Assignment Extraction penicillinx

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Feb 20, 2013 (4 years and 3 months ago)

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EXTRACTION & BIOSEPARATION TECHNOLOGY LABORATORY

(BSB3421)

2011/2012 SEMESTER II


ASSIGNMENT












DATE OF SUBMISSION


2
1
ST

MA
Y
2012

INSTRUCTOR’S NAME


MISS NAZIRA BINTI MAHMUD


MEMBERS OF GROUP:



NAME








ID

1.

RUBINI DEVI SELVARAJOO






























SB09005

2.

KALAISELVI MOHANRAJ
































SB09031

3.

SUMITHALAKSMY GUNASEGARAN























SB09063

4.

CALVIN LEE WENG LEONG






























SB09017















FACULTY OF INDUSTRIAL SCIENCES AND TECHNOLOGY

UNIVERSITI MALAYSIA PAHANG

1.0

INTRODUCTION


A typical fermentation process consists of these steps. Such are;


A.

Production Stage (fermentation)

B.

Cell Harvest, Extraction; Separation and Product Purification


Nevertheless, primarily, the culture media which will be used has to meet the entire
requirement for the bacteria or the fungi to use them up and grow. Therefore selection and
preparation of c
ulture media has to done carefully to ease in the fermentation process.
[1]



2.0

MEDIA CONSIDERATION AND SELECTION


When performing any kind of fermentation, the selection of media is vital to the overall
performance of the fermentation. The aim of the med
ia is to provide all the nutrients and
supports required for the synthesis of cell materials and the formation of the desired product. At
the same time, the media must provide a favorable surrounding for the culture.
[2]



As with any general growth media i
t must supply all the basic constituents for growth/product
formation.



Carbon source



Nitrogen



Phosphates



Trace Elements


Each medium is designed to support a particular stage of the process. Media designed for the
early stage of the process will be generally focusing on achieving optimal spore germination and
strong vegetative growth.
[4]


As antibiotics are secondary metabo
lites, the final production medium will be emphasizing on
the over production of the antibiotic often to the loss of growth and in the case of antibiotic
fermentations, the use of specific precursor molecules in media formulations is substantial.
[2]



2.1

MEDIA FOR THE PRODUCTION OF PENICILLIN G


The production of Penicillin G is greatly enhanced through the introduction of Phenyl
-
acetic acid
as a precursor into the fermentation medium.
[3]


Nitrogen source, generally cheap supplements containing corn steep
liquor (source of penicillin
precursors: phenylalanine and phenethylamine), soya flour and fish meal may often supply
unknown trace elements which help in production. Technical grade glucose and or starch can be
used as a carbohydrate source.
[3]



Many a
ntibiotics are not produced in the presence of surplus carbon sources especially glucose.

In order to repair catabolite repression, the addition of the carbon source to the culture must be
carefully controlled. Fed batch is the most common tactic. Seconda
ry metabolites are
synthesized from primary metabolites. A well
-
organized production of antibiotics basically
requires a steady flow of their precursors.
[3]





3.0

METHODOLOGY



3.1

THE FED
-

BATCH FERMENTATION
PROCESS

[
3][5]



i.

Spores of
Penicillium
chrysogenum

will be used to inoculate 100ml of growth medium in
a 500ml shake flask.

ii.

After 4 days of incubation, contents are transferred into growth medium of a 500L
reactor.

iii.

After incubating for three days, this culture will be used to be inoculated in a

180L
reactors.

iv.

At 24 h intervals an aqueous solution of phenylalanine will be added to each culture.
Growth of the mycelia is observed by phase
-
contrast microscopy.

v.

Final fermentation will be completed in 5
-
6 days where the pH is about 6.5 and the
temper
ature will be in the range of 23
-
28˚C.

vi.

The reaction is stopped by an addition of 0.2 ml of 0.3 M ferric chloride reagent. The
reaction mixture is centrifuged at 20,000 g for 30 min and the sediment is removed.




3.2

CELL DISRUPTION



Since penicillin G i
s produced intracellular by
Penicillium chrysogenum
, the cells are disrupted
by using bead mill homogenizer.

