lignocellulosic materials represent an abundant and inexpensive source of sugars and can be microbiologically converted to industrial products .
xylitol ( c5h12o5 ) , a sugar alcohol obtained from xylose , is generated during the metabolism of carbohydrates in animals and humans .
xylitol was present in fruits and vegetables , at low concentration , which makes its production from these sources economically unfeasible .
its sweetening power was comparable to that of sucrose and is higher than that of sorbitol and mannitol .
in addition to reducing dental caries , xylitol also promotes tooth enamel remineralization by reversing small lesions .
this happens because , when in contact with xylitol , the saliva seems to be favorably influenced ; the chemical composition of xylitol induces the calcium ions and phosphate . for these characteristics , xylitol was a feed stock of great interest to food , odontological , and pharmaceutical industries .
however it was expensive and it requires several steps of xylose purification before the chemical reaction [ 4 , 9 , 10 ] .
xylitol production through bioconversion has been proposed as for utilizing microorganism such as yeast , bacteria , and fungi [ 11 , 12 ] . among these
, yeast has shown to possess desirable properties for xylitol production [ 13 , 14 ] .
therefore , for the present study , yeast strain pachysolen tannophilus was selected for xylitol production .
furthermore studies have shown that nutritional factors including sources of carbon and nitrogen can influence xylitol production .  
corncob is a large volume solid waste for using sweet corn processing industry in india .
they are currently used as animal feed or returned to the harvested field for land application .
corncob contains approximately over 40% of the dry matter in residues   and thus has value has a raw material for production of xylose , xylitol , arabinose , xylobiose , and xylo oligosaccharides .
these carbohydrates mainly consist of the xylose and other minor pentose [ 1820 ] . among various agricultural wastes ,
corncob was regarded as promising agricultural resources for microbial xylitol production . in microbial production of xylitol from corncob ,
the cobs were first hydrolysed to produce from hemicelluloses by acid hydrolysis and the corncob hydrolysate is then used as the medium for xylitol production .
the bioconversion of xylitol is influenced by the concentration of various ingredients in culture medium .
this study also investigates the effect of process variables such as ph , temperature , substrate concentration , inoculum size , and agitation speed on xylitol yield .
response surface methodology ( rsm ) is a mathematical and statistical analysis , which is useful for the modeling and analysis problems that the response of interest is influenced by several variables .
rsm was utilized extensively for optimizing different biotechnological process [ 22 , 23 ] .   in the present study ,
the screening and optimization of medium composition and process variables for xylitol production by pachysolen tannophilus using plackett - burman and rsm were reported .
the plackett - burman screening design was applied for knowing the most significant nutrients enhancing xylitol production .
then , box - behnken design and central composite design ( ccd ) were applied to determine the optimum level of significant nutrients and process variables , respectively .
the yeast strain pachysolen tannophilus ( mtcc 1077 ) was collected from microbial type culture collection and gene bank , chandigarh .
the lyophilized stock cultures were maintained at 4c on culture medium supplemented with 20  g of agar .
the medium composition ( g / l ) was compressed of the following : malt extract : 3.0 ; yeast extract : 3.0 ; peptone : 5.0 ; glucose : 10.0 at ph : 7 .
corncob was collected from perambalur farms , tamil nadu , india , and was dried in sunlight for 2 days , crushed , and sieved for different mesh size ranging from 0.45  mm to 0.9  mm ( 2040 mesh ) and used for further studies .
the composition of the corncob ( g / l ) : xylose : 28.7 , glucose : 5.4 , arabinose : 3.7 , cellobiose : 0.5 , galactose : 0.7 , mannose : 0.4 , acetic acid : 2 , furfural : 0.8 , hydroxymethyl furfural : 0.2 was used for xylitol production .  
