

John D.Douros, Jr., West Chester et.al. in 1965, described a method for

the production of L-Glutamic acid using Nocardia globerula ATTC15076 by

Utilizing hydrocarbons under aerobic conditions at 300c for 48-96 hrs. The

yield obtained was 1-4 g/L for n-decane with in 36 hrs of incubation time.

L-Glutamic acid was produced from unsaturated fatty acids which were

reported by Hisoyoshi Okazaki et.al. in the year 1967. The organisms used

were Bacillus thiogenitalis No. 653 and its Oleic acid requiring mutant D-248.

D-248 utilizes oleic acid in presence of 30 μg/L of biotin and liberating 50

mg/ml of L-Glutamic acid but at the same biotin concentration glutamic acid

production by No.563 was reduced to zero. It was also found that No.563

produces glutamic acid 40mg/ml at 5μg/L of biotin concentration present in

the medium.

The study of Shigeho Ikeda in 1972 revealed that, an artificially induced

mutants like Brevibacterium ketoglutamicum S-10 (ATCC 21533),

Corynebacterium hydrocarboclastus R-17 ,S-15 (ATCC 21534) and Arthrobacter

paraffineus S-4 (ATCC 21535) utilized hydrocarbons and their oxidation

products present in the nutrient medium containing penicillin and liberated

7g/dl of glutamic acid.

 The microorganism Bacillus ammonia genes which required both biotin

and thiamine along with aminoacids like Histidine or Cystine liberated

maximum amount of L-Glutamic acid i.e more than 50% (w/w) of initial sugar

content present in the medium containing wheat bran extract or rice bran

extract [Hong, soon woo, et.al,. 1974]

The mutant strains of genus Brevibacterium produced L-Glutamic acid by

utilizing polyoxy ethylene sorbitan mono palmitate was reported by Takinami

et.al., in 1976. The yield was 21 mg/ml for Brevibacterium lactofermentum AJ

3611, 19 mg/ml for Brevibacterium lactofermentum ATCC 13869, 52 mg/ml

and 50 mg/ml for Brevibacterium flavum AJ 3612 and Brevibacterium flavum

ATCC 14067.

Haruo Momose and Takashi Takagi in 1978 reported that glutamic acid

can be produced by a temperature sensitive mutant strains derived from

Brevibacterium lactofermentum 2256. Among 159 selected mutant strains

produced glutamic acid after a temperature shift from 300C to 370C.One typical

mutant strain, TS-88 produced 2 g/dl of glutamic acid in a biotin rich beet

molasses medium with a temperature shift from 300C to 400C.

A novel fermentation process in which L-Glutamic acid can be produced

with ethanol at increased concentrations from 5 g/L to 25 g/L during fed batch

culture and the yield of L-Glutamic acid reached to 26 g/L. The organism used

was Brevibacterium divaricatum NRRL 2311. This method was first explained

by Kishimoto michimasa et.al. in 1981 using regression analysis.

In 1982, Yoshimura et.al , found out that the mutant strains of

Brevibacterium or Corynebacterium which are resistant to respiratory

inhibitors (or) ADP phosphorylation inhibitors produce high yields of glutamic

acid from 51 g/L to 52 g/L in glucose medium.

The mutants of genus Corynebacterium which were resistant to vitamine-

P compounds like esculetin, coumarin, Dicumarol produce high yields of

glutamic acid. When these resistant strains grown in 10 g/dl of glucose the

yield obtained was in between 5-41% and it was 5-33% when resistant strains

grown in g/dl of cane molasses. But at varied temperatures 31.50C, 350C, 370C

in presence of 3.6 g/dl of cane molasses the yield of glutamic acid was in the

concentration range 26-37% [H.Nakazawa et.al., 1982].

A patented work reported by H. Hiraga et.al. in 1983, described a

method for the production of L-Glutamic acid using mutant strains of

Brevibacterium or Corynebacterium which were resistant to antibiotics like

Decoyinine or Tubercidin produced 15-17.8 g/L of glutamic acid in glucose

medium. The same strains produced 47-50 g/L of glutamic acid when grown in

sugar cane molasses.

