The document discusses Sri Paramakalyani College being reaccredited with an A+ grade by NAAC. It then summarizes the key steps in the microbial fermentation process for producing the amino acid L-lysine using Corynebacterium glutamicum, including feeding the microorganism appropriate carbon and nitrogen sources, controlling the pH and temperature during fermentation, recovering lysine from the broth using ultrafiltration and ion exchange, and crystallizing the final product.

1. SRI PARAMAKALYANI COLLEGE
REACCRITED WITH A+ GRADE WITH A GCPA OF 3.39 IN THE
THIRD CYCLE OF NAAC
AFFILIATED TO MANONMANIYAM SUNDARANAR
UNIVERSITY, TIRUNELVELI.
ALWARKURICHI 627 412
POST GRADUATE AND RESEACH
DEPARTMENT OF MICROBIOLOGY
IV SEM CORE: INDUSTRIAL MICROBIOLOGY
UNIT – 5
Take on:
Submitted by,
T. RAMAR
REG NO: 20211232516121
II M.SC.MICROBIOLOGY
SRI PARAMAKALYANI COLLEGE
ALWARKURICHI.
Submitted to,
GUIDE: Dr.S.VISWANATHAN, Ph.D,
ASSISTANT PROFESSOR & HEAD,
DEPARTMENT OF MICROBIOLOGY
SRI PARAMAKALYANI COLLEGE,
ALWARKURICHI.

3.  Amino acid are building blocks of proteins.
 They are the group of organic components containing two functional groups amino
acid and carboxyl.
 The amino group (-NH2) is basic while the carboxyl group (-COOH) is acidic in
nature.
 And a side chain (R group) specific to each amino acid is also present.

4.  All the 20 aminoacid are not needed to be taken in the diet.
 Based on the nutritional requirements amino acid can be classified into two
classes:.
 Essential amino acid
 The amino acid which cannot be synthesized by the body and need to be supplied
through the diet are called essential amino acid.
 (Eg) 10 amino acid are essential amino acid.
 Arginine, Valine, Histidine, Isoleucine, Leucine, Lysine, Methionine,
Phenylalanine, Threonine, Tryptophan.

5.  Non essential amino acid:
 There are synthesized be the body, and do not need not be supplied in the diet.
 There are – Glycine, Alanine, Serine, Cysteine, Aspartate, Asparagine, Glutamate,
Glutamine, Tyrosine, and Proline.
 Some microorganisms are capable of production certain amino acid such as
glutamine acid and tryptophan.

6.  L – lysine is an essential amino acid for the nutrition of human and other animals. It
is used for supplementing cereal proteins lacking this amino acid. Molecular formula
(C⁶H¹⁴N²O²).
 Thus, protein quality of certain foods (e.g. wheat based food) is improved growth and
tissue synthesis. This amino acid has also bee used medically as a nutrient.
 Kinoshita et al(1958) first reported the fermentative production of L-lysine using
homoserine auxotroph’s of Corynebacterium glutamicum.
 Food source of lysine are high protein foods such as eggs, meat,etc.
 Solubility: sparingly soluble in water.
 Covering more then 90% of total. World amino acid production.
 80% fermentation
 20% chemical synthesis

7.  1889 – Lysine first isolate from casein.
 1956 – Lysine was commercially introduced as feed.
 1978 – The first production of fermented L-lysine was produced by a Japanese
company kyowa Hakko kogyo.

8.  Lysine biosynthesis
 Production of lysine naturally takes place by two biosynthesis pathway
1. The DAP (Diaminopimelic Acid) pathway found in Bacteria
(e.g.Corynebacterium glutamicum), Algae, Higher plant, Oomycota.
2. The AAA (alpha – amino – adipate) Pathway found in Fungi and Euglenoids.

9.  The pathway for the synthesis of l L-lysine is complex.
 As the glucose gets oxidised by glycolysis, phosphoenol pyruvate are formed. Both
these metabolites can be converted to oxaloacetate, a key component of citric acid
cycle. On transamination, oxaloacetate forms Aspartate (aspartic acid). The
enzyme Aspartate kinase converts Aspartate to aspartyl phosphate which later
forms Aspartate semi-aldehyde.
 Aspartate semi-aldehyde has two fates the biosynthesis of lysine and formation of
3 other amino acid (methionine, threonine and Isoleucine). When homoserine
dehydrogenase acts on Aspartate semi-aldehyde, it is diverted for the synthesis of
3 amio acid. The enzyme dihydrodipicolinate synthase converts aspartate semi-
aldehyde (and pyruvate) to piperideine 2, 6-dicarboxylate.
 There are two distinct enzyme succinylase variant (catalyses 4-step reaction) and
dehydrogenase variant (catalyses a single step reaction) that can convert
piperideine 2, 6-dicarboxylate to D,L-diaminopimelate which later forms l L-
lysine.

