Biochemical Engineering Journal 120 (2017) 136145

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Artificial neural network and regression coupled genetic algorithm to

optimize parameters for enhanced xylitol production by
Debaryomyces nepalensis in bioreactor
Sharon Mano J. Pappu, Sathyanarayana N. Gummadi
Applied and Industrial Microbiology Laboratory, Bhupat and Jyoti Mehta School of Biosciences, Department of Biotechnology, Indian Institute of Technology
Madras, Chennai 600 036, India

a r t i c l e i n f o a b s t r a c t

Article history: In this study, prediction ability and optimization ability of regression and artificial neural network (ANN)
Received 6 October 2016 models were compared. The input variables used to predict xylose consumption, biomass and xylitol
Received in revised form 7 January 2017 production by regression analysis and neural network model are temperature, fermentation time, pH,
Accepted 21 January 2017
kL a, biomass and glycerol of previous data points. Determination of coefficient (R2 ) was used to assess the
Available online 23 January 2017
adequacy of the regression model and R2 for xylitol and biomass were 86.56% and 96.43% respectively. A
multi layered feed forward neural network (ANN) of 5-10-2 topology has been developed to predict the
Keywords:
xylitol production. Results showed that prediction accuracy of ANN was apparently higher when com-
Artificial neural network
Genetic algorithm
pared to regression model. Genetic algorithm (GA) was used to find the optimum parameters to enhance
Regression analysis xylitol production. Optimization of fermentation parameters was carried out using hybrid regression GA
Xylitol and hybrid ANN GA with an optimum prediction error of 10% and 3.5% respectively. ANN coupled GA is
Optimization considered to be the better optimization method due to its high accuracy and low prediction error and
recommended to be employed for optimization of fermentative process.
2017 Elsevier B.V. All rights reserved.

1. Introduction lignocellulose wastes [5]. Among the reported microbial strains,

Xylitol is a naturally occurring sugar alcohol with one third
calories lesser than sucrose [1]. In recent years, interest in xyli-
tol has increased considerably, mainly due to many commercial
applications in several industrial sectors like food, dental and
pharmaceuticals. Industrially, xylitol is currently manufactured by
chemical hydrogenation of pure xylose in the presence of metal
catalysts like nickel, palladium and ruthium [2] at elevated tem-
perature (80140 C) and pressure (50 atm) [3]. Chemical method
of xylitol manufacturing is laborious, energy and cost intensive.
Furthermore, it also requires pure substrate (xylose) for hydro-
genation, thus adding the refining cost to the total production
cost. Microbial or enzymatic production of xylitol is becoming a
more sustainable alternative. Biotechnological production of xyli-
tol is gaining more interest as it takes place under mild operating
conditions, ease in purification and relatively economical and safe
process [4].
Microbial process uses bacteria, yeast and fungi for production
of xylitol from xylose or hemicellulose hydrolysate derived from
Corresponding author.
E-mail address: gummadi@iitm.ac.in (S.N. Gummadi).
Candida [6] and Debaryomyces [7,8] are the best known yeast
species for xylitol production. Biotechnological production of xyl-
itol is limited by several factors which includes age and inoculum
concentration, initial substrate concentration [8], pH, temperature
[7], aeration and agitation conditions of the fermentation process
[9,10].
Debaryomyces nepalensis NCYC 3413, a halotolerant yeast strain
was isolated from rotten apple, which is capable of efficiently utiliz-
ing hemicellulose-derived sugars to produce industrially important
metabolites like xylitol, arabitol and ethanol [11]. Being halotol-
erant, D. nepalensis can be considered an ideal strain for xylitol
production using lignocellulose as substrate, since pretreatment
of lignocellulose releases large amounts of inorganic salts that are
inhibitory to microbes [12]. However, the large scale microbial pro-
duction is rendered by physical and operational parameters such as
pH, temperature, initial xylose concentration and oxygen transfer
rate [7,8,10,13,14]. It is necessary to optimize various parameters to
make a process more cost effective and efficient. Statistical method-
ologies such as response surface method and data driven based
models such as neural networks have been preferred for creating a
suitable optimized model of physico-biological parameters because
of their proven advantage [15].

