67
CHAPTER 4
DESIZING OF ORGANIC COTTON FABRICS WITH
INDIGENOUSLY PRODUCED ALPHA-AMYLASE AND
THEIR ENZYME KINETICS
4.1
INTRODUCTION
The desizing of polyvinyl alcohol (PVA) starch based organic
cotton fabric using alpha-amylase enzyme with various process parameters
such as enzyme concentration, temperature and reaction time are discussed in
this chapter. These process variables are selected based on the Box-Behnken
design of experiment and output of experiment was resulted with weight loss
of the fabric and their results are optimized with minimum error. The test
results are analyzed using design-expert software 8.0 to predict the optimum
process parameters to achieve the required desizing efficiency and compared
their results with actual trials. The alpha amylase enzyme kinetics has been
studied at various concentration, temperature and reaction time to optimize
the desizing process parameters. The performance of desizing was assessed
by an iodine test and FTIR results to confirm the degradation of PVA starch
on the desized organic cotton fabrics.
4.2
MATERIALS AND METHODS
4.2.1
Warp Yarn Sizing and Fabrication
The sizing of warp yarns of organic cotton was carried out using
laboratory model yarn sizing machine and the average size add-on on the
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warp yarn was 12.21% and then the sized warp yarns were taken into warp
beam preparation for weaving. The average size add-on on the organic cotton
fabric was measured with respect to warp and weft yarn mass and was found
to be 8.27%. The aerial density of the PVA sized organic cotton grey fabric
after weaving was found to be 130.24 grams per square metre. The size recipe
of PVA starch used for the organic cotton warp yarn was as follow;
Polyvinyl alcohol (PVA) – 4 parts; Vegetable tallow – 2 parts; Anti
static agent – 0.3 parts; Gum tragacanth – 2 parts; Soap oil – 1 part; Thin
boiling starch – 40 parts and Water – required level of pick up.
4.2.2
Enzymatic Desizing
The organic cotton sized fabrics were treated with alpha-amylase
with various process variables such as enzyme concentration, temperature and
reaction time. The process variables were chosen according to the BoxBehnken method of statistical tool for process optimization. In this present
work, a systematic statistical approach has been adopted to obtain optimum
weight loss of the sized fabric with different process conditions. The response
surface methodology was used to develop a mathematical correlation between
the enzyme concentration, temperature and time. The enzymatic desizing
process was carried out at pH 6-7 and then the fabrics was thoroughly rinsed
with hot water and cold water and dried at 800 C using hot air oven and the
fabric was weighted using electronic balance with accuracy ±0.01grams.
4.2.3
Process Optimization- Desizing
4.2.3.1
Design of experiment
Experiments were conducted based on the Box-Behnken second
order design for three variables. In this experimental design enzyme
concentration (X1), temperature (X2), and time (X3) were taken as
69
independent variables. The variables were selected at three levels, which are 1, 0, +1. The response (Y) is given by a second order polynomial as shown
below,
k
Y
bo
k
bi X i
i 1
bii X i
i 1
2
k
k
bij X i X j
(4.1)
i j i 1
where Y-predicted response, b o-offset term, b i-linear effect, bii-squared effect,
bij-interactive effect. The above equation was solved using the design expert
(State-Ease Statistics Made-Easy, version 8.0.2, 2010) to estimate the
response of the independent variables. The actual design experiment and the
corresponding actual values for each variable are listed in Table 4.1 and 4.2
respectively.
Table 4.1
Alpha Amylase Enzyme process conditions - Box-Behnken
experimental design
Trial
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Coded values of the variables
Enzyme
Temperature Time
concentration (X1)
(X2)
(X3)
0
1
-1
0
0
0
-1
0
1
-1
-1
0
-1
1
0
1
0
1
1
-1
0
0
-1
-1
0
-1
1
0
0
0
-1
0
-1
1
0
-1
1
1
0
0
1
1
0
0
0
70
Table 4.2 Actual values of the variables for the coded values
Uncoded values of the variables
Coded
Enzyme
Temperature,
Time,
concentration, %
deg C
min
-1
2
50
30
0
3
55
45
+1
4
60
60
values
4.2.3.2
Effect of desizing process variables - Box-behnken design
The response surface methodology is an empirical modeling
technique, which is used to evaluate the relationship between a set of
controllable experimental factors and observed results. The Box-Behnken
design of experiment was used to study the optimization of process conditions
to get accurate output and predicted % weight loss of desized organic fabrics.