2  g of corncobs at a solid loading of 10% ( w / w ) was mixed with dilute sulfuric acid ( 0.1% ( w / v ) ) and pretreated in an autoclave at 120c with residence time of 1 hour .
the liquid fraction was separated by filtration and the unhydrolysed solid residue was washed with warm water ( 60c ) .
hemicellulose acid hydrolysate was heated at 100c for 15  min to reduce the volatile components .
the hydrolysate was overlined with solid ca(oh)2 up to ph 10 , in combination with 0.1% sodium sulfite , and filtered to remove the insoluble materials .
activated charcoal treatment was an efficient and economic method of reduction in the amount of phenolic compounds , acetic acid , aromatic compounds , furfural , and hydroxymethylfurfural normally found in hemicellulosic hydrolysates .
after centrifugation , the solutions were mixed with powdered charcoal at 5% ( w / v ) for 30 and stirred ( 100  rpm ) at 30c .
the liquor was recovered by filtration , chemically characterized , and used for culture media .
fermentation was carried out in 250  ml erlenmeyer flasks with 100  ml of pretreated corncob hemicelluloses hydrolysate is adjusted to ph 7 with 2  m h2so4 or 3  m naoh and supplemented with different nutrients concentration for tests according to the selected factorial design , were used for fermentation medium and sterilized at 120c for 20  mins .
the flasks were maintained at 30c for agitation at 200  rpm for 48 hours .
after the optimization of medium composition , the fermentation was carried out with different parameter levels ( table 5 ) with the optimized media for tests according to the selected factorial design . during the preliminary screening ,
the experiments were carried out for 5 days and the maximum production was obtained in 48 hours .
sugar and sugar alcohol in the culture broth were measured by high - performance liquid chromatography ( hplc ) , model lc-10-ad ( shimadzu , tokyo , japan ) equipped with a refractive index ( ri ) detector .
the chromatography column used was a aminex hpx-87h ( 300  7.8  mm ) column at 80c with 5  mm h2so4 as mobile phase at a flow rate of 0.4  ml / min , and the injected sample volume was 20  l . the rsm has several classes of designs , with its own properties and characteristics .
central composite design ( ccd ) , box - behnken design , and three - level factorial design are the most popular designs applied by the researchers .
a prior knowledge with understanding of the related bioprocesses is necessary for a realistic modeling approach .
it assumes that there are no interactions between the different variables in the range under consideration .
plackett - burman experimental design is a fractional factorial design and the main effects of such a design may be simply calculated as the difference between the average of measurements made at the high level ( + 1 ) of the factor and the average of measurements at the low level ( 1 ) . to determine which variables significantly affect xylitol production ,
nine variables were screened in 12 experimental runs ( table 1 ) , and insignificant ones are eliminated in order to obtain a smaller , manageable set of factors . the low level ( 1 ) and high level ( + 1 ) of each factor ( 1 , + 1 )
g / l ) : k2hpo4 ( 6.6 , 7 ) , yeast extract ( 1.5 , 5 ) , peptone ( 2 , 5 ) , kh2po4 ( 1.2 , 3.6 ) , xylose ( 9.8 , 10.2 ) , ( nh4)2so4 ( 1 , 4 ) , mgso47h2o ( 0.7 , 1.3 ) , malt ( 2.8 , 3.2 ) , and glucose ( 9.8 , 10.2 ) , and they were coded with a , b , c , d , e , f , g , h , i , respectively . the statistical software package  minitab 16  is used for analyzing the experimental data .
once the critical factors are identified through the screening , the box - behnken design was used to obtain a quadratic model after the central composite design ( ccd ) was used to optimize the process variables and obtain a quadratic model .
the box - behnken design and ccd was used to study the effects of the variables towards their responses and subsequently in the optimization studies .
this method was suitable for fitting a quadratic surface , and it helps to optimize the effective parameters with a minimum number of experiments , as well as to analyze the interaction between the parameters . in order to determine the relationship between the factors and response variables ,
a regression design was employed to model a response as a mathematical function ( either known or empirical ) for few continuous factors , and good model parameter estimates are desired .
the coded values of the process parameters are determined by the following equation : 
 ( 1)xi = xix0x , 
 where xi is coded value of the ith variable , xi is uncoded value of the ith test variable , and x0 is uncoded value of the ith test variable at center point . the regression analysis is performed to estimate the response function as a second - order polynomial : 
 ( 2)y=0+i=1kixi+i=1kiixi2i=1 ,  i < jk1  j=2kijxixj , 
 where y is the predicted response , 0 constant , and i , ii , ij are coefficients estimated from regression .