The method of production of L-Glutamic acid from the waste of a

Mexican lime citrus aurantifolia swingle using Corynebacterium glutamicum

ATCC 13032 which gave the highest yield of 13.7 g/L. The culture medium also

contained 2% glucose [Islas Murguia.L, et.al, 1984].

L-Glutamic acid production using mutant strains of Brevibacterium or

Corynebacterium which have an increased superoxide dismutase activity and

grows in a medium containing daunomycin or methyl viologen and produced

16-18 g/L of glutamic acid. These strains have liberated 49-51 g/L of glutamic

acid in case molasses at 31.50C for 36 hrs of incubation time [Yoshimura et.al,

1985].
 It was reported that, an immobilized Corynebacterium glutamicum grows

in a three phase fluidized bed reactor and achieved a maximum productivity of

glutamic acid 3 g/L/h in a glucose medium during continuous fermentation [

H.J.Henkel et.al., 1990].

In the year 1990, Yong-Fen Li et.al. Developed an immobilized strain of

Corynebacterium glutamicum T6-13 in Eucheuma gel with cellulose acetate was

used for the production of L-Glutamic acid. The conversion of glutamic acid

can reach 6.0% in the medium containing 12% of glucose.

The maximum yield of glutamic acid 6.86 mg/ml was obtained when 2%

glucose medium was fermented for 48 hrs by a Brevibacterium Sp. was reported

[ Nampoothiri et.al1995].

The production of L-Glutamic acid from palm waste hydrolysate by using

a strain Brevibacterium lactofermentum ATCC 13869 which utilizes glucose as

carbon source and produced 88 g/L of glutamic acid [ Das K et.al, 1995].

A novel fermentation process in which L-Glutamic acid production can

be produced by Escherichia Coli strains W3110, GH-1, AJ12628 and AJ12624

which were deficient or low in α- ketoglutaric acid dehydrogenase activity and

have low L-Glutamic acid decomposing ability, were capable of producing L-

Glutamic acid 3.0 g/L, 5.0 g/L, 18.5 g/L and 20.0 g/L respectively [N.Tujimoto

et.al, 1995].

A novel fermentation process was invented during 1995 by Mototsugu-

Shiratsuchi et.al. to establish a simultaneous cultivation method for

production of glutamic acid and lysine using Brevibacterium

lactofermentumAJ12937.The yield of these amino acids was 132 g/L in the

medium containing 6000 U/L of penicillin and 146 g/L in the medium

containing 4 g/L of PESMP[Motatsugu-Shiratsuchi et.al.,1995].

Eijiono et.al. in 1996, isolated a strain Escherichia coli W3110, PGK

designated as E.Coli AJ12949 which was deficient in α-ketoglutarate

dehydrogenase activity and amplified PEP carboxylase and glutamate

dehydrogenase activities produce high yield of L-Glutamic acid 23.3 g/L in

glucose medium.

The study concluded by [Madhavan Nampoothiri, K., and Ashok pandey

in 1996] was that, Brevibacterium sp. can be cultivated on sugarcane baggase

enriched with 10% glucose, urea, mineral salts and vitamins for the production

of L-Glutamic acid using solid state fermentation system. Maximum yield 80

mg glutamic acid/g dry baggase was obtained.

The co immobilized whole cells of Micrococcus glutamicus and

Pseudomonas reptilivora liberated high yields of L-Glutamic acid 37.1 Kg/m3

under optimized medium constituents in glucose medium and optimized

physical parameters was first reported [Sunitha et.al, 1998].

