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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)
H
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.