they represent the linear , quadratic , and cross - products of xi and xj on response . the regression and graphical analysis
are carried out using design - expert software ( version 7.1.5 , stat - ease , inc . ,
the adequacy of the models is further justified through analysis of variance ( anova ) .
lack - of - fit is a special diagnostic test for adequacy of a model that compares the pure error , based on the replicate measurements to the other lack of fit , based on the model performance .
f value , calculated as the ratio between the lack - of - fit mean square and the pure error mean square , is the statistic parameter used to determine whether the lack - of - fit is significant or not , at a significance level .
the statistical model was validated with respect to xylitol production under the conditions predicted by the model in shake - flask level .
samples were drawn at the desired intervals and xylitol production was determined as described above .
plackett - burman experiments ( table 1 ) showed a wide variation in xylitol production .
this variation reflected the importance of optimization to attain higher productivity . from the pareto chart shown in figure 1 the variables ,
namely , peptone , xylose , mgso47h2o , and yeast extract , were selected for further optimization to attain a maximum response .
the levels of factors and the effect of their interactions on xylitol production were determined by box - behnken design of rsm .
the design matrix of experimental results by tests was planned according to the 29 full factorial designs .
twenty - nine experiments were performed at different combinations of the factors shown in table 2 , and the central point was repeated five times .
the predicted and observed responses along with design matrix are presented in table 3 , and the results were analyzed by anova .
the second - order regression equation provided the levels of xylitol production as a function of peptone , xylose , mgso47h2o , and yeast extract , which can be presented in terms of coded factors as in the following equation : 
 ( 3)y=0.70 + 0.053a+0.018b+0.057c+0.054d  + 0.092ab(2.500e003)ac  + ( 1.000e002)ad+0.028bc+0.077bd  0.040cd0.083a20.16b20.076c20.11d2 , 
 where y is the xylitol yield ( g / g ) and a , b , c , and d were peptone , xylose , mgso47h2o , and yeast extract , respectively .
there is only a 0.01% chance that a  model f - value  this large could occur due to noise .
values of  prob > f  less than 0.05 indicate that model terms are significant .
values greater than 0.1 indicate that model terms are not significant . in the present work ,
linear terms of a , c , and  d and all the square effects of a , b , c , and d and the combination of ab , bd , and cd were significant for xylitol production .
the coefficient of determination ( r ) for xylitol production was calculated as 0.9634 , which is very close to 1 and can explain up to 96.00% variability of the response .
the predicted r value of 0.7898 was in reasonable agreement with the adjusted r value of 0.9267 .
the adequate precision value of 16.010 indicates an adequate signal and suggests that the model can navigate the design space .
the above model can be used to predict the xylitol production within the limits of the experimental factors that the actual response values agree well with the predicted response values .
experimental conditions for optimization of the process variables for xylitol yield were determined by ccd of rsm .
the design matrix of experimental results by tests was planned according to the 50 full factorial designs , and the central point was repeated eight times .
the predicted and observed responses along with design matrix are presented in table 6 and the results were analyzed by anova .
the second - order regression equation provided the levels of xylitol production as a function of temperature , substrate concentration , ph , agitation speed , and inoculums size , which can be presented in terms of coded factors as in the following equation : 
 ( 4)y=0.79 + 0.025a+0.043b+0.049c  + 0.030d+0.038e0.029ab(4.063e003)ac  + 0.018ad+(2.187e003)ae(9.688e003)bc  + 0.014bd+(5.312e003)be+(1.562e003)cd  + ( 5.312e003)ce(3.125e004)de0.040a2  0.041b20.046c20.027d20.034e2 , 
 where y was the xylitol yield ( g / g ) and a , b , c , d , and e are temperature , substrate concentration , ph , agitation speed , and inoculums size , respectively .
there is only a 0.01% chance that a  model f - value  this large could occur due to noise .
values of  prob > f  less than 0.05 indicate that model terms are significant .
values greater than 0.1 indicate that model terms are not significant . in the present work , linear terms and all the square effects of a , b , c , d , and e and
the coefficient of determination ( r ) for xylitol production was calculated as 0.9148 , which is very close to 1 and can explain up to 91.00% variability of the response .
