1. Amino acid Fermentation

2. Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon , hydrogen , oxygen , and nitrogen An alpha-amino acid has the generic formula H2NCHRCOOH, where R is an organic substituent. Amino acids are critical to life, and have many functions in metabolism .
One particularly important function is to serve as the building blocks of proteins , which are linear chains of amino acids. Amino acids can be linked together in varying sequences to form a vast variety of proteins.

3. Essential and Nonessential AAs
Histidine Alanine Isoleucine Arginine * Leucine Asparagine Lysine Aspartic acid Methionine Cysteine * Phenylalanine Glutamic acid Threonine Glutamine * Tryptophan Glycine Valine Ornithine * Proline * Selenocysteine * Serine * Taurine * Tyrosine * (*) Essential only in certain cases

4. EAAs
An essential amino acid or indispensable amino acid is an amino acid that cannot be synthesized de novo by the organism being considered, and therefore must be supplied in its diet. The nine amino acids humans cannot synthesize are phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine.

5. Non EAAs
Six amino acids are considered conditionally essential in the human diet, meaning their synthesis can be limited under special pathophysiological conditions, such as prematurity in the infant or individuals in severe catabolic distress. These six are arginine, cysteine, glycine, glutamine, proline and tyrosine .
Five amino acids are dispensable in humans, meaning they can be synthesized in the body. These five are alanine, aspartic acid, asparagine, glutamic acid and serine.

6. Essential and Non Essential AA
Twenty-two amino acids are naturally incorporated into polypeptides and are called proteinogenic or standard amino acids. Of these, 20 are encoded by the universal genetic code . Nine standard amino acids are called "essential" for humans because they cannot be created from other compounds by the human body, and so must be taken in as food.

7. Amino Acid by Fermentation
NOW A DAYS THREE MAJOR AMINO ACIDS ARE BEING PRODUCED ON LARGE SCALE.
GLUTAMIC ACID
LYSINE
METHIONINE
All PRODUCED BY PROCESS OF FERMENTATION USED TO BE PRODUCED BY CHEMICAL SYNTHESIS

8. Why Produced on a Large Scale?
The amino acid business is a multi-billion dollar enterprise, All twenty amino acids are sold, although each in greatly different quantities
Amino acids are used as animal feed additives (lysine, methionine , threonine ), flavor enhancers (monosodium glutamic, serine, aspartic acid) and as specialty nutrients in the medical field.
Glutamic acid, lysine and methionine account for the majority, by weight, of amino acids sold .
GLUTAMATE IS MEDICALY USED AS A NEUROTRANSMITTER.

9. HISTORY RELATED TO GLUTAMIC ACID & LYSINE PRODUCTION
Many microbe-based industries have their origins in traditions that go back hundreds or thousands of years.
The amino acid industry has its roots in food preparation practices in Japan. Seaweeds had been used for centuries there and in other Asian countries as a flavoring ingredient.
In 1908, Kikunae Ikeda of Tokyo Imperial University isolated the flavor enhancing principle from the seaweed konbu (also spelled kombu , Laminaria japonica; related to kelp) as crystals of monosodium glutamate (MSG).
Adding MSG to meat, vegetables and just about any other type of prepared food makes it savory, a property referred to as umami .

10. Fermentation of GA and Lysine
The production of MSG via “fermentation” grew out of the ashes of WWII in Japan. Around 1957, Japanese researchers led by S. Kinoshita at Kyowa Hakko Kogyo Co. isolated soil bacteria that produced large amounts of glutamic acid.
In the early 1960s , workers at the same company found that C. glutamicum homoserine auxotrophs produced lysine thus providing the first viable fermentation process for lysine production.

11. The usual culture medium for glutamic acid fermentation contains a carbon source such as glucose, the acid hydrolysate of starch, molasses, or a mixture of these substances. A nitrogen source such as urea, and other chemicals is present. The prepared culture medium is sterilized in a fermenter by steam. When the temperature of the medium cools down to 30°C, the micro-organism is added to the fermenter in a proper inoculum size.

12. In this method, the micro-organism, Micrococcus glutamicum or some other glutamic acid producing micro-organisms are used. The micro-organism is incubated for thirty-six to forty-eight hours during which time the pH, temperature, and aeration rate are carefully controlled. When the fermentation is finished, the fermentation broth is hydrolyzed with hydrochloric acid. Glutamic acid is obtained in a process analogous to that for the recovery from the protein hydrolysate.