http://dx.doi.org/10.1016/j.bej.2017.01.010
1369-703X/ 2017 Elsevier B.V. All rights reserved.
 S.M.J. Pappu, S.N. Gummadi / Biochemical Engineering Journal 120 (2017) 136145
Mathematical models could be used for prediction of micro-
bial development, production of metabolites and optimization of
growth conditions in fermentation process. Information gained
from modelling might direct the adjustment of process parame-
ters and model-based simulations proposes regulation of process
parameters, prediction of substrate and product concentrations
[16]. Regression analysis is a statistical technique for investigating
and modelling the relationship between variables. In the recent
years, complex biotechnological process has been modelled and
optimized using artificial intelligence techniques to attain high
operational performance [17,18]. Artifical neural network (ANN)
and genetic algorithm (GA) are more suitable for analyzing data in
which the process variables project non-linear relation to output
response or when it is too difficult to develop deterministic models.
ANNs offer many advantages such as adaptive solutions and re-
estimation of model parameters in relatively simpler forms when
compared to other modelling approaches. ANN model predicts the
model output of the process using the same input as the process
after training and no additional measurements are needed during
the prediction phase. An artificial intelligence based stochastic non-
linear optimization technique, genetic algorithm (GA) is used to
optimize the input vector of regression and ANN model. GA mim-
ics the principles of biological evolution survival of the fittest and
random exchange of data during propagation [19]. GA has been
proved to be an ideal technique for solving non-linear optimization
problems [20].
No modelling studies have been published to date that address
the complete interaction of fermentation parameters accounting
biomass and by-product concentration as variables influencing
production of metabolites. Indeed, many existing models have been
developed using less number of input variables and trained with
reduced number of experimental data points [21,22]. However,
inclusion of biomass and byproduct concentration of previous data
point could improve the prediction capability of the model. In this
study, regression models and ANN were evaluated as predictive
tools for production of xylitol by Debaryomyces nepalensis in biore-
actor and to compare the performances of statistical and artificial
intelligence based optimization techniques.
2. Materials and methods
2.1. Microorganism and fermentation medium
Debaryomyces nepalensis NCYC 3413, isolated from rotten apple,
was maintained on a solid YEPP medium containing yeast extract
10 g L1 , peptone 20 g L1 and pectin 5 g L1 at pH 7.0 and incubated
at 30 C for 24 h and stored at 4 C. A single colony was transferred
from an overnight-grown culture plate into the YEPD medium
(50 mL) containing yeast extract 10 g L1 , peptone 20 g L1 and dex-
trose 20 g L1 and incubated for 12 h at 30 C at 180 rpm 8% (v/v)
seed culture was used to inoculate the fermentation medium in
the stirred tank reactor. Semi-synthetic medium containing xylose
100 g L1 ; (NH4 )2 SO4 3 g L1 ; MgSO4 0.1 g L1 ; K2 HPO4
6 g L1 ; Na2 HPO4 3 g L1 ; yeast extract 1 g L1 ; CaCl2 2H2 O
147 mg L1 ; citric acid 6.9 mg L1 ; FeCl3 10 mg L1 ; MnSO4 H2 O
3.4 mg L1 ; ZnSO4 7H2 O 4.3 mg L1 ; CuSO4 5H2 O 0.25 mg L1 ;
3N H3 PO4 and 3N NaOH were used to adjust pH. All the components
were autoclaved separately and mixed subsequently as described
earlier [23].
2.2. Batch fermentation
The batch fermentation was carried out under varying opera-
tional conditions as indicated in Table 1, in 2.5 L round bottomed
cylindrical glass vessel bioreactor (Minifors, Infors HT, Switzerland)
equipped with a dissolved oxygen probe (Hamilton, Switzerland),
137
pH probe (Mettler Toledo, Switzaerland) and a temperature sen-
sor. The dimensions of the bioreactor and the impeller were as
follows: Total volume: 2.5 L, working volume 1 L, tank height
270 mm, diameter of the tank 110 mm, Number of impeller
2, Type of impeller Rushton flat blade, Number of blades 6,
impeller diameter 46 mm, height of the impeller blade 11 mm,
width of the impeller blade 11 mm, Number of baffles 3. Sam-
ples were collected at regular time intervals and centrifuged at
10,000 rpm for 10 min. The supernatant was used for substrate and
metabolite concentrations and the cell pellet was used to measure
biomass concentration. The aeration and agitation rates were var-
ied to obtain different kL a values using dynamic gassing out method
[9]. Prediction of rate of absorption of gaseous materials in a stirred
tank are usually based on correlations of overall volumetric mass
transfer coefficient (kL a) with mechanical agitation power per unit
volume (Pg /VL ) and gas sparging rate expressed as the superficial
gas velocity (Vg ).
The following correlation is frequently found in the literature
[24]:
kL a = ! (Pg /VL )" (Vg )c , (1)
where P is the mechanical agitation power in gas liquid dispersion
(W), VL is the working volume of the reactor (m3 ), Vg is the super-
ficial gas velocity (m/s) which was calculated by dividing the gas
flow rate (m3 /s) by internal tank area (m2 ), ! is the constant, " and
c are exponents.
Since accurate measurement of power input is difficult, power
number (NP = P3 5 ) was used to calculate the ungassed power
!N D
i i
(Ni Stirrer speed, Di Impeller diameter). As the six blade rushton
turbine is employed for agitation, Np = 6 was used in the calculation
as reported in the literature [25]. An expression for the ratio of
gassed to ungassed power as a function of operating conditions as
given in Eq. (2) was used to calculate the power consumption with
sparging [26].
Pg Fg
! "0.25 # N 2 D4 $0.20
i i
= 0.10 (2)
P0 Ni V gWi V 2/3
where Pg is power consumption with sparging, P0 is power con-
sumption without sparging, Fg is volumetric gas flow rate, Ni is
stirrer speed, V is liquid volume, Di is impeller diameter, g is gravita-
tional acceleration and Wi is impeller blade width. Experimentally
calculated kL a for varying agitation speed and aeration rate was
correlated to Pg /VL and Vs according to Eq. (1). Parameter values of
! = 0.34, " = 0.12 and c = 0.52 was estimated using feval and fmin-
search inbuilt functions of MATLAB. (Supplementary file: Estimated
KL a values for different values of agitation speed and aeration rate).
2.3. Analytical methods
Cell pellet was resuspended in water and A600 was measured.
The cell concentration was calculated from pre-calibrated graph of
A600 vs cell dry weight (experimentally determined). A600 of 1.0
corresponds to 0.34 g cell dry weight per liter culture as mentioned
earlier [27]. The concentration of xylose and metabolites (xylitol
and glycerol) were analyzed by High Performance Liquid Chro-
matography (HPLC, Jasco, Japan) equipped with refractive index
detector and Aminex HPX-87H column (Bio-Rad, Richmond, USA).
The mobile phase used for the analysis was 0.01N H2 SO4 at a
flow rate of 0.6 mL min1 and oven temperature was maintained
at 45 C.
138 S.M.J. Pappu, S.N. Gummadi / Biochemical Engineering Journal 120 (2017) 136145

Table 1
Summary of fermentation conditions and responses for enhanced production of xylitol by Debaryomyces nepalensis NCYC 3413.

