Lysine is an essential amino acid that is used in the biosynthesis of proteins. Lysine is required for the nutrition of animals and humans. Lysine is useful as medicament, chemical agent, food material (food industry) and feed additives (animal food). It's demand has been steadily increasing in recent years. Several thousand tones of L-lysine are annually produced worldwide, almost by microbial fermentation. ±For Education Purpose Only

1. Production of Amino Acids (Lysine)
by Fermentation Technology
By: Harshita Mishra
Jyoti Dangwal
Kuldeep Sharma
Devashish Somani

2. Introduction
What are amino acids?
Amino acids are building blocks of proteins.
Amino acids are a group of organic compounds
containing two functional groups amino and
carboxyl

3. • The amino group (-NH2) is basic while the
carboxyl group (-COOH) is acidic in nature.
• with a side-chain (R group) specific to each
amino acid is also present
• all the 20 amino acids need not be taken in
the diet. Based on the nutritional
requirements amino acids are grouped into
two classes essential and nonessential

4. Facts and History
• French chemists isolated a compound in
asparagus that was named asparagine, the first
amino acid to be discovered.
• Cystine was discovered in1810
• Glycine and leucine were discovered in 1820.
• The last of the 20 common amino acids to be
discovered was threonine in 1935 by William
Cumming Rose.
• who also determined the essential amino acids

5. Essential amino acids
• The amino acids which cannot be synthesized by
the body and need to be supplied through the
diet are called essential amino acids.
• They are required for proper growth and
maintenance of the individual.
The ten amino acids listed below are essential for
humans:
Arginine, Valine, Histidine, lsoleucine,
Leucine, Lysine, Methionine, Phenylalanine,
Threonine, Tryptophan.

6. There’s a catch
• The two amino acids arginine and histidine
can be synthesized by adults and not by
growing children, hence these are considered
as semi-essential amino acids.
Thus, 8 amino acids are absolutely essential
while 2 are semi-essential.

7. Non essential amino acids
• The body can synthesize 10 amino acids to
meet the biological needs, hence they need
not be consumed in the diet. These are-
glycine, alanine, serine, cystein e, aspartate,a
sparagni e, glutamate,glutamine, tyrosine and
proline.
• Some microorganisms are capable of
producing certain amino acids such as lysine,
glutamic acid and tryptophan.

8. • There are 20 naturally occurring amino acids ,
which are required for the synthesis of variety of
proteins besides other biological functions
• However, these 20 amino acids need not to be
taken in diet .
• Based on nutrition requirements amino acids are
grouped into 2 classes :essential & non essential.

9. Types of amino acids……
Essential amino acids: the amino acids which cannot be synthesized
by the body and therefore, need to be supplied through the diet .
Non Essential amino acids : the amino acids which can be
synthesized by the body and therefore, need not to be supplied
through the diet.

10. History….
In 1889 , first it was isolated from casein.
Lysin was commercially introduced as a feed around 1956.
In 1978, first fermented L- lysin was produced by japanese
company “kyowa Hokko Kogyo”.
Recent method of lysin production is based on fementation of
carbohydrates.

11. Lysine production
plant of the BASF
AG located in
Gunsan, South
Korea with an
annualcapacity of
about 100 000 tons.
Copyright BASF
AG—The chemical
company (2003).

12. Three amino acids which are produced at large scale includes,
L-lysine
L-glutamic acid
DL- methionine , arginine, phenylalanine.
So, lysine is one of amongst them which covering more then 90% of
total world amino acid production
Synthesis of lysine 80% by Fermentation , 20% by chemical synthesis .
Two routes
-DAP pathway
- α -aminoadipate pathway

14. DAP pathway

15. α -aminoadipate pathway

16. UPSTREAM PROCESSING

17. 1. Fermentation Process
 Submerged Fermentation
 Aerobic Fermentation
2. Mode of Operation
 Batch Process
 Fed-Batch Process
3. Fermenter Type
 Stirred Tank Reactors
 Air – Lift Bioreactors

18. 4. Microorganism
Corynebactrium glutamicum (ATCC 13287)
• Gram positive bacterium
• Soil Bacterium
• Non-Motile
• Rod shaped
• Non – spore producing
• Non-pathogenic bacterium
Electron micrograph of C. glutamicum
(kromer et al., 2006).