In bead milling, a large number of minute glass
, ceramic or
steel beads are vigorously agitated by shaking or stirring. Disruption occurs by the crushing
action of the glass beads as they collide with the cells. Compared to ultrasonic and high
-
pressure
methods of cell disruption wet bead milling is low

in shearing force. The method has been used
for years to disrupt the microorganisms. It is considered the method of choice for disruption for
Penicillium chrysogenum
, as it works successfully.


Initially, to a cell suspension is added an equal volume of b
eads. Special high energy
electromechanical agitators have been developed for this process. Bead agitation by these
devices is either by shaking or stirring. The mixture is agitated at top speed for ten or more
minutes. Now these disrupted cells in the cel
l suspension is then filtered to obtain the penicillin
G and antibiotic Y.



3.
3

FILTRATION
(HARVESTING
)


At the time of harvesting, the fermentation broth is analyzed and it is discovered that the
specific product is present at a low concentration in an
aqueous solution that contains intact
micro
-
organisms, cell fragments, soluble and insoluble medium components and other metabolic
products. In the first stage, the main objective is to remove large solid particles and microbial
cells by filtration. Filtra
tion is the most versatile and most efficient method for removing
insoluble from our broth.



In filtration, the micro
-
organisms are captured in a concentrated cake, which looks like
sand, sludge or paste. Many factors influence the choice of filtration th
at takes place such as
viscosity and density of filtrate, solid: liquid ratio, size and shape of particles, scale of operation,
need for aseptic conditions, need for batch or continuous operation and the need for pressure or
vacuum suction to ensure an suf
ficient for rate for liquid. The Rotary Vacuum Filter is the most
common type of equipment used for the extraction of penicillin, and is used in continuous
processing. Rotary Vacuum Filter designs vary, but usually outline as follows:


• The Filter Drum:
Cylindrical, hollow drum which carries the filter cloth. On the inside it is
segmented into rows to which a vacuum can be applied or shut off in sequence as the drum
slowly revolves.

• Trough: Filter is partially immersed in through which contains the pen
icillin broth. The trough
is sometimes fitted with an agitator to maintain solids in suspension.

• Discharge Nodes: Filter cakes are produced from the filtration of to penicillin broth. Because of
this a node is devised to scrap off the cake after filtrat
ion. When this happens the vacuum is
broken.
[1]



The filter drum, partially submerged in the trough of broth, rotates slowly. Filtrate and
washings are kept separate by the segments in the drum. The liquid is drawn through the filter
and a cake of solids

builds up on the outer surface. Inside the drum, the filtrate moves from the
end of the cylindrical drum onto a storage tank. As our penicillin cells move from the broth, the
vacuum is used to remove as much moisture as possible from the cake, and to hold

the cake on
the drum. The section at the node/knife, which scrapes off the filtrate can get air pressure to burst
out, helping contact with the node.
[6
]


2. Filtrate Cooled



From filtration, the penicillin rich solution is cooled to 5°C. As penicillin G

only has a half
-
life
15 minutes at pH 2 at 20°C, this helps reduce enzyme and chemical degradation during the
aqueous two
-
phase extraction step later.


3. Further Filtration

Further filtration again takes place using the Rotary Vacuum Filter. In additio
n, we know that:


Rate of filtration = Driving force/resistance



Resistance can be caused by the filter cloth, which also adds to the resistance of the filter
cake as it accumulates. Pre
-
coats and filter aids were used to assist the filtration. The
addition of
a pre
-
coat/filter aid increases the strength of the filter cake and minimises compaction. Perlite, an
exploded rock, or diatomaceous earths are such materials added. This substance is built up over
the conventional filter, and each time the dru
m completes a cycle the shave
-
off gear moves
slightly nearer the drum. This continuous shaving away of contaminated earth prevents the filter
becoming clogged, and means that there is always a clean filter starting the next cycle. The pores
of their skelet
ons take up greasy materials also. Their addition to poor filters will increase the
rate of filtration greatly
.

[7
]





3.
4

SEPARATION (HARVESTING)



After harvesting product which contains the Penicillin G is filtered, Penicillin G needs to
be separated
from other filtrate which includes antibiotic Y. This can be done via the aqueous
phase separation using the PEG
-
dextran system. The PEG
-
dextran system is prepared consisting
the phases that are immiscible to one anot
her.
The upper phase is formed by the
m
ore

hydrophobic

polyethylene glycol (PEG), which is of lower

density

than the lower phase,
consisting of the more
hydrophilic

and denser dextran solution. Although PEG is inherently
denser than water, it occupies the upper layer. This is believed to be due to its solvent 'ordering'
properties, whi
ch excludes excess water, creating a low density water environment
.