Several factors influence the desizing of PVA starch such as alpha-amylase
concentration, temperature and time play a vital role. To study the effect of
these variables Box-Behnken design is used. Treatments are carried out based
on the experimental design given in Table 4.1. The limits for the design in
terms of enzyme concentration, temperature and time was fixed after careful
consideration based on size add-on in warp threads during weaving operation
which playing a significant degradation level of starch in enzymatic desizing.
The effect of enzyme concentration and temperature on weight loss of PVA
desized organic fabric at various time intervals of (a) 30 min, (b) 45 min and
(c) 60 min are shown 3D surface plot in Figure 4.1.
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72
The empirical model was fitted to the response and the polynomial
equation derived from the design of experimental is given below taking into
account the significant interaction effects as given in Table 4.3.
Final Equation in Terms of Coded Factors:
Weight loss (%) = +7.60 + (0.80*X 1)+ (0.21* X2) +(0.74*X3) +
(0.001*X1* X2)-(0.15*X1*X3)-(0.025*X2*X3)2
2
2
(0.19*X1 ) + (0.088*X2 )) -(0.26*X3 )
(4.2)
where X1-Enzyme concentration (%); X2-Temperature in oC; X3- Time in
minutes.
The model F-value was 21.07 which implied that the model was
significant and there was only 0.18% chance that a “Model F-value” of this
large value could occur due to noise. The predicted R2 value was 0.9743 and is
in reasonable agreement with the Adjusted R2 of 0.9281. Adequate precision
which measures the signal to noise ratio was 16.132, which is greater than 4
indicating that the model can be used to navigate the design space. From the
Table 4.3 it is noticed that the enzyme concentration in the degradation of PVA
size starch in the desizing process was noticed significant differences at F actual
>Fcritical (F2,14 values of 93.94>21.07) at 95% confidence level. With respect to
the design of experiments, there is no significant differences found between
temperature levels of 50 oC to 60oC at Factual <Fcritical (F2,14 values of 6.63<21.07)
at 95% confidence level, it may be due to better stability and kinetics of
enzyme at selected temperature ranges in the desizing process. With respect to
reaction time in the desizing process, it was noticed significance differences at
Factual >Fcritical (F2,14 values of 79.84>21.07) at 95% confidence level, it may be
due to the enzyme reaction time on the degradation of PVA starch depends
mainly the selected range of times to achieve required weight loss of the PVA
starch on the sized fabric.
73
Table 4.3 Analysis of variance for the response surface
4.2.3.3
Source
F
P (Prob > F)
X1-Enzyme concentration
93.94
0.0002
X2-Temperature
6.63
0.0498
X3-Time
79.84
0.0003
X1 X2
1.000
0.0064
X1 X3
0.255
0.075
X2 X3
0.838
0.0037
Predicted Vs Actual
The predicted and actual weight losses of the PVA sized fabric
during desizing with alpha amylase are shown in Figure 4.2. From the test
results it was observed that the error % of the predicted and actual value of the
experimental design was noticed within 2%. It shows that the selected
experiment design of the box-behnken and the actual weight loss of the
experiment were no significant differences at 95% confidence level.
Figure 4.2
Relationship between the actual and predicted weight loss of
PVA desized organic cotton fabric
74
4.2.3.4
Effect of temperature and time
Figure 4.3 shows the weight loss of PVA desized organic cotton
fabric at various enzyme concentrations. It can be seen that at any given time,
the weight loss of the fabric increases with increasing temperature and it can
also be observed from the same figure that with increasing time, the desizing
efficiency is increasing at higher alpha amylase enzyme concentrations. At
higher concentration, the degradation of the PVA starch increases and it can be
attributed to the significant weight loss of the sized organic cotton fabric during
desizing as result of starch hydrolysis. Moreover, it should be mentioned that at
higher enzyme concentration, the increase in temperature and time aids in
faster hydrolysis resulting quick conversion of polymer chains into water
soluble products. Similar kind of results has also been observed by other
researchers (Tatsuma et al 1999) and has attributed to the autocatalytic effect
after certain time duration, thereby resulting in higher weight loss.