19. • Homoserine auxotrophs strains
• Genetically Engineered strains
I. Overproducing genes
 dapA (Dihydrodipicolinate Synthase)
 lysC (Aspartate Kinase)
 dpaB (Dihydrodipicolinate Reducatse)
II. Lysine excreting mutants
 lysE (lysine export carrier gene)

20. 5. Fermentation Media
 Carbon Source : Cane Molasses
 Nitrogen Source: Corn steep liquor / Soybean meal
 Minerals and Salts: KH2PO4/K2HPO4, CaCO3
 Trace Elements: Corn steep liquor
 Antifoaming Agents: PEG-2000, Silicone based oils
6. Process Parameters
• Optimum pH : 7.2
• Optimum Temperature: 35-37 °C
• Time: 100 hours production cycle

21. 7. Fermentation Process
I. Seed Inoculum preparation
The typical medium composition includes;
Glucose 100g/L
Urea 5 g/L
Biotin 0.01 g/L
Leucine 0.4 g/L
MgSO4 2.85 g/L
KH2PO4/K2HPO4 0.5 g/L
MnSO4 0.016 g/L
CaCO3 20 g/L
(NH4)2SO4 46 g/L
II. Main Fermenter (upto 500 m3 volume)

22. DOWNSTREAM PROCESSING

23. PROCESS FLOW CHART

24. Product Recovery
• The fermentation broth is sent to an
ultrafiltration system for the removal of cell
debris and other suspended solids.
Subsequently, the liquor from ultrafiltration is
fed to ion-exchange columns, where L-lysine is
selectively adsorbed. The adsorbed L-lysine is
eluted from the ion-exchange resins by
washing with an aqueous ammonia solution.

26. The lysine has an amino group on its side chain,
its isoelectric point is at pH=9.7. At pH=1 the
lysine possesses two positive, at pH=5.6 two
positive and one negative, at pH=9.7 one
positive and one negative and at pH=11 one
negative charge.

27. Product Concentration & Drying
• The L-lysine eluted from the ion-exchange columns is
mixed with mother liquor from the product filtration
step and concentrated by evaporation. The
concentrated lysine solution is acidified with
hydrochloric acid, and free L-lysine is converted to L-
lysine HCl.
• The L-lysine HCl solution is then sent to the
crystallizer, and lysine salt is filtered. The mother
liquor is recycled to the evaporator and the wet cake
is conveyed to dryers. Final dry L-lysine-HCl (98.5
wt.%) is obtained and sent to a packaging line before
being stored in bags.

28. CRYSTALLIZER

29. SPRAY DRYER

32. • As the drum rotates, it is partially submerged in
the feed slurry.
• Vacuum draws liquid through the filter medium
(cloth) on the drum surface which retains the
solids.
• The vacuum pulls air (or gas) through the cake
and continues to remove moisture as the drum
rotates.
• If required, the cake can be washed to remove
impurities or to extract more product. Additional
drying of the cake follows washing.

33. • Finally, the cake is discharged from the drum to
a conveyor or chute to the next process step.
• The filtrate and air pulled through the medium
flow through internal filtrate pipes and pass
though the rotary valve and into the filtrate
receiver.
• The liquid stream is separated from the vapor
stream in the receiver.
• Liquid filtrate is then pumped to the next step
in the process.
• Vacuum is applied using a liquid ring vacuum
pump or other means.

34. Regulation of Lysine production
• The pathway leading to lysine biosynthesis is
initiated with the conversion of aspartate to
aspartyl-P via the enzyme aspartate-kinase (AK).
• The phosphorylated aspartate is then converted to
aspartyl-semialdehyde (ASA) that can converted to
homoserine by homoserine dehydrogenase (HSD)
or to diaminopimelic acid (DAP) by a series of five
enzymatic conversions, and hence to lysine.
• Lysine feedback inhibits the activity of aspartate
kinase

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