Run # pH Agitation Aeration Temperature Time (h) Biomass Xylitol Productivity Product yield Number of data
(rpm) (vvm) ( C) (g L1 ) (g L1 ) (g L1 h1 ) coefficient YP/S g points
of xylitol (g of
xylose)1

1 0.290 0.623 0.388 0.179 84 24.73 19.35 0.230 0.198 13

( 6.0) ( 550) ( 1.00) ( 30)
2 0.230 0.050 0.658 0.179 132 16.08 32.08 0.243 0.327 18
( 5.5) ( 200) ( 1.60) ( 30)
3 0.050 0.214 0.478 0.179 96 16.61 55.01 0.573 0.551 14
( 4.0) ( 300) ( 1.20) ( 30)
4 0.110 0.541 0.748 0.179 84 25.66 32.16 0.383 0.322 12
( 4.5) ( 500) ( 1.80) ( 30)
5 0.410 0.132 0.298 0.179 100 9.99 48.39 0.482 0.492 16
( 7.0) ( 250) ( 0.80) ( 30)
6 0.470 0.459 0.568 0.179 54 18.22 51.47 0.953 0.517 10
( 7.5) ( 450) ( 1.40) ( 30)
7 0.350 0.295 0.838 0.179 102 17.51 73.09 0.716 0.748 14
( 6.5) ( 350) ( 2.00) ( 30)
8 0.170 0.377 0.208 0.179 54 18.37 39.28 0.727 0.397 14
( 5.0) ( 400) ( 0.60) ( 30)
9 0.050 0.214 0.613 0.179 84 17.28 49.86 0.575 0.504 16
( 4.0) ( 300) ( 1.50) ( 30)
10 0.290 0.214 0.163 0.179 120 12.59 42.91 0.358 0.429 14
( 6.0) ( 300) ( 0.50) ( 30)
11 0.050 0.541 0.163 0.179 84 26.84 26.49 0.315 0.265 13
( 4.0) ( 500) ( 0.50) ( 30)
12 0.290 0.541 0.613 0.179 60 23.73 28.42 0.474 0.288 10
( 6.0) ( 500) ( 1.50) ( 30)
13 0.370 0.099 0.748 0.179 108 23.16 38.01 0.348 0.388 16
( 6.6) ( 230) ( 1.80) ( 30)
14 0.314 0.230 0.500 0.179 84 18.12 34.48 0.410 0.345 12
( 6.2) ( 310) ( 1.25) ( 30)
15 0.398 0.066 0.523 0.179 108 12.40 23.09 0.218 0.236 16
( 6.9) ( 210) ( 1.30) ( 30)
16a 0.290 0.295 0.163 0.179 72 21.60 47.38 0.657 0.480 6
( 6.0) ( 350) ( 0.50) ( 30)
a
17 0.170 0.173 0.163 0.179 120 12.45 42.53 0.354 0.431 16
( 5.0) ( 275) ( 0.50) ( 30)
a
18 0.410 0.295 0.163 0.179 120 25.40 53.67 0.446 0.538 11
( 7.0) ( 350) ( 0.50) ( 30)
19a 0.290 0.214 0.163 0.307 120 19.20 37.82 0.315 0.379 10
( 6.0) ( 300) ( 0.50) ( 30)
20a 0.290 0.295 0.163 0.179 120 21.61 52.19 0.434 0.533 11
( 6.0) ( 350) ( 0.50) ( 30)
a
21 0.290 0.541 0.163 0.179 120 26.43 9.361 0.078 0.094 10
( 6.0) ( 500) ( 0.50) ( 30)
22a 0.350 0.295 0.163 0.179 120 21.16 44.74 0.373 0.448 10
( 6.5) ( 350) ( 0.50) ( 30)
23a 0.290 0.377 0.163 0.179 108 22.32 37.48 0.346 0.383 8
( 6.0) ( 400) ( 0.50) ( 30)
24a 0.290 0.377 0.275 0.179 120 23.58 20.73 0.173 0.210 11
( 6.0) ( 400) ( 0.75) ( 30)
a
25 0.290 0.377 0.086 0.179 120 17.28 24.39 0.203 0.246 11
( 6.0) ( 400) ( 0.33) ( 30)
26a 0.290 0.377 0.118 0.179 120 15.67 25.89 0.216 0.262 13
( 6.0) ( 400) ( 0.40) ( 30)
27a 0.179 0.214 0.050 0.050 120 13.89 13.82 0.115 0.138 15
( 5.0) ( 300) ( 0.25) ( 25)
a
Data points taken from Kumdam and Gummadi [9]. Values in the parenthesis represent the original values of the parameter. Total number of data points: 340

2.4. Modelling approaches as given in Eq. (4), since it allows description of the response in a
relatively wide area of change in input variables [29],
2.4.1. Regression model % % %
All the experimental data were normalized as follows: y = 0 + i xi + ii xi2 + ij xij (4)

(Xori Xmin ) 0.9 where Y represents response variable (xylitol(i+1) , biomass(i+1) ),

