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Abstract
Response surface methodology were used to study the cumulative effect of the culture conditions and to enhance the production of
extracellular ribonuclease in shake flask fermentation by Aspergillus niger. These conditions considered include initial pH, inoculum size,
total carbon, ratio of glucose to corn powder, NH4 NO3 and K2 SO4 . The relative importance of these conditions on ribonuclease production
was investigated by using the Plackett–Burman experimental design. Initial pH, ratio of glucose to corn powder and NH4 NO3 were found
to significantly influence the ribonuclease production. For obtaining the mutual interaction between the variables and optimizing these
variables, a 23 factorial central composite design using response surface methodology was employed. Initial pH has a significant positive
effect on the ribonuclease production. NH4 NO3 has a significant negative effect. The mutual interactions have no significant effect. The
ribonuclease production was increased by 23.2% compared to that under unoptimized conditions. The fermentation time was also shortened
after optimization.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Ribonuclease; Aspergillus niger; Optimization; Response surface methodology
1369-703X/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2004.04.010 转载
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28 Y.-H. Xiong et al. / Biochemical Engineering Journal 21 (2004) 27–32
design using response surface methodology were employed 2.3.1. Plackett–Burman design
for optimization. Plackett–Burman experimental design [18] based on the
first order model:
2. Materials and methods Y = β0 + βi xi (1)
The optimization of culture conditions for RNase produc- where y = predicted response, β0 = offset term, βi = linear
tion by A. niger SA-13-20 was carried out in two stages. effect, βii = squared effect, and βij =interaction effect.
Table 1
Plackett–Burman experimental design matrix for screening of culture conditions of RNase production by Aspergillus niger SA-13-20
Run no. Variables/levels RNase (U ml−1 )
X1 X2 X3 X4 X5 X6 X7
1 + + + + − − − 1256.9
2 − + + + + − − 1118.3
3 − − + + + + − 1200.1
4 − − − + + + + 1074.1
5 + − − − + + + 1206.5
6 + + − − − + + 1467.8
7 + + + − − − + 1143.5
8 − − − − − − − 1045.8
X1 , initial pH at a high level of 2.50 and a low level of 2.20; X2 , inoculum size at a high level of 12.5% (v/v) and a low level of 10.0 (v/v); X3 , dummy
variable; X4 , total carbon at a high concentration of 187.5 g l−1 and a low concentration of 150.0 g l−1 ; X5 , ratio of glucose to corn powder at a high
level of 1:3 and a low level of 1:4; X6 , NH4 NO3 at a high concentration of 2.50 g l−1 and a low concentration of 2.00 g l−1 ; and X7 , K2 SO4 at a high
concentration of 0.110 g l−1 and a low concentration of 0.087 g l−1 .
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Table 2
Central composite design matrix of independent variables with corresponding experimental and predicted value
Run order no. Initial pH Ratio of glucose to corn powder NH4 NO3 (g l−1 ) RNase activity (U ml−1 )
The Design Expert was used for regression analysis of the above explanation. So, the analytic method may be reliable,
data obtained and to estimate the coefficients of the regres- especially after 2-day fermentation.
sion equation. Isoresponse contour plots were also obtained Analyses were carried out in duplicate. The data given
by using Design Expert. here are the average of the measurements.
Table 3
Results of the screening experiment for RNase production by Aspergillus
niger SA-13-20 Table 5
The least-squares fit and the parameter estimates (significance of regres-
Variable Exi β t-values Confidence level (%) Model Parameter Degree of Computed t P (P > |t|)
sion coefficient). The number in parentheses is the standard error
term estimate freedom
X1 636.4 318.2 8.440 **
X2 460.0 230.0 6.101 * Intercept 1495.2 (56.16) 1
X4 −214.2 −107.1 −2.841 x1 87.2 (37.26) 1 2.340 0.0412
X5 −315.0 −157.5 −4.178 * x2 55.6 (37.26) 1 1.492 0.1668
X6 380.0 190.0 5.093 * x3 −149.0 (37.26) 1 −3.999 0.0025
X7 270.3 135.1 3.585 x1 x2 40.3 (48.68) 1 0.828 0.4268
x1 x3 −25.2 (48.68) 1 −0.518 0.6160
x2 x3 10.1 (48.68) 1 0.207 0.8402
x12 −89.7 (36.27) 1 −2.473 0.0329
Significant levels of regression coefficient are given as (**) 90% and (*) x22 40.0 (36.27) 1.103 0.2964
85% by t-test. x32 −127.1 (36.27) 1 −3.504 0.0057
3.2. Optimization of the screening culture conditions for
RNase production
Because 12.5% (v/v) of inoculum size was already high pared. Usually, the higher the value of CV, the lower is
enough, the other variables (initial pH, ratio of glucose to the reliability of experiment. Here, a lower value of CV
corn powder and NH4 NO3 ) showing confidence levels above (10.02%) indicates a greater reliability of the experiments
85% in the Plackett–Burman design were selected and op- performed.