13. GLUTAMIC ACID PRODUCTION
GLUTAMIC ACID PRODUCING MICRORGANISMS: BACTERIA PRODUCING GLUTAMIC Acid.
First discovered was Micrococcus glutamicum
Now a days Coryneform SPECIESES are being used. Others are Brevibactirium

14. In order to be useful, glutamate producers must do two things well:
1. they must overproduce glutamate in excess of their normal metabolic needs
2. they must excrete it into culture broth.
METHODS USED FOR COMPLETING THESE TWO REQUIREMRNTS:
-Use of minimum conc. of biotin
-Addition of penicillin
-Use of surfactants
-Use of the bacteria lacking alpha-KETOGLUTARIC ACID DEHYDROGENASE enzyme
-Increased permeability of the bacterial cell membrane results in over production of glutamic acid

15. WHY WE ARE USING BIOTIN IN LESS CONCENTRATION?
Biotin is a cofactor (a “vitamin”) used by enzymes that carboxylate substrates. One such enzyme is acetyl- CoA carboxylase that converts Acetyl- CoA + CO2 to Malonyl-CoA in the first step of fatty acid biosynthesis.
Biotin auxotrophs growing in biotin deficient medium were proposed to have altered membranes due to suboptimal fatty acid biosynthesis.

16. WHY USE PENICILLIN AND SURFACTANTS?
Because both have selective actions on the bacterial cell wall and alter the permeability of the Bacterial cell wall and thus allowing more of the glutamic acid to excrete.

17. REASON FOR USING alpha- Ketoglutaric acid dehydrogenase lacking Bacteria:
All the bacteria synthesize glutamic acid from carbohydrate sources Or indirectly from alpha- keto glutaric acid (an intermediate product of glucose metabolism).
Now this alpha keto glutaric acid has two optional metabolic pathways: It will convert to L- glutamic acid through amination
Or it will convert in succinic acid with the help of enzyme alpha Keto Glutarate dehydrogenase ,which further goes in TCA CYCLE.
SO IF WE AVOID THE SECOND OPTION ,THEN MORE OF THE L- glutamic acid can be produced.

18. The metabolic pathway for biosynthesis of L- glutamic acid GLUCOSE -->ACETYL CoA CITRIC ACID Alpha ketoglutaric acid
AMINATION: GLUTAMATE DEHYDROGENASE (M.Glutamicus possesses this enzyme) L –GLUTAMIC ACID

19. DIRECT/ ONE STAGE FERMENTATION
UPSTREAM PROCESS: Inoculum development: Use of single microbe which produces L- glutamic acid directly. Commonly employed are M.glutamicum
Sterlization :is achieved by employing steam under pressure i.e.autoclaving.
Growth media contains: Ingredient concentration
Cane molasses 20.0%
Soyabean meal hydrolysate 1.8%
Biotin %
NaCl 0.25% (NH₄)₂SO₄
2% Homoserine Peptone
1% Meat extract 0.5%
Distilled water q.s

20. Process Parameters
Process control parameters: Production is carried out by submerged culture method-fed batch fermenter .
Aerobic process, high aeration required
pH b/w
Temp should be controlled at C
The process involves intermittent addition of urea, ammonia or ammonium salts which serves as source of ammonium ions
Time period for completion i.e when maximum yield is obtained is little less than 2 days.
The obtainable yield is 30g/l

21. DOWNSTREM PROCESS:
RECOVERY AND PURIFICATION: FILTRATION for removal of microbial cell mass
Concentration of the filtered broth Adsorption on ion exchange resins
Elution Concentration of the eluent
Crystallization and recrystallization to obtain L- glutamic acid in purified form.

22. INDIRECT/TWO STAGE FERMENTATION
IN THE FIRST STAGE THE INTERMEDIATE, ALPHA KETO GLUTARIC ACID IS PRODUCED BY ONE MICROBE.
THEN IN THE SECOND STAGE,IT IS CONVERTADE TO L-GLUTAMIC EMPLOYING ANOTHER MICROBE

23. Two Stage Fermentation
MICROBES USED IN SECOND STAGE are: Bacterium Alphaketoglutaricum, Bacillus, E.Coli Erwinia, E.Freundii, Kluyvera citrophila, Hansenula, Pseudomonas fluroscenns, Mycotorula
After 14 hours of growth, the temperature is increased from about 32-33°C to 38°.
Sugar is fed in as the fermentation proceeds up to about 36 h.
During the course of the fed-batch process about 160 g or more of glucose per liter, or its equivalent, is fed into the bioreactor.

24. Glutamic Acid
Ikeda in 1908, working on the flavouring component in kelps
Discovered GLUTAMIC ACID (L-glutamate) after acid hydrolysis and fractionation of kelp and neutralization with caustic soda.
These treatments enhance the taste of kelp
Gave rise to the birth of:
MONO SODIUM GLUTAMATE (MSG), flavor enhancing compound.
It was extracted from soy and wheat. Now micro-organisms (Corynebacterium glutamicum) are used for MSG production.
Commercial production of MSG is the largest and biggest industries world over.
Commercial Production
Glutamic acid > lysine > methionine > threonine > Aspartic acid
The market is growing steadily by about 5–10% per year.

