Biomedical Engineering Reference
In-Depth Information
center points for replication. The range and coding values selected for all independent
variables in this experimental design are listed in Table 2.
After this previous optimization, a new second-order orthogonal design was used to study
the effect of three factors (total sugars concentration, inoculum size and moisture content) on
amylase production in the optimal conditions calculated before (particle size, pH and
incubation temperature). In this case, the design consisted of 20 experiments with eight (2
3
)
factorial points, six axial points to form a central composite design with
α
= 1.525 and six
center points for replication.
The medium was supplemented with 2.5 mL of the basal salt solution and an appropriate
volume of distilled water containing different amounts of soluble potato starch to give both
the different moisture contents and total sugars concentration in the medium (mg of total
sugars per g of dry support) defined by the experimental design (Table 3). The media were
autoclaved (121ºC for 15 min), cooled and inoculated with 1.25 mL of a cell suspension
containing the appropriate amount of spores to obtain the inoculum rate (spores/g of dry sugar
cane bagasse) defined by the experimental design (Table 3). The media were then incubated
at 32.5ºC for 96 h.
All the solid state fermentations were done in triplicates and the data presented are mean
values of each experiment. Results were analyzed by Experimental Design Module of the
Statistica software package (Statistica 5.1 for Windows computer program manual; StatSoft
Inc. Tulsa, OK, USA). The response surfaces were plotted using the DeltaGraph software,
version 4.0 (SPSS, Inc., Chicago, IL, USA).
Table 2. Experimental domain and codification of the variables used in the factorial
design to test the effect of temperature (T) and pH on amylase production by A. oryzae
FQB-01 in SSF
Natural values
Coded values
T
(ºC)
pH
- 1.267
23.4
3.7
-1
25.0
4.0
0
31.0
5.3
+ 1
37.0
6.5
+ 1.267
38.6
6.8
Table 3. Experimental domain and codification of the variables used in the factorial
design to test the effect of total sugars (TS in mg/g of dry support (gds)), moisture
content (M in %) and inoculum size (IS in log (number of spores/gds)) on amylase
production by A. oryzae FQB-01 in SSF
Natural values
Coded values
TS
M
IS
- 1.525
9.5
58.5
4.2
-1
20.0
63.0
5.0
0
40.0
71.5
6.5
+ 1
65.0
80.0
8.0
+ 1.525
70.5
84.5
8.8