XNorm = + 0.05
Xmax
where XNorm represents normalized value of the variables, Xori
denotes the original value of the variables before normalization,
Xmin and Xmax represents the minimum and maximum value of
the variable considered for analysis [28]. The experimental data
obtained was fitted to the quadratic polynomial model equation
(3)
"0 is intercept coefficient, "ii is the coefficient of squared terms,
"ij is the coefficient of interaction terms and xi represents inde-
pendent variables considered for modelling (x1 temperature
( C), x2 time (h), x3 pH, x4 kL a, x5 biomass(i) , x6
glycerol(i) , x7 xylose(i) ). Subscript i represents values of vari-
ables at ith h (where i = 0,6,. . .114) and i+1 represents data at
(i+6)th h (i+1 = 6,12,. . .120). The effect of the parameters and their
 S.M.J. Pappu, S.N. Gummadi / Biochemical Engineering Journal 120 (2017) 136145
Fig. 1. Overall schematic representation of regression and artificial neural network coupled genetic algorithm for optimization of fermentation conditions to enhance xylitol
production by Debaryomyces nepalensis NCYC 3413 in batch reactor.
interaction on the output response has been analyzed by conduct-
ing significance tests and analysis of variance (ANOVA). Coefficient
of determination (R2 ) values was used to statistically assess the
adequacy of polynomial model and significance of the regression
coefficient was tested by using Students t-test. Regression analysis
of the experimental data and statistical analysis of the model were
carried out using MINITAB 16 software.
2.4.2. Artificial neural network (ANN) model
Artificial neural network methodology employed in this study
was a multilayer perceptron model. Parameters (time, pH, temper-
ature, kL a, biomass(i) , xylose(i) , glycerol(i) ) were used to construct
ANN topology to identify the non-linear relationship between input
variables and output response. Transfer functions tan sigmoid
(f1: tansig) and pure linear (f2: purelin) were used in the hidden
and output layer respectively. Predicted output response can be
described as follows:
Response = f 2[wo f 1 (wH Inputvector + bH ) + bo ]
where wH , bH and wo , bo are weights and biases of hidden and
output layer respectively. LevenbergMarquardt back propagation
(BP) algorithm was used to train the network. In this training algo-
rithm, the error between the predicted and observed values was
calculated and propagated backward through the network to the
weights of each layer. The algorithm autonomously adjusted the
connection weights in the direction of reducing errors between pre-
dicted and observed until the errors met the requirement. Trained
ANN must be evaluated by testing samples to determine the pre-
diction capability. Adequacy of the developed model was estimated
using mean squared error (MSE) and normalized root mean squared
error (NRMSE) as represented in Eqs. (6) and (7).
n
1 %& '2
MSE = YExp,i YPred,i
n
i=1
( represents the weight vectors; x denotes the input vector, the
1
)n & '2
n i=1
YExp,i YPred,i
NRMSE = & '
YExp,min YExp,max
139
where n is the number of experimental data points used for test-
ing; YExp,i is the experimental value; YPred,i is the predicted values
obtained from the model. The lower values of NRMSE means a
better performance of the developed ANN [22].
2.5. Optimization by genetic algorithm (GA)
Regression equation and trained neural network model were
used as the fitness function of GA for optimization of fermenta-
tion parameters to enhance the production of xylitol. GAs operate
simultaneously on a population of potential individuals employ-
ing the principle of natural evolution i.e., survival of the fittest
strategy, to produce better and better approximation to a solu-
tion of the problem. The major operations of GA is summarized as
follows: (i) Initialization: initial population of a set of individuals
(Input variables) was generated randomly, (ii) Evaluation: using the
predefined objective function (Regression equation/ANN weights
and biases), the fitness of individuals were evaluated, (iii) Selec-
(5) tion: ranking method was used in this study to select individuals
based upon its fitness such that the better individuals have an
increased chance of being selected, (iv) Genetic operators: new
set of individuals were generated from the existing individuals in
the populations by using genetic operators such as cross-over
with a probability of 0.8, (v) Termination: GA is terminated after
a pre-specified number of generations and the resulted solution is
tested against objective function. Overall schematic representation
of regression and ANN coupled GA is shown in Fig. 1.
The objective function of hybrid regression GA (Eq. (8)) and
hybrid ANN-GA (Eq. (9)) can be defined as follows:
Maximize y = f(x, regression model);
xiL xi xiU , i = 1, 2, . . ..n (8)
Maximize y = f(x, W);
xiL xi xiU , i = 1, 2, . . ..n (9)
(6)
where f represents the objective function (regression model); W
fermentation parameters to be optimized; y refers to the xylitol
(7) concentration; n denotes number of input variables; and xiL and
xiU represents the lower and upper bounds on xi respectively. Each
140 S.M.J. Pappu, S.N. Gummadi / Biochemical Engineering Journal 120 (2017) 136145
individual of population is evaluated based on the following fitness
function:
1 biomass(i) , x6 glycerol(i) , x7 xylose(i) ). Subscript i represents
Errorj = 1 ; j = 1, 2...N (10)
j values of variables at ith h (where i = 0,6,. . .114) and i+1represents
ypred
where Errorj denotes the fitness value of the candidate solution
j
and ypred denoted the model predicted xylitol concentration for
particular individual. A constrained based GA optimization was
implemented, which searches for the optimal only in the feasible
region and the optimization is subjected to the constraint that tem-
perature of fermentation process is maintained at 30 C. Training
of artificial neural network and optimization by genetic algorithm
was performed using MATLAB 2013.
3. Results and discussion
Xylitol production in bioreactors was carried out at different