25. USES OF AMINO ACIDS IN INDUSTRIAL APPLICATIONS
Food industry: 65%
Feed Additives: 30%
Pharmaceutical: 5%
FOOD INDUSTRY
Flavor enhancers, MSG, glycine, alanine. Tryptophan and histidine act as antioxidants to preserve milk powder. For fruit juices cysteine is used as an antioxidant.
Aspartame, dipeptide (aspartyl-phenylalanine-methyl ester) produced by combination of asp and Phe is 200 times sweeter than sucrose. Used as low calorie artificial sweetener in soft drink industry
Essential amino acids are those deficient in plant based foods like lys, met, thr, Trp improves nutritional quality of food and feed additives (animal). Bread: lysine, soy products or soyabean meal (pigs/animals): methionine

26. Used as medicines, infusions to patients with post operative treatment
PHARMACEUTICAL INDUSTRY
Used as medicines, infusions to patients with post operative treatment
CHEMICAL INDUSTRY
Used as a precursor for production of several cpds
Glycine used to manufacture GLYPHOSATE
Threonine used for AZTHREONAM (herbicide)
Poly methyl glutamate: manufac. Of synthetic leather
N-acyl derivatives of amino acids used for making cosmetics

27. METHODS FOR PRODUCTION OF AMINO ACIDS
EXTRACTION: hydrolysis of proteins to isolate amino acids like cys, tyr, leu
CHEMICAL SYNTHESIS: can result in racemic mixture (D and L amino acids), most applications are for L-form sometime DL or D maybe required.
MICROBIOLOGICAL SYNTHESIS
Direct fermentation: MO use carbon sources and produce aa. Carbon like glu, fructose, alkanes, ethanol, glycerol, molasses, starch, methanol etc.
Conversion of metabolic intermediates to amino acids:
Use of enzymes (microbial) or immobilized cells: resting cells, crude cell extracts, immobilized cells can be used.

28. Regulatory control has to be removed
STRAIN IMPROVEMENT METHODS FOR AA PRODUCTION
Because of regulatory control of metabolic reactions natural over production is rare
Regulatory control has to be removed
Mutagenesis and screening for mutants are done
Auxotrophic mutants: lack of formation of regulatory end product (repressor or effector molecule). Intermediates accumulate and get excreted.
Genetic recombination: for overproduction (recombinant molecules created) or protoplast fusion to develop hybrids
Recombinant DNA Technology: gene cloning, gene engineering
Functional genomics: whole chromosome sequencing data

29. L-GLUTAMIC ACID
Corynebacterium glutamicum, is a short, aerobic, Gram-positive rod capable of growing on a simple mineral salt medium with glucose, provided that biotin is also added.
Production of L-glutamic acid by C. glutamicum is maximal at a critical biotin concentration of 0.5 mg g-1 of dry cells, which is suboptimal for growth
Detergents like Tween-40,
addition of penicillin,
use of Glucose,
Glucose-6P,
CO2,
fatty acid auxotrophic strains, or
addition of ethambutol- inhibiting arabinogalactan synthesis.

30. Phosphoenol pyruvate carboxylase
L-GLUTAMIC ACID
Metabolic pathway
C.glutamicum used glycolysis, PPP and Citric acid cycle
Krebs cycle is replenished the Glu produced is high in amount
biotin
biotin
PDH
Phosphoenol pyruvate carboxylase
Glutamic acid bacteria have high activity of Glutamate dehydrogenase and low activity of a-ketoglutarate dehydrogenase
Isocitrate
dehydrogenase
Microbacterium
Brevibacterium
Arthrobacter

31. Glutamate dehydrogenase (high activity)
L-GLUTAMIC ACID
Regulatory control:
Good supply of glucose and efficient conversion of phosphoenol pyruvate to oxaloacetate
Phosphoenol pyruvate carboxylase and pyruvate carboxylase, pyruvate dehydrogenase
a-ketoglutarate dehydrogenase (low activity by adding penicillin, surfactants)
Glutamate dehydrogenase (high activity)
1 mole of glucose should produce 1 mole of glu
In practice, efficiency is 70%

32. Glu is synthesized intracellularly
Carrier mediated processess
Biotin is essential co factor (for Acetyl CoA carboxylase), deficiency of biotin affects fatty acid biosynthesis, membrane formation alters, permeability is affected and intracellular export of glu is altered
FACTORS INFLUENCING PRODUCTION
Carbon sources
Nitrogen source: ammonia for carbon to glu pH control
Growth factors :biotin
O2 supply: high conc inhibits growth and low O2 leads to lactic acid production and succinic acid, Afftects Glu production in both cases

33. Nutrients Glucose (12%) 38oC 30-35h Dissolving tank Sterlizer
Buffer tank
FERMENTER
Cell separator
Anion exchanger
Evaporation
Crystallization
Ammonia, pH control (7.8)
(ammonium acetate 0.5%)
Inoculum
Sterile air
Eluted in NaOH
100g/L