INDUSTRIAL LYSINE PRODUCTION

1. LYSINE PRODUCTION
BY FERMENTATION PROCESS
1
SOURAV MONDAL
MSC MICROBIOLOGY

2. INTRODUCTION
AN OVERWIEW OF AMINO ACIDS AND LYSINE

3. 3
• AMINO ACIDS ARE THE MONOMERIC SUBUNITS BUILDING BLOCKS OF PROTEINS. ALL
PROTEINS IN OUR WORLD ARE MADE FROM THE UBIQUITOUS SET OF 20 AMINO ACIDS
• AMINO ACIDS ARE ORGANIC COMPOUNDS, THEY HAVE TWO FUNCTIONAL GROUPS, A
CARBOXYL GROUP (-COOH) AND AN AMINE GROUP (-NH2) BONDED TO THE SAME
CARBON ATOM (THE α CARBON)
• THE AMINO GROUP IS BASIC IN NATURE AND THE CARBOXYL GROUP IS ACIDIC IN NATURE
• THEY ALSO CONTAIN A HYDROGEN ATOM AND A SIDE CHAIN (R GROUP) WHICH IS
SPECIFIC TO EACH AMINO ACID.
• THE KEY ELEMENTS OF AN AMINO ACID ARE CARBON (C) , HYDROGEN (H) , OXYGEN (O) ,
AND NITROGEN (N).
• ACCORDING TO THE REQUIREMENTS OF THE AMINO ACIDS IN DIET , THEY ARE DIVIDED
INTO TWO CLASSES - ESSENTIAL AMINO ACIDS AND NON ESSENTIAL AMINO ACIDS.
WHAT IS AN AMINO ACID ?

4. 4
AMINO ACID
PRODUCED IN
THIS PHASE

5. WHAT IS LYSINE ??
5
H
I
H2N—C—COOH
I
CH2
I
CH2
I
CH2
I
CH2
I
NH2
LYSINE
(Lys, K)
• L- LYSINE (2,6-DIAMINOHEXANOIC ACID) IS AN ESSENTIAL AMINO
ACID FOR THE NUTRITION OF HUMANS AND OTHER ANIMALS.
OTHER NAMES OF LYSINE ARE Lys , K AND HAVING MOLECULAR
FORMULA (C6H14N2O2)
• IT IS USED FOR SUPPLEMENTING THE CEREAL PROTEINS LACKING
THIS AMINO ACID.
• THIS AMINO ACID IS USED MEDICALLY AS A NUTRIENT IN FORM OF
SUPPLEMENTS AND MEDICINES
• LYSINE IS PRESENT IN TWO FORMS , L - FORM AND D - FORM

6. HISTORY…
6
1889
• LYSINE FIRST ISOLATED FROM CASEIN
1956
• LYSINE WAS COMMERCIALLY INTRODUCED AS FEED
1978
• THE FIRST PRODUCTION OF FERMENTED
L-LYSINE WAS PRODUCED BY A JAPNESE COMPANY
KYOWA HAKKO KOGYO

7. 7
PRODUCTION OF
LYSINE
OVERVIEW OF LYSINE PRODUCTION
NATURALLY AND INDUSTRIALLY

8. 8
• PRODUCTION OF LYSINE NATURALLY , TAKES PLACES BY TWO BIOSYNTHETIC PATHWAYS
1. THE DAP (DIAMINOPIMELIC ACID) PATHWAY
FOUND IN BACTERIA (e.g. Corynebactrium glutamicum), ALGAE , HIGHER PLANTS AND OOMYCOTA
2. THE AAA ( α – AMINO – ADIPATE) PATHWAY
FOUND IN FUNGI AND EUGLENOIDS
NATURALLY (LYSINE BIOSYNTHESIS)

9. 9

10. 10
I U F D O
INBOUND
LOGISTICS
UP STREAM
PROCESSING
FERMENTATION DOWN STREAM
PROCESSING
OUTBOUND
LOGIDTICS
• RAW MATERIALS
• MICROBIAL
CULTURE
• MEDIA
• CULTURE
OPTIMIZATION
• MEDIA
FORMULATION
• MEDIA
UTILIZATION &
LYSINE
PRODUCTION
• SEPERATION
• EXTRACTION
• PURIFICATION
• PACKAGING
OF L- LYSINE

11. 11
INBOUND
LOGISTICS

12. 12
• FERMENTATION MEDIA
CARBON SOURCE  CANE MOLASSES
NITROGEN SOURCE  CORN STEEP LIQUOR / SOYABEAN MEAL
MINERAL AND SALTS  KH2PO4 / K2HPO4 / CaCO3
TRACE ELEMENTS  CORN STEEP LIQUOR
ANTI FOAMING AGENT  PEG-2000, SILICONE BASED OIL.
RAW MATERIALS
• INSTRUMENTS

13. 13
• IT IS A GRAM POSITIVE BACTERIUM
• IT IS ROD SHAPED BACTERIUM
• THEY LIVES IN CHAINS OR IN CLUSTERS
• THE BACTERIUM IS USUALLY PRESENT IN SOIL
• IT IS NON MOTILE
• THE BACTERIUM IS A NON SPORE FORMER
• THE BACTERIUM IS A NON PATHOGENIC
CULTURE ( Corynebacterium glutamicum ATCC 21526)