11.  Aspartate kinase:
 This enzyme is controlled by feedback inhibition of the end products.
 By genetic manipulation, it has been possible to create mutants with an altered
Aspartate kinase that are insensitive to feedback regulation by l L-lysine.
 Dihydrodipicolinate synthase:
 Overexpression of dihydrodipicolinate synthase has been shown to increase the
production of L-lysine.
 A strain with a decreased homoserine dehydrogenase activity (so that diversion
for the synthesis of methionine, threonine and Isoleucine is minimised).

12.  Gram positive.
 rod shaped bacterium.
 They live in chain or in clusters, usually present in soil.
 Non motile.
 Non sporing.
 Non pathogenic bacterium.

13.  In batch fermentation microorganisms grows until one or essential nutrients is
exhausted or until fermentation conditions become unfavorable (e.g. product
inhibition, oxygen limitation, pH decrease in shake flasks uncontrolled
fermentation etc.).
 In fed- batch fermentation one or more nutrients are continuously or
intermittently supplied to the culture medium, either from the beginning of
fermentation or after the culture has reached a certain age, or when nutrient(s)
are exhausted .The microorganisms grows at growth rate dictated by the rete or
timing of nutrient feed.
 The continuous fermentation uses continuous feeding of a complete medium, while
culture fluid is continuously or semi-continuously withdrawn in such a way that
the working fermenter volume remains constant.

14.  The fermentation is carried out at 28’C and is allowed upto 60 hours.
 L-lysine fermentation is an aerobic process demanding large amounts of oxygen.
 The pH is a very important factor strongly influencing microbial fermentations.
Basic componds such as sodium hydroxide, potassium hydroxide, ammonium
hydroxide, calcium carbonate, urea, ammonia and gaseous ammonia, or inorganic
acid compounds such as phosphoric or sulfuric acid and organic acids are utilized
for controlling pH in L-lysine cultures at a pH ranging 5 to 9.
 Foam production in the aerated culture can be controlled by adding suitable
antifoam agent.

15.  The fermentation broth is sent to an ultra filtration system for the removal of cell
debris and other.
 Subsequently, the liquor form ultra filtration is fed to ion exchange columns,
where L-lysine is selectively absorbed.
 The absorbed L-lysine is eluted from the ion exchange resin by washing with an
aqueous ammonia solution.

16.  The L-lysine eluted from the ion-exchane column 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 is
converted to lysine HCL.

17.  Crystallization is also a chemical solids – liquid separation techniques.
 In which mass transfer of solute from the liquid solution to a pure solid
crystallization phase occur.
 Chemical engineering crystallization occurs in a crystallizer.
 TYPES:
 Cooling
 Concentration
 Reaction (or) equilibrium displacement.

18.  The most commonly used carbon sources for lysine manufacture is molasses (cane
or sugar beet), starch hydro lysates or source.
 The other sources like acetate, ethanol or alkanes are used to a lesser extent.

19.  Inorganic salts of various metals, like magnesium (e.g.magnesium sulfate),
calcium, potassium, sodium, iron (e.g. iron sulfate), manganese, and zinc or traces
of other metals.
 Protein hydro lysates are added to supply certain amino acids (L-methionine, L-
homoserine, L-threonine).
 The protein hydro lysates also supply growth factors such as biotin.

20.  Inorganic compounds such as gaseous and aqueous ammonia, ammonium salts of
inorganic or organic acids such as ammonium sulfate, ammonium nitrate,
ammonium phosphate, ammonium chloride, ammonium acetate and ammonium
carbonate.
 Alternative, natural nitrogen containing organic materials like soyabean –
hydrolyzate, soyprotein HCl – hydrolyzate, soy bean meal, soybean cake
hydrolysate, corn steep liquor, casein hydrolysate, yeast extract, meat extract,
malt extract, urea, peptones and amino acid may also be utilized.

21.  Succinylase and dehydrogenase variants:
 The conversion of piperideine 2,6-dicarboxylate to D, L-diaminopimelate
(precursor for the synthesis of L-lysine) is carried out by these two enzyme.
 At the start of the fermentation, dehydrogenase variant predominantly acts, and
later succinylase variant comes into picture for the biosynthesis of L-lysine.