A biotin dependent surfactant temperature sensitive mutant strains of

Corynebacterium or revibacterium capable of producing L-Glutamic acid

during shift up temperatures 340C, 370C, 390C and the yield was 0.0, 0.5, 0.9

g/dL for ATCC 13869 and it was 5.8, 8.3, 9.2 g/dL for the strain AJ13029 by

utilizing glucose as carbon source. But during time intervals 8hrs, 12hrs,

16hrs produced 0.5, 0.1, 0.0 g/dL of glutamic acid by ATCC 13869 and 8.3,

7.0, 5.4 g/dL by AJ 13029.These strains when subjected to enhancement of

gene expression of glutamic acid biosynthesis system with different plasmids

expression systems produces L-Glutamic acid in the range 33 to 41 g/L

[Kimura et.al., 2003]

The investigated reports of K.Madhavan Nampoothiri and Ashok Pandey

in 1999 described that the strain Brevibacterium Sp DSM. 20411 utilized

cassava starch hydrolysate and accumulated 21g/L of L-Glutamic acid. In fed

batch fermentation using 5% w/v sugar concentration L-Glutamic acid

produced was 25 g/L.

Yoshioka et.al. in 1999, invented a method of producing L-Glutamic acid

with different mutant strains of Corynebacterium and Brevibacterium using

continuous fermentation. Brevibacterium lactofermentum ATCC13869 when

grown in production medium containing glucose, Biotin, Polyoxyethylene

sorbitan monopalmitate etc yielded 56% of L-glutamic acid and the productivity

was 5g/L with in 40 hrs. The strain Brevibacterium lactofermentum AJ12821

liberated 56% of L-glutamic acid with a productivity of 6.6g/L in 40hrs and the

same strain yields 55% with a productivity of 8.3 g/L in 100hrs.

L-Glutamic acid production by Delaunay.S, et.al, 1999, had revealed the

importance of PEP carboxylase and pyruvate carboxylase in Corynebacterium

glutamicum metabolism during temperature triggered glutamic acid

fermentation. In absence of PEP Carboxylase and pyruvate carboxylase activity

was sufficient and 70% of glutamic acid was liberated. In glucose medium

containing optimized biotin concentrations.

An aminoacid auxotrophic strain Bacillus methanolicus ATCC 55403 by

utilizing Methanol & Vit-B12 produced L-Glutamic acid at a concentration of

5g/L which was reported [ Hanson Richard. S. et.al. 2000].

 Corynebacterium glutamicum 2262, was subjected to several temperature

shift up from 330C to 370C, 380C, 390C, 400C and 410C causes the

accumulation of 80 g/L of L-Glutamic acid in glucose medium under fed batch

fermentation [Delaunay, et.al. in 2002].

In 2004, choi et.al,reported that the strain Brevibacterium sp.Tc452

during the shift-up temperature from 300C to 380C at 25 h of cultivation

produce maximum yield of L-Glutamic acid 41.42 g/L in glucose medium[choi

et.al. 2004].

The investigations carried out by Jyothi et.al. in the year 2005, revealed

that L-Glutamic acid can be produced by submerged fermentation of cassava

starch using Brevibacterium divaricatum.Under optimized parameter conditions

the highest glutamate yield of about 3.86% was obtained.

The experimental study carried out by yugandhar N.M et.al. During 2007

revealed that the maximum yield of L-Glutamic acid 40.5 mg/ml was obtained

with Brevibacterium roseum free cells under optimum parameters. The glutamic

acid at a concentration of 37.2 mg/ml and 39.6 mg/ml were obtained with

immobilized Brevibacterium roseum and co-immobilized Brevibacterium roseum

and Escherichia intermedia type 1 strain in a glucose media.

Amin G et.al. In 2007, described the production of L-Glutamic acid from

sugarcane baggase using Corynebacterium glutamicum ATCC13022 entrapped

into carrageenan gel beads. The best yield was obtained 75.7% at a

concentration of 73 g when immobilized bioreactor was operated continuously.

S. Y. Pasha et.al in the year 2011, Comparative studies were carried out

on glutamic acid production with wild type cells, mutants, immobilized cells
and immobilized mutants of Corynebacerium glutamicum. Immobilization was

carried out by sodium alginate method; physical mutagenesis was performed

by U.V irradiation and chemical mutagenesis with nitrosoguanidine. Five

physical mutants and five chemical mutants were selected for study.