physiological conditions of pH, temperature, aeration and agita-
tion rates as shown in Table 1. The samples were collected at every
6 h interval resulted in 340 data points (27 runs). The fermenta-
tion conditions, xylitol concentration, productivity, time at which
maximum xylitol concentration was produced, product yield coef-
ficient (YP/S ) and the number of data points for each run that were
considered for ANN model training were tabulated in Table 1. All
340 data points from Table 1 were used to train ANN model (Sec-
tion 3.2). Experiments were performed at various combinations of
pH (4.0, 5.0 and 6.0), temperature (25 C, 30 C and 35 C) and kL a
(0.14 h1 , 0.28 h1 and 0.56 h1 ) were considered as test data. To
check the robustness of the model, these set of experiments were
not included in the training data. Fermentation profiles for fermen-
tation run# (1,7 and 13) were provided as Supplementary figures
(Supplementary Fig. S1ac).
3.1. Regression model for prediction of xylitol production by
Debaryomyces nepalensis NCYC 3413 in batch reactor
The experimental conditions and experimental values (Table 1)
were used to build a second order polynomial model by a multi-
ple regression technique to examine the individual and combined
effect of input variables on xylitol and biomass production. The
coefficients of each term (normalized) of the second order polyno-
mial equation for biomass and xylitol were given in Eqs. (11) and
(12).
Xylitol(i+1) = 0.167 1.214 x1 1.930 x2 + 1.067 x 3 + 0.461 x 4
+ 3.777 x 5 + 2.323 x 6 0.300 x 7 + 11.490 x 1 x2 16.078 x 1
x5 6.278 x 1 x6 + 1.651 x 1 x7 + 0.593 x 2 x3 + 1.171 x 2
x4 0.282 x 2 x5 0.586 x 2 x6 0.188 x 2 x7 0.455 x 3
x4 0.494 x 3 x5 1.476 x 3 x6 1.053 x 3 x7 2.024 x 4
x5 + 1.246 x 4 x6 0.256 x 4 x7 1.533 x 5 x6 0.322 x 5
x7 0.301 x 6 x7
Biomass(i+1) = 0.345 1.264 x 1 0.599 x 2 + 0.0402 x 3
+ 0.096 x 4 + 2.106 x 5 0.825 x 6 0.386 x 7
+ 3.10346 x 1 x2 6.433 x 1 x5 + 4.741 1 x6 + 1.551 x
0.399 x 2 x3 + 0.201 x 2 x4 0.021 x 2 x5 0.023 x 2
1 x7
x6 + 0.0351 x 2 x7 0.029 x 3 x4 + 0.377 x 3 x5 + 0.197 x 3
x6 0.0423 x 3 x7 0.311 x 4 x5 0.215 x 4 x6 0.009 x 4
x7 0.0593 x 5 x6 + 0.177 x 5 x7 0.104 x 6 x7
where x1 temperature ( C), x2 time (h), x3 pH, x4 kL a, x5
data at (i+6)th h (i+1 = 6,12,. . .120).
Positive and negative signs of the coefficients in the regression
model equations indicate that any increase in that particular factor
leads to a statistically-significant decrease or increase in p-value
[30]. Adequacy and statistical significance of the regression models
thus obtained was evaluated through analysis of variance (ANOVA).
Table 2 depicts analysis of variance of xylitol regression model, in
which the F and p values of the model were observed to be 78.06 and
<0.0001 respectively, which implied that the model was significant
at 95% confidence level (p < 0.05). Terms in the model equation with
p < 0.05 exhibits a significant contribution to the output response.
In the regression model for xylitol, linear terms of factors (tem-
perature, time, pH, kL a, biomass and glycerol) were found to be
significant. Interactions terms involving pH and kL a, pH and xylose
and interaction of kL a with biomass and glycerol were found to
have p < 0.05 which implies that pH, agitation and aeration affects
the production of xylitol. Lack of fit with high t-value and low
p-value denoted that the model is significant relative to the pure
error. Further to test the model adequacy, determination of coef-
ficient (R2 ) was evaluated which was observed to be 86.56% that
indicated the model could explain 14% of the variability. Regres-
sion models for biomass prediction exhibits high adequacy with
R2 value of 96.43% and F and p values of the model were observed
to be 327.8 and <0.0001 respectively (Table 3), which implied that
the model was significant at 95% confidence level (p < 0.05). It has
been reported that xylose to xylitol conversion by microorganisms
is strongly affected by oxygen supply. In the presence of excess aer-
ation, NADH is reoxidized by respiratory chain, catalyzed by NAD+
dependent xylitol dehydrogenase (XDH) and xylitol is consumed
for growth [31]. pH of the medium also plays a vital role in the
enhanced production of xylitol as pH affects the transport of xylose
across the cell membrane [32]. In addition to aeration, agitation and
pH, the activities of enzyme responsible for microbial production
of xylitol are very sensitive to the substrate concentration. since
the real substrates for xylitol producing microorganisms are ligno-
cellulosic hydrolysate which often contain a variety of sugars [33].
These results suggest that pH of the medium, aeration and agita-
tion rates are very much crucial for enhanced xylitol production in
bioreactor.
3.2. Development of ANN model for prediction of xylitol
production in batch reactor
Experimental data from 27 fermentation batches (Table 1) were
integrated (Total number of data points: 340) and then divided
randomly into two divisions: training data (75%) and validation
data (10%). The mean square error (MSE) between predicted and
experimental values was set at 0.005 and the maximum number of
(11)
training epochs was 1000. A set of data which is different from the
training data is used for validating the network during the training
phase. When the network begins to over fit the data, the error on
the validation set begins to increase typically. When the validation
error increases for a specified number of iterations (set n = 6), the
network training is stopped and the weights and biases of the net-
work at the minimum of the validation error were obtained. The test
data set, which are not used for training and validation were used
to examine the trained network. ANN topology was identified by
training with different neural networks varying the number of neu-
rons in the input and hidden layer. The NRMSE values of the neural
network N/w I (6-10-3), N/w II (4-10-3) and N/w III (5-10-2) for
(12) the test runs were tabulated in Table 4. In order to check the model
 S.M.J. Pappu, S.N. Gummadi / Biochemical Engineering Journal 120 (2017) 136145 141