14. 14
UP STREAM
PROCESS

15. 15
MEDIA FORMULATION
INGREDIENTS AMOUNT (GRAM / LITRE )
GLUCOSE 100 g
UREA 5 g
BIOTIN 0.01 g
LEUCINE 0.4 g
MgSO4 2.85 g
KH2PO4 / K2HPO4 0.5 g
MnSO4 0.016 g
CaCO3 20 g
(NH4)2SO4 46 g
DISTILLED WATER 1000 L
MEDIA PREPRATION FOR GROWTH OF CULTURE AND INOCULLUM DEVELOPEMENT

16. 16
• DONE BY GENOME
SHUFFLING
• RIBOZOME ENGINEERING
• PROTOPLAST FUSION
CULTURE OPTIMIZATION
DONE TO OVEREXPRESS THE EXPRESSION OF INDIVIDUAL BIOSYNTHETIC
GENE Lys C AND Dap A

17. INOCULLUM DEVELOPEMENT
17
500
ml
1 litre
L . F
5 L
SEED
FERMENTER
50 L
PILOT
FERMENTER
100 L
100
ml
PRE CULTURE
SLANT
TO PRODUCTION
FERMENTER

18. 18
FERMENTATION

19. CHARACTERISTICS OF THE FERMENTATION
19
FERMENTATION
PROCESS
SUBMERGED
FERMENTATION
AEROBIC
FERMENTATION
MODE OF
OPERATION
BATCH
PROCESS
FED-BATCH
PROCESS
FERMENTER
TYPE
STIREDTANK
FERMENTER
AIRLIFT
FERMENTER
PROCESS
PARAMETERS
OPTIMUM PH 
7.2
OPTIMUM
TEMPERATURE
 (35 – 37)° C
TIME  100 HRS
PRODUCTION
CYCLE

20. 20
• CULTURE MEDIA PREPATED BY MIXING
PROCESSED WATER, GLUCOSE & NUTRIENTS
• FERMENTATION PERFORMED IN FED-BATCH
MODE AND UNDER AEROBIC CONDITION
• IN BATCH PHASE SEED CULTURE IS FED INTO
THE FERMENTER PREVIOUSLY FILLED WITH
FERMENTATION BATCH MEDIUM
• WHEN GLUCOSE IS EXHAUSTED, THE BATCH
FERMENTATION PHASE ENDS AND FED-BATCH
FERMENTATION PHASE STARTS
THE FERMENTATION PROCESS
CULTURE
CARBOHYDRATE
SOURCE
PRODUCE
AMINO
ACID

21. 21
DOWN STREAM
PROCESSING

22. 22
SEPERATION
• CELL REMOVAL
• L-LYSINE
RECOVERY
EXTRACTION • FURTHER REMOVAL OF IMURITIES
• CONCENTRATION OF L-LYSINE
PURIFICATION
• PURIFIED L-LYSINE READY
FOR PACKAGING
D S P - SCHEMATIC

23. SEPERATION OF L-LYSINE
23
LIQUID
L-LYSINE
L-LYSINE SEPERATED
FROM CELLS BY
ULTRAFILTRATION
L-LYSINE
SELECTIVELY ABSORBED
BY ION-EXCHANGE
CHROMATOGRAPHY
L-LYSINE ELUTED
FROM CHROMATOGRAPHY
RESINS BY WASHING
WITH AQUEOUS AMMONIA
SOLUTION

24. 24
L – LYSINE HCL
CONCENTRATED
(ACIDIFIED L-LYSINE)
L - LYSINE
MOTHE
R
LIQUOR
HCL
LIQUID
L - LYSINE
MOTHER
LIQUOR
(CANE
MOLASSES)
HCL
EXTRACTION OF L-LYSINE

25. PURIFICATION OF L-LYSINE
25
ACIDIFIED
L - LYSINE
PURE
L – LYSINE
LIQUID
OR
POWDERED L- LYSINE
OR
CRYSTALINE L-LYSINE

26. 26
OUTBOUND
LOGISTICS

27. 27
PACKAGING OF L-LYSINE
25 KG BAG
50 g BOTTLE
PURIFIED
L-LYSINE

28. 28
USES OF L-LYSINE
FIELD OF APPLICATION USED IN
 IN FOOD NUTRITIONAL SUPPLEMENTS, FOOD DECORANTS
 IN BEVERAGES NUTRITIONAL SPORTS DRINK
 IN PHARMACEUTICAL PREVENTING AND TREATING COLD SORES
CAUSED BY HERPES SIMPLEX LABIALIS VIRUS
 IN HEALTH AND PERSONAL CARE COSMETICS, PERSONAL CARE PRODUCTS, BABY PRODUCTS,
BATH PRODUCTS, EYE MAKEUP, SAVING KITS
 IN AGRICULTURE / ANIMAL FEED /
POULTRY
ANIMAL FEED AND USED IN FERTILIZERS
 IN OTHER INDUSTRIES AS INTERMEDIATE TO PRODUCE OTHER ORGANIC CHEMICALS

29. 29
THANK YOU
FOR
WATCHING