Fermentation was carried out for a period of six days at 300C at 200 rpm.

Highest amount of Glutamic acid was produced with immobilized chemical

mutants. The maximum yield was wild type 14 g/l, physical mutant at 32.6 g/l

and chemical mutant 36.8 g/l.

The study of Mahmud Tavakkoli et.al. in the year 2012 concluded the

fact that date waste juice was the best substrate for the production of glutamic

acid. They used Corynebacterium glutamicum CECT 690 culture and response

surface methodology to predict the effect of fermentation parameters for L-

Glutamic acid production. The maximum yield was 39.32 mg/ml which was

determined by this model and in the second stage the yield was 118.75 mg/ml,

142.25 mg/ml and 95.83 mg/ml at three different air flow rates.

2.8 Importance of fermentation medium

Apart from physical parameters like pH, agitation, aeration rate,

temperature, dissolved oxygen and foaming, medium composition is a very

important factor and strongly influencing fermentation processes. The culture

medium must satisfy the requirements of microbial growth and production

[N.M yugandhar et.al.,2007].

Defined media comprising nutrients and essential additives or

alternatively undefined media containing natural organic substances such as

peptone or beef extract can be used for the production of glutamic acid .Normal

fermentation medium for the L-Glutamic acid fermentation contain various

carbon , nitrogen sources, inorganic ions and trace elements etc.

2.8.1 Influence of carbon source

Corynebacterium glutamicum and its mutants or related microorganisms

facilitates the inexpensive production of amino acids from cheap renewable

carbon sources by direct fermentation. Varieties of carbohydrates are utilized

individually or as a mixture for the production of L-Glutamic acid such as

glucose, fructose, sucrose etc.., are used [Kinoshita et.al, 1981].

2.8.2 Influence of nitrogen source

Various numbers of sources of nitrogen are utilized individually or as a

mixture for the commercial and pilot scale production of L-Glutamic acid,

including inorganic compounds such as ammonium salts, urea, ammonium

nitrates, peptones and other amino acids may also be utilized. [Nishida et.al.,

1979].

2.8.3 Influence of salts, trace elements and growth factors

Further components are added to the fermentation media at the

initiation or intermittently during the process of fermentation, such as

inorganic salts of various metals like magnesium sodium, potassium and also

added biotin, sources of phosphorous for the production of L-Glutamic acid

[Delauney s.et.al.,1999].
2.9 L-Glutamic acid fermentation technology

In the 1950s Corynebacterium glutamicum was found to be a very efficient

producer of L-Glutamic acid.Since this time biotechnological processes with

bacteria of the species Corynebacterium developed to be among the most

important in terms of tonnage and economical value. L-Glutamic acid and L-

lysine are bulk products nowadays. L-Valine, L-isoleucine, L-threonine, L-

aspartic acid and L-alanine are among other amino acid produced

by Corynebacteria. Applications range from feed to food and pharmaceutical

products. The growing market for amino acid produced with Corynebacteria led

to significant improvements in bioprocess and downstream technology as well

as in molecular biology. During the last decade big efforts were made to

increase the productivity and to decrease the production costs [Westrin I.A,

1990].

The discovery of the soil bacterium, Corynebacterium glutamicum, which

is capable of producing L-Glutamic acid with high productivity from sugar,

paved the way for the success of the fermentation technique in amino acid

production. It was advantageous here that the wild strain could be used on an

industrial scale under optimized fermentation conditions for mass production

of glutamate. Glutamate biosynthesis and methods for improving production

strains have been investigated in depth [Minoru Yoshimura et.al., 1982]. The

fermentation process is in principle very simple: A fermentation tank is charged

under sterile conditions with a culture medium containing a suitable carbon

source, such as sugar cane syrup, as well as the required nitrogen, sulfur, and

phosphorus sources, and some trace elements. A culture of the production

strain prepared in a pre fermenter is added to the fermentation tank and

stirred under specified conditions (temperature, pH, aeration). The L-Glutamic

acid released by the microorganism into the fermentation solution is then

obtained by crystallization in the recovery section of the fermentation plant.

