Date Published: June 3, 2015
Publisher: Springer Berlin Heidelberg
Author(s): Abhishek Walia, Preeti Mehta, Shiwani Guleria, Chand Karan Shirkot.
The effects of yeast extract (X1),
NH4NO3 (X2), peptone
(X3), urea (X4), CMC (X5),
Tween 20 (X6), MgSO4 (X7), and
CaCO3 (X8) on production of xylanase from Cellulosimicrobium cellulans CKMX1 were optimized by statistical analysis
using response surface methodology (RSM). The RSM was used to optimize xylanase production by
implementing the Central composite design. Statistical analysis of the results showed that the
linear, interaction and quadric terms of these variables had significant effects. However, only the
linear effect of X4, X5, interaction effect of
X5X8 and quadratic effect of X32, X52 and X72 found to be insignificant terms in the quadratic model and had no response at significant
level. The minimum and maximum xylanase production obtained was 331.50 U/g DBP and 1027.65 U/g DBP,
respectively. The highest xylanase activity was obtained from Run No. 30, which consisted of yeast
extract (X1), 1.00 g (%);
NH4NO3 (X2), 0.20 g (%);
peptone (X3), 1.00 g (%); urea (X4), 10 mg (%); CMC
(X5), 1.00 g (%); Tween 20 (X6), 0.02 mL (%);
CaCO3 (X7), 0.50 g (%) and
MgSO4 (X8), 9.0 g (%). The optimization resulted in
3.1-fold increase of xylanase production, compared with the lowest
xylanase production of 331.50 U/g DBP after 72 h of incubation in stationary flask experiment.
Application of cellulase-free xylanase in pulp biobleaching from C.
cellulans CKMX1 under C–EP–D sequence has been shown to bring
about a 12.5 % reduction of chlorine, decrease of 0.8 kappa points (40 %), and gain in brightness
was 1.42 % ISO points in 0.5 % enzyme treated pulp as compared to control.
Xylan is the major hemicellulosic constituent of hard and soft wood and is the next
most abundant renewable polysaccharide after cellulose. This complex heteropolysaccharide consists
of a main chain of 1,4-β-d-xylose monomers and short chain
branches consisting of O-acetyl, α-l-arabinofuranosyl and α-d-glucuronyl residues.
Xylanases and associated debranching enzymes produced by a variety of microorganisms including
bacteria, yeast and filamentous fungi, bring about the hydrolysis of hemicelluloses (Maheshwari et
al. 2000). Xylanolytic enzymes are receiving increasing
attention because of their potential application in pulp bleaching (Goluguri et al. 2012; Singh et al. 2013)
and bioconversion of lignocelluloses into feedstocks and fuels (Kim et al. 2000). The xylan degrading system includes endo-1,4-xylanases
(1,4-β-xylan xylanohydrolase; EC 220.127.116.11), which release long and short xylo-oligosaccharides, and
other xylanases that attack only longer chains, and β-d-xylosidase (1,4-β-xylan xylohydrolase; EC 18.104.22.168), which remove d-xylose residues from short xylo-oligosaccharides (Gomez et al.
2008; Saha 2003).
The kappa number (Tasman and Berzins 1957) of enzyme pre-treated wheat straw pulp was lower than control. At an enzyme
dosage of 0.2 and 0.5 %, the enzymatic pre-treatment decreased kappa number by 0.9 points or 7.69 %
and 1.4 points or 11.96 %, respectively, as compared with control. In addition to this, enzymatic
pre-treatment of 0.2 % of pulp increases the brightness points to 1.1 % ISO, while enzyme dose of
0.5 % of pulp increases the brightness points to 2.2 % ISO. Cellulase-free xylanase from C. cellulans CKMX1 under C–EP–D sequence has been shown to bring about a
6.10 % reduction or savings of chlorine in 0.2 % enzyme treated pulp and 12.5 % reduction or savings
of chlorine in 0.5 % enzyme treated pulp as compared to control treatment where no enzyme
pre-treatment was given, when enzymatically prebleached pulp was charged with 7.4 % of total
chlorine. Decrease of 0.5 kappa points or 25 % was observed in treatment where enzyme dose was 0.2 %
of pulp, and decrease of 0.8 kappa points or 40 % was found in enzyme treatment of 0.5 % of pulp as
compared to control. Paper sheets were prepared using 60 g of pulp on OD basis. Enzyme dose of 0.2 %
of pulp increased brightness to 84.71 % ISO points and enzyme dose of 0.5 % of pulp increased the
brightness to 85.2 % ISO points as compared to control treatment where brightness was observed to be
83.78 % ISO points. Gain in brightness points was 0.93 % ISO in enzymatic treatment of 0.2 % of pulp
and 1.42 % ISO points in enzymatic treatment of 0.5 % of pulp.