the predicted r value of 0.6867 was in reasonable agreement with the adjusted r value of 0.8561 .
the adequate precision value of 12.951 indicates an adequate signal and suggests that the model can navigate the design space . in both designs
the interaction effects of variables on xylitol production were studied by plotting 3d surface curves against any two independent variables , while keeping another variable at its central ( 0 ) level .
the 3d curves of the calculated response ( xylitol yield ) and contour plots from the interactions between the variables were obtained .  
this evidence from above figures shows the dependency of xylose , mgso47h2o , yeast extract on xylitol production .
the optimal operation conditions of peptone , xylose , mgso47h2o , and yeast extract for maximum xylitol production were determined by response surface analysis and also estimated by regression equation .
the predicted values from the regression equation closely agreed with that obtained from experimental values .
the xylitol production increased with increase in temperature to about 36c , and thereafter xylitol production decreased with further increase in temperature .
this evidance from above figures shows the dependency of ph , substrate concentration , agitation speed , and inoculum size on xylitol production .
the optimal operation conditions of temperature , substrate concentration , ph , agitation speed , and inoculum size for maximum xylitol production were determined by response surface analysis and also estimated by regression equation .
the predicted values from the regression equation closely agreed with that obtained from experimental values .
validation of the experimental model was tested by carrying out the batch experiment under optimal operation conditions which are ( g
/ l ) : peptone : 6.03 , xylose : 10.62 , mgso47h2o : 1.39 , yeast extract : 4.66 established by the regression model . under optimal process variables levels are temperature ( 36.56c ) , ph ( 7.27 ) , substrate concentration ( 3.55 
g / l ) , inoculum size ( 3.69  ml ) , and agitation speed ( 194.44  rpm ) .
the xylitol production ( 0.80  g / g ) obtained from experiments was very close to the actual response ( 0.78  g / g ) predicted by the regression model , which proved the validity of the model .
in this work , plackett - burman design was used to test the relative importance of medium components on xylitol production . among the variables , peptone , xylose , mgso47h2o , and yeast
g / l ) : peptone : 6.03 , xylose : 10.62 , mgso47h2o : 1.39 , and yeast extract : 4.66 . then the influence of various process variables , namely , temperature , ph , substrate concentration , agitation speed , and inoculum size on the xylitol production was evaluated by ccd
. the optimum levels of process variables are temperature ( 36.56c ) , ph ( 7.27 ) , substrate concentration ( 3.55 
g / l ) , inoculum size ( 3.69  ml ) , and agitation speed ( 194.44  rpm ) .
this study showed that the corncob is a good source for the production of xylitol . using the optimized conditions , the xylitol yield reaches 0.80  g / g .
 optimization of the culture medium and process variables for xylitol production using corncob hemicellulose hydrolysate by pachysolen tannophilus ( mttc 1077 ) was performed with statistical methodology based on experimental designs . 
 the screening of nine nutrients for their influence on xylitol production was achieved using a plackett - burman design . 
 peptone , xylose , mgso47h2o , and yeast extract were selected based on their positive influence on xylitol production . 
 the selected components were optimized with box - behnken design using response surface methodology ( rsm ) . the optimum levels ( 
 g / l ) were peptone : 6.03 , xylose : 10.62 , mgso47h2o : 1.39 , yeast extract : 4.66 . 
 the influence of various process variables on the xylitol production was evaluated . 
 the optimal levels of these variables were quantified by the central composite design using rsm , for establishment of a significant mathematical model with a coefficient determination of r2 = 0.91 . 
 the validation experimental was consistent with the prediction model . 
 the optimum levels of process variables were temperature ( 36.56c ) , ph ( 7.27 ) , substrate concentration ( 3.55  
 g / l ) , inoculum size ( 3.69  ml ) , and agitation speed ( 194.44  rpm ) . 
 these conditions were validated experimentally which revealed an enhanced xylitol yield of 0.80  g / g . 
1. Introduction 
2. Materials and Methods 
3. Results and Discussions
4. Conclusion