Table 2
Analysis of variance (ANOVA) for regression model of xylitol by Debaryomyces nepalensis NCYC 3413 in batch reactor.

Source DF Seq SS Adj SS Adj MS F value p value

Regression 26 14.30 14.30 0.550 78.06 0.000

x1 1 0.133 0.000 0.000 3.022 0.081a
x2 1 5.174 0.036 0.036 5.095 0.025a
x3 1 0.502 0.024 0.024 3.454 0.064a
x4 1 1.406 0.016 0.016 2.942 0.105a
x5 1 0.000 0.067 0.067 9.521 0.002a
x6 1 1.276 0.008 0.008 2.566 0.093a
x7 1 2.702 0.001 0.000 0.070 0.792
x1 *x2 1 0.052 0.044 0.044 6.267 0.013a
x1 *x5 1 0.638 0.044 0.043 6.166 0.014a
x1 *x6 1 0.008 0.002 0.002 0.279 0.598
x1 *x7 1 0.000 0.000 0.000 0.047 0.829
x2 *x3 1 0.009 0.006 0.006 0.842 0.360
x2 *x4 1 0.073 0.067 0.067 9.492 0.002a
x2 *x5 1 0.060 0.007 0.007 0.979 0.323
x2 *x6 1 0.698 0.020 0.020 2.838 0.093a
x2 *x7 1 0.222 0.005 0.005 0.754 0.386
x3 *x4 1 0.061 0.018 0.018 2.520 0.113
x3 *x5 1 0.043 0.001 0.001 0.182 0.670
x3 *x6 1 0.037 0.056 0.056 7.976 0.005a
x3 *x7 1 0.000 0.025 0.025 3.589 0.059a
x4 *x5 1 0.864 0.177 0.177 25.14 0.000a
x4 *x6 1 0.181 0.142 0.142 20.13 0.000a
x4 *x7 1 0.006 0.002 0.002 0.327 0.568
x5 *x6 1 0.141 0.149 0.149 21.11 0.000a
x5 *x7 1 0.018 0.015 0.015 2.162 0.142
x6 *x7 1 0.004 0.004 0.004 0.545 0.461
Error 314 2.214 2.214 0.007
Lack-of-Fit 299 2.213 2.213 0.007 158.5 0.000
Pure Error 15 0.001 0.001 0.000
Total 340 16.52

x1 Temperature, x2 Time (h), x3 pH, x4 kL a, x5 Biomass(i) , x6 Glycerol(i) , x7 Xylose(i) ; DF Degree of freedom; Seq SS Sequential sum of squares; Adj SS Adjusted
sum of squares; Adj MS Adjusted mean square.
a
Represent the significant terms of the model.

Table 3
Analysis of variance (ANOVA) for regression model of biomass production by Debaryomyces nepalensis NCYC 3413 in batch reactor.

Source DF Seq SS Adj SS Adj MS F P

Regression 26 13.60 13.60 0.523 327.8 0.000

x1 1 0.130 0.000 0.000 0.156 0.693
x2 1 6.251 0.004 0.004 2.756 0.101a
x3 1 0.012 0.000 0.000 0.037 0.847
x4 1 1.075 0.002 0.002 2.886 0.099a
x5 1 6.055 0.022 0.022 13.49 0.000a
x6 1 0.001 0.001 0.001 0.582 0.446
x7 1 0.007 0.001 0.001 0.434 0.510
x1 *x2 1 0.000 0.003 0.003 2.112 0.147a
x1 *x5 1 0.003 0.007 0.007 4.296 0.039a
x1 *x6 1 0.001 0.001 0.001 0.640 0.424
x1 *x7 1 0.000 0.000 0.000 0.241 0.624
x2 *x3 1 0.002 0.005 0.005 2.852 0.092a
x2 *x4 1 0.023 0.003 0.003 2.776 0.101a
x2 *x5 1 0.009 0.000 0.000 0.007 0.935
x2 *x6 1 0.000 0.000 0.000 0.016 0.900
x2 *x7 1 0.003 0.000 0.000 0.268 0.605
x3 *x4 1 0.001 0.000 0.000 0.037 0.847
x3 *x5 1 0.005 0.003 0.003 2.052 0.153
x3 *x6 1 0.001 0.001 0.001 0.670 0.414
x3 *x7 1 0.000 0.000 0.000 0.048 0.827
x4 *x5 1 0.011 0.007 0.007 4.078 0.044a
x4 *x6 1 0.006 0.005 0.005 2.969 0.086a
x4 *x7 1 0.000 0.000 0.000 0.097 0.755
x5 *x6 1 0.000 0.000 0.000 0.121 0.728
x5 *x7 1 0.003 0.004 0.004 2.792 0.101a
x6 *x7 1 0.001 0.001 0.001 0.721 0.397
Error 314 0.501 0.501 0.002
Lack-of-Fit 299 0.501 0.501 0.002 236.9 0.000
Pure Error 15 0.000 0.000 0.000
Total 340 14.10
a
Represent the significant terms of the model.
142 S.M.J. Pappu, S.N. Gummadi / Biochemical Engineering Journal 120 (2017) 136145

Table 4
Normalized root mean square error (NRMSE) for artificial neural network N/w I, N/w II and N/w III.