MSG (1.5 million tons) is currently produced each year by this method, making

L-Glutamic acid the number one amino acid in terms of production capacity

and demand [Amin G.A.et.al., 2007].

For almost 50 years now, biotechnological production processes have

been used for industrial production of amino acids. Market development has

been particularly dynamic for the flavor-enhancer glutamate and the animal

feed amino acids L-lysine, L-threonine, and L-tryptophan, which are produced

by fermentation processes using highperformance strains of Corynebacterium

glutamicum and Escherichia coli from sugar sources such as molasses, sucrose,

or glucose. But the market for amino acids in synthesis is also becoming

increasingly important, with annual growth rates of 5–7%. The use of enzymes

and whole cell biocatalysts has proven particularly valuable in production of

both proteinogenic and nonproteinogenic L-amino acids, D-amino acids, and

enantiomerically pure amino acid derivatives, which are of great interest as

building blocks for active ingredients that are applied as pharmaceuticals,

cosmetics, and agricultural products. Nutrition and health will continue to be

the driving forces for exploiting the potential of microorganisms, and possibly

also of suitable plants, to arrive at even more efficient processes for amino acid

production [Tamaska et.al., 1995].

2.10 Importance of L-Glutamic acid

Glutamic Acid is sometimes referred to as Glutamate or a negative ion

form. Glutamic acid is a nonessential amino acid that functions as an

important metabolic intermediate. Glutamic acid can be synthesized from

oxoglutaric acid, formed in the metabolism of carbohydrates, so it does not

require direct dietary sources. Glutamic acid is biosynthesized from a number

of amino acids including ornithine and arginine. Glutamic acid is a

nonessential amino acid that the body uses to build proteins. It is also the

most common excitatory (stimulating) neurotransmitter in the central nervous

system when aminated, glutamic acid forms the important amino acid

glutamine. Because it has a carboxylic acid moiety on the side chain, glutamic

acid is one of only two amino acids (the other being aspartic acid) that have a

net negative charge at physiological pH. This negative charge makes glutamic

acid a very polar molecule and it is usually found on the outside of proteins

and enzymes where it is free to interact with the aqueous intracellular

surroundings. On a molar basis, glutamic acid is incorporated into proteins at

a rate of 6.2 percent compared to the other amino acids. Glutamic acid is also

a precursor of GABA, an important neurotransmitter in the central nervous

system. Glutamic acid helps transport potassium into the spinal fluid and is

itself an excitatory neurotransmitter. Glutamic acid has been used to treat

mental retardation, epilepsy, Parkinson's disease, muscular dystrophy and

alcoholism. It is widely distributed in protein foods and even some plant

proteins yield as much as 45% of their weight as glutamic acid. Glutamic Acid

is a natural occurring amino acid in many proteins. Rich sources of glutamic

acid are soy, meat, poultry, fish, eggs, and dairy products. When glutamic acid

combines with ammonia, a waste product of metabolism is converted into

Glutamine. [Susan.G 1998 and Willaert et.al., 1994].

2.10.1 Sources and uses of Glutamic acid

Good sources of Glutamic acid are

1. Dairy products

2. Meat

3. Poultry

4. Fish

Considered to be natural Brain food by improving mental capacities and

is used by the body to build proteins. It can attach itself to nitrogen atoms in

the process of forming glutamine, and this action also detoxifies the body of

ammonia. Glutamate is the most common excitatory (stimulating)

neurotransmitter in the central nervous system and is also important in the

metabolism of sugars and fats. It helps with the transportation of potassium

across the blood brain barrier, although it does not pass this barrier that

easily. It also shows promise in the future treatment of neurological

conditions, ulcers, hypoglycemic come, muscular dystrophy, epilepsy,

Parkinson's, and mental retardation. The fluid produced by the prostate gland
contains significant amounts of glutamic acid, and this amino acid may play a

role in normal function of the prostate. Glutamic acid may have protective

effects on the heart muscle in people with heart disease. Monosodium

glutamate (MSG), the form of glutamic acid that is used as a flavor enhancer,

has been reported in anecdotal studies to have a number of different adverse

effects (including headache, fatigue, and depression) [Reeds P.J. et.al, 2000].