[
9]


The filtrate is poured into the system. We can observe that the compound that moves or
partition towards the lower phase of the system which is hydrophilic. The compound would be
the Penicillin G. In this process, the penicillin G will partition more to th
e dextran in the system.
The separation occurs on the basis of partition coefficient of the solutions or compounds.
The
degree of polymerisation of PEG also affects the

phase separation

and the partitioning of
molecules during extraction.
[10]





3.
5

PENICILLIN G PURIFICATION


Recovery of the organic phase from the aqueous raffinate is also
crucial

to minimize costs
and environmental impact.
The aqueous

two phase
extract recovered in the previous stage is
carefully extracted back with aqueous sodium hydroxide. This is followed by charcoal treatment
to eliminate pyrogens

and by sterilization. Pure metal salts of penicillin can be safely sterilized
by dry heat, if desired. Thereafter, the aqueous solution of penicillin is subjected to
crystallization.

Then the crystals are filtrated, washed and dried.




Problems that can

arise in the process include the formation of emulsions during
extraction from the presence of surface
-
active impurities in the filtered broth. This e

ect can be
minimized by introducing appropriate surfactants that can also reduce the accumulation of soli
ds
in the extraction equipment. In addition, other organic impurities are present that can be co
-
extracted with the penicillin. It has been found that a number of these can be removed by
adsorption onto active carbon. Most of the penicillin is used as inte
rmediates in the production
of, for example, cephalosporin, but if it is necessary to produce pure penicillin for
pharmaceutical use, it can be purified by re
-
extraction at pH 2

2.5 and further stripping with a
phosphate solution at pH 6.
[8]











Figure 1:
Purification Process of Penicillin G

Aqueous t
wo
phase
extraction

4.0

REFERENCES



[1]

Antibiotic Production [online] 20
th

May 2012

Retrived from http://www.dcu.ie/~oshead/BE401/brewing/lecture4.pdf

[2]

Belter
, P. A.
, Cussler
, E.L.
,
Shou Hu
, W.(
1988).
Bioseparations : downstream processing
for biotechnology
.
New York : Wiley

[3]

Elmer L., & Gaden Jr. (2000). Fermentation Process Kinetics.
Journal Of Biochemical
And Microbiological Technology And Engineering
, 1(4)
, 413
-
419.

[4]

Jarvis, F. G., and Johnson,

M. J.
(
1947
).

T
he role of the constituents of synthetic media
for penicillin production. J.Am. Chem. Soc., 69, 3010
-
3017.

[5]

Keating, C. (2012). Aqueous Phase Separation as a Possible Route to
Compartmentalization of Biological Molecules. Acc. Chem. Res.,
Article ASAP.

[6]

Kurzatkowski W., Kurytowicz W., & Paszkiewicz A. (1982). Penicillin G Production by
Immobilized Fungal Vesicles.

European Journal of Applied Microbiology and
Biotechnology, 15
, 211
-
213.

[7]

Morikawa Y., Karube I., & Suzuki S. (1979). Penicillin G

production by immobilized
whole cells of Penicillium chrysogenum.
Biotechnology and Bioengineering, 21(2),

261
-
270.

[8]

Stanbury
, P.F.,

and Whitaker
, A. (1084).

Principl
es of fermentation technology.
1st ed

New York ; Oxford [Oxfordshire] : Pergamon Press

[9]

St
enekes,

R.,

Franssen
, O.,
van

Bommel
, E.,

Crommelin,

D.,

Hennink,

W.

(1999).

The

use

of

aqueous

PEG/dextran

phase

separation

for

the

preparation

of

dextran

microspheres.

Volume

183,

Issue

1,

Pages

29
-
32.

[10]

Taskin E., Eltem R., & Soyak

E. (2010). Enhancement of solid state fermentation
Penicillin for Production of Penicillin G on Sugar Beet Pulp.
BioResources, 5(1),

268
-

275.