full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 5 (3) 1325 -1337 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)
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and incubated in an orbital incubator shaker at
28 ± 2ÚC for 88 h. All experiments were carried
out in triplicates.
Optimization of fermentation medium using
one factor at-a-time method
Using the production medium selected for
the optimization study, the following parameters
were optimized
Effect of various carbon sources: The effect of
various carbon sources on production of
cephamycin C was studied by substituting
glucose in the production media with other
carbon sources such as fructose, galactose,
glycerol, lactose, maltose, sucrose, starch.
Several oils such as soybean oil, groundnut oil,
mustard oil, coconut oil and sesame oil were also
evaluated for their effect on cephamycin C
production. Each carbon source was used at 10
g/l. Reducing sugars were autoclaved separately
and added before inoculation. The carbon source
which supported maximum production of
cephamycin C was used for further studies.
Effect of inorganic and organic nitrogen
sources: Here ammonium chloride was
substituted with other nitrogen sources such as
yeast extract, peptone, malt extract, ammonium
nitrate, urea, ammonium sulphate, (NH 4
) 2
HPO 4
and (NH 4
)H 2
PO 4
to check their suitability for
cephamycin C production. Each nitrogen source
was used at 1 g/l. The nitrogen source which
supported maximum production of cephamycin
C was used for further studies.
Production profile of cephamycin C and growth
curve of N. lactamdurans NRRL 3802: To
obtain production profile for cephamycin C and
growth curve of N. lactamdurans NRRL 3802,
three flasks were taken out each day for a period
of 5 days and processed to determine cephamycin
C content and dry cell weight. All other
conditions were maintained as previously
described. The incubation period that gave
maximum production of cephamycin C was
chosen for subsequent experiments.
Effect of initial pH: In order to investigate the
effect of initial pH of the medium on cephamycin
C production, N. lactamdurans was cultivated
at different initial pH of medium (pH 5.0 - 8.0).
The pH was adjusted using 1N hydrochloric acid
or 1N sodium hydroxide. The fermentation was
carried out at 28 ± 2°C for 72 h. The optimum
pH was used for further studies.
Effect of inoculum size: To study the effect of
inoculum size on cephamycin C production, by
N. lactamdurans, the production media was
inoculated with 1 ml of 10 9 ,10 8 ,10 7 and 10 6 CFU/
ml in 50 ml of autoclaved medium and studied
for production of cephamycin C.
Effect of various minerals: Here each
component from the production medium was
removed one at-a-time (except glycerol, yeast
extract and L-glutamic acid) to check its effect
on cephamycin C production.
Optimization of fermentation medium using
statistical methods
Optimization using the L 16
-orthogonal array:
Taguchi design was used to determine the most
significant factors which affect the production
of cephamycin C. The design for the L 16
-
orthogonal array with 8 factors at two levels to
give a total of 16 experiments was developed
and analyzed using “MINITAB 13.32” software.
The factors selected were glycerol, yeast extract,
L-glutamic acid, K 2
HPO 4
, NaCl, MgSO 4
, FeSO 4
and Na 2
S 2
O 3.
Table 1 depicted L 16
-orthogonal
array design, which was used in the present study.
All experiments were performed in triplicates.
Optimization of concentrations of the selected
medium components by RSM: To examine the
Sequential optimization of cephamycin C