(a)
Figure 4.3 (Continued)
75
(b)
(c)
Figure 4.3
Effect of reaction time and temperature on weight loss of
PVA desized organic fabric at various enzyme concentrations
of (a) 2%, (b) 3% and (c) 4%
76
4.2.3.5
Effect of enzyme concentration and time
Figure 4.4 shows the contour plot of weight loss of the PVA starch
sized organic cotton fabric at different temperatures. It can be seen that the
weight loss of the fabric increases with the increase in concentration of the
enzymes used in both lower and higher temperatures. Moreover, it can be seen
from the figures that the amount of hydrolysis of size starch by the enzymatic
reaction is higher at higher temperature of 60 oC indicating hydrolysis of starch
chain groups into water soluble groups at the elevated temperature, thereby
increasing the weight loss. However, it was noted that the effect of time is more
pronounced at elevated temperature and can be seen by skew in the contour
lines.
(a)
Figure 4.4 (Continued)
77
(b)
(c)
Figure 4.4
Effect of enzyme concentration and reaction time on weight
loss of PVA starch desized organic fabric at various
temperatures of (a) 50 deg C, (b) 55 deg C and (c) 60 deg
78
4.2.3.6
Effect of enzyme concentration and temperature
Figure 4.5 presents the effect of enzyme concentration and
temperature on the weight loss of the PVA sized organic cotton fabrics at
various reaction times. With increase in enzyme concentration and temperature
there is an increase in desizing efficiency at both lower and higher reaction
time intervals but at higher time duration there is higher rate of PVA starch
hydrolysis with increase in enzyme concentration. Another interesting
observation that occurred during the trials was that specimen was noticed
higher desizing rate at higher enzyme concentration, time and temperature of
maximum weight loss of 8% and above.
(a)
Figure 4.5 (Continued)
79
(b)
(c)
Figure 4.5
Contour graphs represent the effect of enzyme concentration
and temperature at various reaction times of (a) 30 minute
(b) 45 minutes (c) 60 minutes
80
4.2.3.7
Process optimization
The desizing process variables such as alpha-amylase enzyme
concentration, temperature and reaction time was optimized using BoxBehnken experimental design and their output values are executed using
Design-Expert software 8.0. The software of design-expert was executed to get
various options / predicted process parameters to achieve required weight loss
of the fabric 8.0% during desizing of PVA starched organic cotton fabric. The
software was processed and it resulted the desirability and FDS-Fraction of
Design Space of design model of process is shown in Figure 4.6 and 4.7
respectively. The output result of the design-expert software to achieve the
desired weight loss of 8.0% in the PVA starch desizing process is predicted the
process variables of 9 solutions which is shown in Table 4.4, out of which the
software opted best process conditions of alpha-amylase enzyme concentration
of 3.37% at temperature of 550 C and reaction time 48 minutes with 1.0%
desirability which is shown in Figure 4.8. From the best opted test results, the
actual weight loss of the desizing process of the organic cotton fabric was
achieved 7.90% with error of 1.07%.
Figure 4.6
Desirability of process optimization of PVA starch desized
organic cotton fabric for achieving 8% weight loss
81
Figure 4.7
Fraction of Design Space (FDS) of design model of PVA
desizing process
Table 4.4 Output of design-expert software
Solution
no.