2.10.2 Biomedical uses of L-Glutamic acid

It is an important excitatory neurotransmitter, and glutamic acid is also

important in the metabolism of sugars and fats. It helps with the

transportation of potassium across the blood brain barrier, although itself does

not pass this barrier that easily. It also shows promise in the future treatment

of neurological conditions, ulcers, hypoglycemic come, muscular dystrophy,

epilepsy, Parkinson's, and mental retardation. Glutamic acid can be used as

fuel in the brain, and can attach itself to nitrogen atoms in the process of

forming glutamine, and this action also detoxifies the body of ammonia. This

action is the only way in which the brain can be detoxified from ammonia. The

fluid produced by the prostate gland also contains amounts of glutamic acid,

and may play a role in the normal function of the prostate [ Delauney .S et.al.,

2002].

2.10.3 Therapeutic uses of L-Glutamic acid

Glutamic acid is classified as a non-essential amino acid. It is an

important excitatory neurotransmitter and required for lipid and glucose

metabolism.

Dosage based on the results from clinical studies with positive results,

daily dosage of Glutamic Acid ranges from between 2 - 15 grams (high doses
 may produce symptoms like Headache and Neurological problems) required

for lipid and glucose metabolism [Reeds P.J. et.al. ,2000]. Therapeutic Uses

are as follows

1. Childhood behavioral disorders.

2. Neurological conditions such as, epilepsy, mental retardation, muscular
dystrophy, Parkinson's disease
3. Glutamic Acid injections (I.V.) have been shown to increase exercise
tolerance and heart function in population with stable angina pectoris
4. BPH (Benign Prostate Hyperplasia)
2.11 Immobilization for L-Glutamic acid production

With the advent of new uses and the growing markets of amino acids,

amino acid production technology has made significant progress during the

latter half of the 20th century. Amino acid industry has been expanding and

this industrial growth will surely continue because of incessant efforts to

improve the established production processes also can be expected to

further reduce the production costs, thereby increasing the world rapid

progress in biotechnology including strain wide market. The development of

low cost fermentation processes for many kinds of amino acids and the recent

improvement technology, progress in biochemical engineering and

downstream processing indicate that fermentation attains the key position in

the amino acid industry. Fermentation technology has played crucial roles

over a period of time and currently the amino acid produced by fermentation

represent chief products of biotechnology in both volume and value

[Eggeling.L et.al., 2003].

 The recent biotechnological development of industrial processes for the

production of amino acids like L-Glutamate reveals the importance of

immobilized cells .To obtain better economics , the fermentation with

immobilized cells, in batch mode has better operational convenience

compared to the process using free cells in batch mode. The entrapment of

cells in sodium or calcium alginate gel beads is very useful procedure because

simplicity of the method, low price and non toxicity [Devlin T.M, 2002].

Due to high demand of L-Glutamic acid all over the world, for

application in various fields. It is very much essential to produce L-Glutamic

acid by immobilized fermentation process. It is evident from the literature, the

most research work confined to free cell fermentation gap existing between

free cell batch reactor studies and immobilized cell reactor studies. Since

significant research has not been carried out in immobilized cell reactor there

is a vast scope to carry out research work for producing L-Glutamic acid by

immobilized cell system.

The present studies have undertaken to carry out immobilized reactor

studies by using Corynebacterium glutamicum species. It is essential to

evaluate the optimum process parameters of immobilized reactor studies and

batch reactor studies.