timized using central composite design. The experimental The Student t-distribution and the corresponding
and predicted response of RNase production at 5 days of P-values, along with the parameter estimate, are given in
cultivation by A. niger SA-13-20 are given in Table 2. By Table 5. The P-values are used as a tool to check the sig-
applying multiple regression analysis on the experimental nificance of each of the coefficients which, in turn, are nec-
data, the following second order polynomial equation was essary to understand the pattern of the mutual interactions
found to explain the RNase production: between the best variables. The smaller the magnitude of
the P, the more significant is the corresponding coefficient.
y = 1495.2 + 87.2x1 + 55.6x2 − 149.0x3 − 89.7x12 The parameter estimate and the corresponding P-values
+40.0x22 − 127.1x32 + 40.3x1 x2 − 25.2x1 x3 + 10.1x2 x3 (Table 5) suggest that among the independent variables
initial pH (x1 ) and NH4 NO3 (x3 ) have a significant effect
(4)
on RNase production. The quadratic term of these two
where y = predicted response; x1 , x2 , and x3 are the coded variables also have a significant effect. But the independent
values of initial pH, ratio of glucose to corn powder and variable, ratio of glucose to corn powder (x2 ), has no effect.
NH4 NO3 , respectively. And there is no interaction. The effect of NH4 NO3 (x3 ) is
Statistical testing of the model was done by the Fisher’s greater than that of pH (x1 ). It was also found that RNase
statistical test for analysis of variance (ANOVA) and the re- production increased with the increase of initial pH and
sults are shown in Table 4. The analysis of variance of the with the decrease of NH4 NO3 concentration.
quadratic regression model demonstrates that the model is A representative contour plot which is more or less spher-
highly significant, as the computed F-value (Fmodel = 4.92) ical is shown in Fig. 1. Fig. 1 shows the relative effect of
is much greater than the tabular F10,9 value (3.02) at the initial pH and NH4 NO3 on RNase production. The regres-
5% level. The closer the value of R (multiple correlation sion equation (Eq. (4)) was solved by using Design Expert.
coefficient) to 1, the better the correlation between the ob- The optimal values of test variables in the coded units were
served and predicted values. Here the value of R (0.9032) x1 = 0.95, x2 = 1.68, and x3 = −0.61. At these values,
indicates a good agreement between the experimental and the initial pH, the ratio of glucose to corn powder and the
predicted values. The coefficient of variation (CV) indicates concentration of NH4 NO3 were 2.98, 0.33, and 2.20 g l−1 ,
the degree of precision with which the treatments are com- respectively. The maximum predicted value of RNase yield
obtained was 1815.4 U ml−1 .
Table 4 A repeated fermentation of RNase under optimal condi-
Analysis of variance (ANOVA) for the selected quadratic modela
tions was carried out. The result is given in Fig. 2. The
Source Sum of Degrees of Mean F-value P>F maximal RNase level obtained was 1750.0 U ml−1 . This
squares freedom square value was found to be 3.6% less than the predicted value.
Model 839700 9 93305.3 4.92 0.0102 This discrepancy might be due to the slight variation in ex-
Error 189600 10 18960.5 perimental conditions. The optimization resulted in 23.2%
increase of RNase production (from 1420 U ml−1 of maxi-
Corrected 1029000 19
total
a Coefficient of variation (CV) = 10.02%; coefficient of determination mal RNase level at the sixth day under unoptimized condi-
(R2 ) = 0.8158; correlation coefficient (R) = 0.9032, and adjusted R2 tions to 1750 U ml−1 at the fifth day under optimized con-
= 0.6500. ditions) and 19.3% increase of dry cell weight. The fer-
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Y.-H. Xiong et al. / Biochemical Engineering Journal 21 (2004) 27–32 31
3.30 4. Conclusion
1370. 0
1423. 2 initial pH and with the decrease of NH4 NO3 concentration.
2.50 The optimization enhanced the RNase production by 23.2%
and shortened the fermentation time.
156 7.2
2.10
1529. 6 Acknowledgements
1468. 1 1423 .2
1.70
We are grateful to the National Natural Science Founda-
tion of China, the Natural Science Foundation of Guangdong
1.66 2.08 2.50 2.92 3.34
Province for their financial support.
Initial pH
Fig. 1. Isoresponse contour plot of RNase production for initial pH and
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