76 N. Parvez et al.
Validation of Optimization of Media
For validation studies, periodic analysis of chitin-
ase activity up to 5 days was performed by tak-
ing six separate 20 ml fermentation systems for
unoptimized fermented KBCG broth with 1.5 %
colloidal chitin v/v at 29 °C and compared with
periodical responses in optimized media pro-
vided by the design. Log CFU/ml and antifungal
activity of matured broth at the time period of
maximum chitinase activity were also analyzed.
Results and Discussion
Effects of crude glycerol and inducers chitinase
assay (Uml−1) were performed from supernatants
of cultured broth for 84 h. Among all the eight,
which had amendments in basal KB media and
KB media with crude glycerol (KBCG), super-
natant of cultured broth in presence of colloi-
dal chitins showed significantly higher 3.51and
2.72 Uml−1 chitinase activity, respectively. Inhi-
bition zone and microbial biomass were higher
in case of colloidal chitins with KB and KBCG,
respectively (Table 5 ). Colloidal chitin proved
to be the most significant inducer for chitinase
production in P. aeruginosa p-15 after 84 h. In
presence of altered carbon source as crude glyc-
erol as carbon source in KB media showed higher
chitinase activity and inhibition diameter and ex-
hibited significantly higher microbial biomass
(Fig 1 ).Screening of Important Medium
Components via Plackett–Burman
Design
To enhance the production of chitinase, Plack-
ett–Burman design was employed as a statistical
approach for the screening of suitable medium
components. Table 2 represents the independent
variables and their respective high and low con-
centrations used in the optimization study, while
Table 3 shows the Plackett–Burman experimen-
tal design for 12 trials with two levels of concen-
trations for each variable and the corresponding
chitinase activity in terms of (Uml−1) of the cul-
ture medium. The variables X 1 −X 6 represent the
experimental variables, whereas D 1 −D 5 represent
the dummy variables. Table 6 represent the ef-fect, S.E., t( (^) xi), and P-values of each process vari-
able for chitinase production.
The significant process variables were
screened at probability value ( P ≤ 0.05). The
probability value of crude glycerol, colloidal
22
Actual chtinase activity U/ml
lm/UytivitcaesanitihcdetciderP
1.30
1.88
2.45
3.02
3.60
1.35 1.90 2.45 3.00 3.55
Fig. 1 Deviations in predicted and observed response