Run # Fermentation conditions Biomass Xylose Xylitol

N/w I N/w II N/w III N/w I N/w II N/w I N/w II N/w III

1 Temperature 30 C pH 4.0 0.012 0.012 0.010 0.007 0.008 0.025 0.030 0.028
2 kL a 0.28 h1 pH 5.0 0.019 0.020 0.016 0.015 0.014 0.017 0.017 0.012
3 pH 6.0 0.016 0.017 0.012 0.012 0.015 0.018 0.019 0.008

4 pH 4.0 T 25 C 0.029 0.025 0.021 0.044 0.046 0.014 0.015 0.011

5 kL a 0.28 h1 T 35 C 0.018 0.018 0.013 0.012 0.013 0.029 0.028 0.019

6 pH 4.0 kL a 0.14 h1 0.009 0.010 0.006 0.005 0.009 0.013 0.014 0.012
7 Temperature 30 C kL a 0.56 h1 0.008 0.008 0.005 0.017 0.018 0.010 0.012 0.005

N/w I 6-10-3; N/w II 4-10-3; N/w III 5-10-2

predictability, the model was trained at fermentation temperature
of 30 C (Table 1, Run # 126, 325 data points) and one single run of
fermentation temperature 25 C (Table 1 run # 27, 15 data points).
To check this, test experiments were performed at the following
conditions: varying pH (4.0, 5.0, 6.0) but at constant temperature
of 30 C and kL a 0.28 h1 (Run# 13); varying temperature (25, 30
and 35 C) but at constant pH 4.0 and kL a 0.28 h1 (Run# 1, 4 and
5); and varying kL a (0.14, 0.28 and 0.56 h1 ) but at constant pH 4.0
and temperature 30 C (Run# 1, 6 and 7) (Table 4). The experimen-
tal conditions and data considered for testing the model (Table 4)
were not included in the training data set (Table 1).
The effective network topology for xylitol prediction was 5-
10-2, where 5 represents the input variables (temperature, kL a,
biomass(i) , glycerol(i) , xylose(i) ), 10 as hidden neurons and 2 as out-
put responses (biomass(i+1) , xylitol(i+1) ). The performance of the
models was depicted in Fig. 2, which presents the prediction val-
ues versus experimental values for training, validation and test data
for network III run# 1. Figures representing the regression values
of training, validation and test data of network III (Run# 27) as
tabulated in Table. 4 were provided as supplementary material in
MATLAB.FIG format. Even though ANN model was trained with only
15 data points at 25 C, the network III could predict the xylitol
production effectively at temperatures where the model was not
trained (35 C).
The weight matrix of each network can be employed to deter-
mine the relative importance of input variables on the output
response. For this purpose, Eq. (13) is used in the partitioning of
connection weights.
*# $ +
)Nh |W ih |
jm ho |
m=1 ) Ni |W mn
|W ih |
Ij = , *#k=1 kmih $ +-
)Ni )Nh |W |
jm ho |
k=1 m=1 ) Ni ih
|W mn
k=1
|W
km
| with artificial intelligence technique GA for optimizing the fer-
where Ij is the relative importance of the jth input variable on the
output response, Ni and Nh are the number of neurons in the input
and hidden layer respectively, W is the connection weights, the
superscripts i, h and o represents input, hidden and output lay-
ers respectively, and k, m and n refers to input, hidden and output
layer neuron number respectively. Relative importance of different
ANN was calculated and tabulated in Table 5. Agitation and aeration
shows equal percentage of contribution in predicting the output
response. When temperature was included as an input parame-
ter, the contribution of agitation and aeration were halved which
indicates that aeration and temperature are inter dependent. Con-
tribution of fermentation time is reduced to 15% when biomass(i) is
included as an input variable in prediction of xylitol which signifies
the fact that the variation in biomass concentration as fermentation
progresses accounts for the fermentation time. The contribution
of fermentation time is further reduced to 8% when both biomass
and glycerol were considered as input variables in training the net-
work. Since, glycerol is a by-product in the fermentation of xylose,
the concentration of biomass(i) and glycerol(i) of the previous data
point aids in better prediction of xylitol production. Network with
6-10-3 topology shows equal contribution of temperature (21%),
kL a (22%), biomass(i) (22%), glycerol(i) (20%) and time (6%) and pH
(9%). Network II (N/w II 4-10-3) was trained by excluding input
variables time and pH and shows increased contribution of
biomass(i) (31%). Network III (N/w III 5-10-2) was trained by
including xylose concentration of previous data point as input vari-
able and exhibits contribution of temperature as 21%, kL a 23%,
biomass(i) 26%, glycerol(i) 13% and xylose(i) 17%. The pre-
diction capability of N/w I 6-10-3, N/w 4-10-3 and N/w
5-10-2 were compared using normalized root mean square error
(NRMSE). Network II and III were also trained with same number
of data points with reduced number of input variables and tested
for different fermentation conditions. NRMSE values for test data
of seven fermentation runs were tabulated in Table 4 and no sig-
nificant difference was observed in NRMSE values of network IIII.
This result suggests principal components to be included in the
prediction network were temperature, kL a, biomass(i) , xylose(i) and
glycerol(i) .
3.3. Optimization of fermentation parameters for enhanced
production of xylitol by Debaryomyces nepalensis NCYC 3413 in
batch reactor using genetic algorithm (GA)
Experimental runs were carried under fermentation conditions
(time (h), pH, agitation (rpm), aeration (vvm), biomass(i-1) (g L1 )
and glycerol(i-1) (g L1 )) optimized based on hybrid regression GA
and hybrid ANN GA and the profiles of xylose consumption, biomass
(13) and xylitol production were shown in Fig. 3a and b respectively.
Regression equation and weights and biases of ANN were coupled
mentation operational conditions to enhance xylitol production.
The comparison of xylitol production at optimized fermentation
conditions using different approaches (Hybrid regression-GA and
Hybrid ANN-GA) are given in Table 6. Optimized fermentation
conditions pH 5.2, agitation speed 400 rpm, aeration rate
1.0 vvm by hybrid regression GA approach predicts 58.70 g L1 of
xylitol at 78 h of fermentation, however optimized fermentation
conditions pH 4.9, agitation speed 500 rpm, aeration rate
0.5 vvm by hybrid ANN GA approach predicts xylitol concentra-
tion to be 65.73 g L1 at a fermentation time of 82 h. Experiments
at the optimized fermentation conditions of regression coupled GA
results in higher biomass yield (28.38 g L1 ) and low xylitol produc-
tion (52.88 g L1 ) whereas experiments at fermentation conditions
optimized by ANN coupled GA results in high xylitol production
(63.42 g L1 ).
Regression model has predicted xylitol concentration of
58.70 g L1 at optimized conditions. Experimental validation has
given a xylitol concentration of 52.88 g L1 . Similarly predicted and
 S.M.J. Pappu, S.N. Gummadi / Biochemical Engineering Journal 120 (2017) 136145 143