Enzyme
concentration, %
Temperat
ure, C
Time,
min
Weight
loss, %
1
3.37
55.34
47.72
7.99999
2
3.27
55.21
49.62
7.99998
3
3.05
50.57
59.29
8.00001
4
3.36
52.07
50.79
7.99999
5
3.03
58.29
49.63
8.00001
6
3.14
51.53
56.08
7.99999
7
3.94
53.65
41.90
8.00001
8
3.05
52.31
58.17
8.00002
9
3.24
58.41
45.72
8
82
Figure 4.8
Predicted optimization of process variables for achieving 8%
weight loss of PVA desized organic fabric (Output results of
Design-Expert software 8.0)
4.2.4
Characteristics of the Desized Organic Cotton Fabrics
4.2.4.1
Iodine test
The desizing efficiency of the PVA starch sized fabric was assessed
by the presence of starch on the fabric in the iodine test which indicates the
depth of color of the starch. The various enzyme concentration treated organic
cotton fabrics at 2%, 3% and 4% levels compared with grey organic sized
cotton fabric which are shown in Figure 4.9. The absence or pale white color of
the desized fabric is the indication of removal of starch during enzymatic
desizing.
83
(a)
(c)
Figure 4.9
(b)
(d)
Iodine test for assessing the desizing efficiency at (a) grey
organic cotton fabric (b) 2% enzyme conc. (c) 3% enzyme
conc. (d) 4% enzyme concentration
84
4.2.4.2
FTIR spectroscopic analysis
The FTIR spectra of the grey organic cotton fabric (without sizing)
and sized with PVA starch are shown in Figure 4.10. It can be clearly
understood the presents and integrity of the PVA and other sizing compounds
in the sized organic cotton fabric. The hydrolysis of the PVA starch during
enzymatic desizing at 8% level of weight loss of the organic cotton fabric
sample shows the removal of PVA groups in the specimen after desizing of 60 0
C reaction temperature and 60 min time at 4% enzyme concentration at 3315
cm-1 which is responsible for –OH group stretching. The test results, the
transparency (%) of the sized organic cotton fabrics are noticed lower level
when compared to grey fabric which is due to the starch and other ingredients
present in the size paste. The residual size components are analyzed after
desizing using FTIR reports by differentiating the transparency (%) wave
length of the grey and desized organic cotton fabrics are given in Table 4.5 in
which at 1058cm-1, 1112cm-1 and 2362cm-1 groups are responsible for
C-C
stretch from phenyl ring, -CH 2 symmetric stretching and C-H stretching in the
desized organic cotton fabrics.
85
86
Table 4.5
FTIR test results of Organic cotton fabrics and their
transparency values
Transparency, %
Wave length,
cm-1
After
Sized
desizing
fabric,
Reduction Difference,
%
%
Grey
Sized
desized
fabric
fabric
fabric
559
45.8
39.2
45.8
14.41
0.00
617
45.3
38.4
45.2
15.23
-0.22
667
46.4
38.6
45.8
16.81
-1.29
898
53.2
44.2
53.4
16.92
0.38
1058
29.3
30.2
34.8
3.07
18.77
1112
31.3
29.8
35.3
4.79
12.78
1165
36.2
32.4
37.5
10.50
3.59
1371
39.8
35.2
45.0
11.56
13.07
1431
44.2
36.4
44.8
17.65
1.36
1612
51.4
37.5
52.2
27.04
1.56
2362
53.5
36.3
46.8
32.15
-12.52
2901
36.3
28.4
36.3
21.76
0.00
3415
22.2
20.4
22.7
8.11
2.25
87
4.3
CONCLUSION
The process optimization of desizing of PVA starch sized organic
cotton fabric have been studied and the process variables such as alpha
amylase enzyme concentration, temperature and reaction time was optimized
to achieve the required desizing efficiency in terms of weight loss % of fabric
and degradation of PVA starch during desizing process on the fabrics was
assessed by iodine test and FTIR test results. The alpha-amylase enzymes are
better active and catalyze the degradation of PVA starch at temperature range
of 50-550 C and time of 30-40 min to achieve required level of 8% size
removal efficiency. The pH of the desizing bath is major influence for better
reaction of enzyme to catalyze the hydrolysis of starch groups. The higher
enzyme concentration at 4% level and higher temperature of 60 oC were
noticed lesser time to achieve required desizing efficiency. Process variables
are optimized using design-expert software 8.0 and it will pave the way to
predict the enzyme kinetics at various concentrations, temperature and
reaction time to achieve required desizing efficiency with minimum error %.
This study will be helpful to the organic cotton processors for the eco-friendly
and sustainable textile wet processing using alpha amylase enzyme in desizing
of PVA starch based desizing operations.