5 mins Discussion on

Screening of microbes producing Industrial metabolites

Paper used

“Screening for Lysine Production by

Bacteria Isolated From Nigerian Soil”

Okpalla J, Ekwealor I.A

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 L-Lysine
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L-Lysine is an essential amino acid as it is not synthesized by humans and
hence has to be obtained from the diet.(Jose et al., 2005).
Cereals and vegetables also have low lysine content and are often fortified
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with lysine supplements.

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Microorganisms are capable to synthesizing several amino acids, some of
them in excess of their need. These amino acids are excreted out of the cells
and this fact can be exploited in the industrial production of amino acids.
(Shah et al., 2002)

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 Procedure
Isolation of Bacteria from Soil.
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Screening of bacteria for lysine production on solid agar medium

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Lysine production in submerged culture.

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Inoculum preparation
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Fermentation
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Quantitative determination of lysine

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Determination of growth of bacterial isolates

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Identification of bacterial isolates

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Effect of medium volume on growth and lysine production

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Effect of inoculum size

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 Isolation of Bacteria from Soil.
One gram of soil sample was collected.
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Tenfold serial dilution was done and aliquots of 0.1ml of
10-6 of dilution were used to inoculate plates of Nutrient
agar (Fluka) and Tryptone Soy agar (Oxoid).
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Incubated at 300C for 24h. Colonies that developed were
subcultured and pure cultures preserved at 40C on sterile
nutrient agar slants.

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 Screening of bacteria for lysine production on
solid agar medium
minimal medium
Glucose, 4.0g; (NH4)2SO4,2.0; K2HPO4, 0.5g; KH2PO4,
0.5; MgSO4.7H2O, 0.001g;FeSO4.7H2O, 0.0O1g;
MnSO4.4H20, 0.00lg; CaCO3, 2g; Agar, 15.0g, water, 1 litre
of water with pH adjusted to 7.2.
The medium which was seeded with a 24h broth culture of
the lysine auxotroph, Escherichia coli (NCCB 1841), was
inoculated with the isolate by spreading technique.

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This technique is employed for the
detection and isolation of
microorganisms capable of
producing certain extracellular
substances such as growth
stimulating factors like amino acids,
vitamins etc. A test organism with a
definite growth requirement for the
particular metabolite is used in this
method.

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Auxotrophs only grow in the presence of
L-lysine
1. Source 1
2. Source 2
3. Source 3
4. Control
lawn grown inoculation with auxotrophic E.coli for L-Lysine.
Then another agar plate is placed on top on which our sample

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How Auxotrophic Screening works.

L-lysine
producing

Auxotroph

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 Lysine production in submerged culture
For LAB scale testing of strain.
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For Secondary Screening.

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Inoculum preparation
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The seed medium consists of peptone, 10.0g; yeast
extract, 10.0g; NaCl, 5.0g; water, 1litre; pH adjusted to 7.2.

FERMENTATION
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 Quantitative determination of lysine
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Lysine in the broth culture was determined by acidic ninhydrin method of Chinard
(1952).
The isolate's culture broth was centrifuged at 5000 x g for 20 min with a volume of
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5 ml, and the cell-free supernatant was collected and tested for lysine production.
In a test tube, 1ml of glacial acetic acid was added to 1ml of supernatant.
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1 ml of a reagent solution which contains an acid mixture,0.4ml of 6M

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orthophosphoric acid, 0.6ml of glacial acetic acid and 25mg of ninhydrin, was also
added to the supernatant in the test tub
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The optical density of the reacting mixture was read against the blank at 515nm
in a spectrophotometer.

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Effect of medium volume on growth and lysine production
Various volumes (20–40ml) of fermentation medium consisting of: KH2PO4, 0.5g; K2HPO4, 0.5g; MgSO4.7H2O, 0.001g; MnSO4.H2O, 0.001g; FeSO

Effect of inoculum size

Various inoculum sizes(4-20%) were inoculated into Erlenmeyer flasks (100ml) containing 20ml of the medium composed of KH2PO4, 0.5g; K2HPO4,

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