Nowadays, there is growing acceptance of the use of statistical experimental
designs in biotechnology to optimize culture medium components and conditions (Khucharoenphaisan et
al. 2008; Wang et al. 2008; Coman and Bahrim 2011). Many
studies have reported satisfactory optimization of xylanase production from microbial sources using
a statistical approach (Silva and Roberto 2001; Li et
al. 2007a, b; Coman and Bahrim 2011). RSM and CCD
were employed to optimize a fermentation medium for the production of xylanase by C. cellulans CKMX1 at pH 8.0. The optimization resulted in 3.1-fold
increase of xylanase production, compared with the lowest xylanase production of 331.50 U/g DBP
(Table 2). Dobrev et al. 2006 also showed that the xylanase activity obtained with the optimized nutrient
medium is 33 % higher than the activity, achieved with the basic medium. The application of
statistical design for screening and optimization of culture conditions for the production of
xylanolytic enzymes allows quick identification of the important factors and the interactions
between them (Katapodis et al. 2007; Vasconcelos et al.
2000). The RSM applied to the optimization of xylanase
production in this investigation suggested the importance of a variety of factors at different
levels. A high degree of similarity was observed between the predicted and experimental values,
which reflected the accuracy and applicability of RSM to optimize the process for enzyme production
(Techapun et al. 2002; Vasconcelos et al. 2000). The ANOVA (F test) shows
that the second model was well adjusted to the experimental data. The CV indicates the degree of
precision with which the treatments were compared (Wang et al. 2008; Vasconcelos et al. 2000). Usually,
the higher the value of CV, the lower the reliability of experiment is. Here, a lower value of CV
(4.13) indicated a better precision and reliability of the experiments. The precision of a model can
be checked by the determination coefficient (R2) and correlation coefficient (R).
The determination coefficient (R2) implies that the sample variation of 97.59 % for xylanase production
was attributed to the independent variables, and only about 2.41 % of the total variation cannot be
explained by the model (Table 3). Normally, a regression
model having an R2 value higher than 0.9 is considered to have a very high correlation.
The closer the value of R (correlation coefficient) to 1, the
better the correlation between the experimental and predicted values (Li et al. 2007b). Here, the value of R
(0.9971) for Eq. (1) indicates a close agreement between the
experimental results and the theoretical values predicted by the model equation. Linear and
quadratic terms were both significant at the 1 % level. Therefore, the quadratic model was selected
in this optimization study. There have been reports on optimization of culture media using
statistical approaches for a few bacterial xylanases processes but not for cellulase-free,
alkali-stable xylanases in SSF of apple pomace (Li et al. 2007a, b). The statistical optimization
approach is efficient and has been applied successfully to SSFs that have overcome the limitations
of classical empirical methods (Yu et al. 1997; Ellouze
et al. 2008). A response surface method with
three-factor-three-level design has been used to optimize the medium components and its pH, for
maximum xylanase production by Bacillus circulans D1 in submerged
fermentation (SmF), which resulted in a maximum concentration of 22.45 U/mL (Bocchini et al.
2002; Senthilkumar et al. 2005). Similarly, xylanase production by Schizophyllum
commune and Thermomyces lanuginosus has been maximized
by CCRD method, and the maximum xylanase yields were 5.74 and 2.7 U/mL, respectively, in SmF
(Haltrich et al. 1993; Purkarthofer et al. 1993). The results of CCD indicate the significance of yeast extract
(X2), peptone (X3), Tween 20
(X6), CaCO3 (X7), and
MgSO4 (X8) on production of xylanase by C. cellulans CKMX1. Despite some interactions, maximum interactions of
different variables i.e. X1,X2,
X3, X6, X7,
X7X8, X12, X22, X42, X62, X82, respectively, in the present investigation were found to be significant.
Statistical optimization of cultivation conditions using the central composite
appeared to be a valuable tool for the production of xylanase by C.
cellulans CKMX1. The predicted and actual xylanase activity under optimal conditions in
stationary flasks experiments were 1041.93 U/g DBP and 1027.65 U/g DBP, respectively. A scale-up of
the fermentation process was carried out in a aluminium trays to reconfirm the maximum xylanase
activity of 1150.37 U/g DBP after 72 h cultivation under optimized conditions. Cellulase-free
xylanase from C. cellulans CKMX1 under
C–EP–D sequence has been shown to bring about a 12.5 % reduction of chlorine,
decrease of 0.8 kappa points (40 %) and gain in brightness was 1.42 % ISO points in 0.5 % enzyme
treated pulp as compared to control where no enzyme pre-treatment was given, when enzymatically
prebleached pulp was charged with 7.4 % of total chlorine. From the present studies, it is clear
that C. cellulans CKMX1 xylanase is having the characteristic
suited for an industrial enzyme.