Superbugs and its production and application

1. MICROBIAL
BIOTECHNOLOGY
Genetically Engineered Microorganism
(Super bug)
Presentation by:
A.Abinaya Kalyani
15SUMB01
III B.Sc Microbilogy

2. Genetically Engineered Microorganism :
An organism whose genetic characteristics have
been altered by the insertion of a modified gene or
a gene from another organism using the
techniques of Genetic Engineering.

3. Super bug :
‘Superbug’ is a constructed
bacterium, Pseudomonas putida that
can degrade hydrocarbons found in
petroleum wastes.
It is a multiplasmid strain developed
by using genetic engineering
technique.
Pseudomonas putida

4. Super bug was developed by Anand Chakrabarty et
al. in 1979.
It is used to treat oil spills as a measure to control oil
pollution.
Petroleum products contain cycloalkenes(octane),
napthenes, xylene, tolune and aromatic
hydrocarbons. Since these compounds are not
easily biodegradable, oil wastes become a major
pollutant on the soil and water.
Chakrabarty et al. took attempts to degrade oil
wastes using micro organisms.
They developed superbug to control oil pollution.
Anand Chakrabarty

5. Construction of Superbug :
The construction of superbug involves the following steps :
Identification of parent strains
Conjugative transfer of plasmids
Selection of superbug
Mass culture

6. Strains
CAM plasmid OCT plasmid XYL plasmid NAH plasmid
Isolation and characterization of parent strains
Isolatian step followed
by selection

7. Identification of parent strains :
Chakrabarty et al. had succeeded in isolating different strains of P.putida that could
degrade camphor,octane,xylene and napthenes from polluted soils. They found out
that each strain has a special type of plasmid. Thus four types of plasmids are
recognized in the bacterial strains.
They are:-
1.CAM plasmid
2.OCT plasmid
3.XYL plasmid
4.NAH plasmid
The bacterial strain containing CAM plasmid could degrade camphor compounds.
Those strain containing OCT plasmid could degrade octane,hexane and decane.
The strain containing XYL plasmid could degrade xylene and toluene. And the
strain harbouring NAH plasmid could degrade napthenes.

8. Conjugative Transfer of plasmids :
The strain A containing CAM plasmid is mated with strain B containing OCT
plasmid. During this process, CAM plasmid is transferred from strain A to B.
Inside the bacterial strain B, CAM plasmid undergo homologous
recombination, so that a large CAM-OCT plasmid is formed. Thus the strain
B becomes strain E.
The strain C containing XYL plasmid is mated with strain D containing NAH
plasmid. During this conjugation XYL of strain C enters the strain D. Thus
strain D becomes strain F containing both XYL and NAH plasmids.

9. The strain E and strain F are mated to transfer
CAM-OCT plasmid into the strain F. Because
of the plasmid transfer, strain F becomes
strain G.
The strain G has three plasmids, namely
CAM-OCT plasmid, XYL plasmid and NAH
plasmid. It can degrade camphor, octane,
xylene and naphthenes. It is named as
superbug because of its high metabolic
capabilities.
Mixture of
Pseudomonas
strains
Colonies of
superbug
Subculture
Mixture+camphor,
octane,xylene&
napthene

10. Selection of Superbug:
Because of repeated mating, the Pseudomonas culture has strain A,
strain B, strain C ,strain D, strain E, strain F and strain G (Super
bug).
From this mixture of strains, strain G is selected by culturing the strains
in the presence of all the four pollutants, namely camphor,
octane,xylene and napthenes. The superbug alone can grow in the
medium. It is subcultured in fresh medium lacking the pollutants for
future use.

12. Mass culture of Superbug:
The selected colonies of superbug is mass
cultured in a suitable liquid medium in a
large bioreactor for mass production.
Pseudomonas putida on
Blood agar plate

13. Application of Superbug :
A patent was given to Chakrabarty regarding the construction and use of
superbug. The American Government, in 1990, allowed to use the superbug to
clean up oil spills in the water of Texas state.
The mass culture of superbug is sprinkled over paddy straw and the straw is dried
in shade.
The bacteria inoculated straw can be stored for more than an year until we are in
need.
To treat oil spill, the straw is spread over the oil slicks and is left as such for a
week or more.
During this time, the straw soak up the oil and the bacteria living on it break the oil
into non-polluting materials. As a result, the oil wastes become harmless to other
organisms living on soil or water polluted with petroleum oil.

14. Superbug
culture
Mixing with
paddy straw
Drying
Spreading on
oil slicks
Microbial
degradation
of oil wastes
Flow chart showing stages in the use of superbug in the treatment of oil wastes

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