Fig. 2. Regression plots of training, validation and test data of artificial neural network III (Topology 5-10-2). Fermentation conditions of test run: Temperature 30 C, pH
4 and kL a 0.28 h1 .

Table 5
Relative importance of input parameters on output responses of artificial neural network in prediction of xylitol production by Debaryomyces nepalensis NCYC 3413 in batch
reactor.

Topology Output Relative importance of input parameters (%)

structure of ANN responses
(Ni -Nh -No )

Time pH Agitation Aeration Temperature kL a Biomassi Glyceroli Xylosei

(h) (rpm) (vvm) ( C) (h1 ) (g L1 ) (g L1 ) (g L1 )

4-10-1 Xyti 18 17 30 35
5-10-2 BMi , 23 17 19 19 22
Xyti
6-10-2 BMi+1, 15 12 18 18 17 20
Xyti+1
7-10-3 BMi+1, 8 10 15 18 18 16 15
Xyli+1,
Xyti+1
6-10-3 BMi+1, 6 9 21 22 22 20
Xyli+1,
Xyti+1
4-10-3 BMi+1, 22 27 31 19
Xyli+1,
Xyti+1
5-10-2 BMi+1, 21 23 26 13 17
Xyti+1

BM Biomass; Xyl Xylose; Xyt Xylitol.

Table 6
Optimized parameters for enhanced xylitol production by Debaryomyces nepalensis NCYC 3413 in batch reactor using hybrid regression GA and hybrid ANN-GA.

Approach Variables Xylitol concentration (g L1 )

Time (h) pH Agitation (rpm) Aeration (vvm) Biomass (i-1) (g L1 ) Glycerol (i-1) (g L1 ) Predicted Experimental

Hybrid regression GA 78 5.2 400 1.0 28.38 5.070 58.70 52.88 0.32
Hybrid ANN-GA 82 4.9 500 0.5 24.82 8.652 65.73 63.42 0.27
144 S.M.J. Pappu, S.N. Gummadi / Biochemical Engineering Journal 120 (2017) 136145
Fig. 3. Fermentation profiles of xylitol production by Debaryomyces nepalensis NCYC 3413 under optimized fermentation conditions based on (a) Hybrid regression GA (b)
Hybrid ANN GA. ! Biomass (g L1 ), " Xylose (g L1 ), ! Xylitol (g L1 ), " Glycerol (g L1 ).
experimental concentration of xylitol by hybrid ANN-GA approach
were 65.73 g L1 and 63.42 g L1 respectively. Under optimized fer-
mentation conditions, production of xylitol was improved when
compared to the previous studies in which the concentration of xyl-
itol was found to be 59.4 g L1 after optimization by multi-response
analysis [34]. The prediction error at optimum condition by hybrid
regression-GA and hybrid ANN-GA were 10% and 3.5% respectively.
The difference between predicted and experimental values can be
contributed to the extent deviation in predictive capacity of the
model [35]. ANN is better equipped to reach the global optimum
due to its accuracy in expressing non-linear relationship and gen-
eralized model than regression. In case of regression, the search
window needs to be narrowed down approximately. Search process
of GA is highly dependent on input space and the objective func-
tion. The limitation of regression models can be easily overcome
by ANN since it can inherently capture any form of non-linearity
existing between input and output variables.
4. Conclusions
In the present work, the predictive capabilities of regression
model and ANN model were compared and optimization of fermen-
tation conditions was carried out by coupling with GA. With same
number of data points for training ANN showed better accuracy
than regression models. ANN has shown high accuracy in finding
optimum fermentation condition to enhance xylitol production in
bioreactor. The production of xylitol was significantly increased
(from 59.4 g L1 (multi response analysis) to 65.73 g L1 ) when arti-
ficial intelligence based technique was used for optimization. Thus
it can be concluded that ANN coupled GA is a better optimization
method than regression models and is recommended to be used
during the optimization of fermentation process.
Acknowledgement
The authors would like to acknowledge Department of Biotech-
nology, Government of India for funding this research work.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bej.2017.01.010.
References
[1] T.B. Granstrm, K. Izumori, M. Leisola, A rare sugar xylitol. Part II:
biotechnological production and future applications of xylitol, Appl.
Microbiol. Biotechnol. 74 (2007) 273276, http://dx.doi.org/10.1007/s00253-
006-0760-4.
[2] J.-P. Mikkola, H. Vainio, T. Salmi, R. Sjholm, T. Ollonqvist, J. Vyrynen,
Deactivation kinetics of Mo-supported Raney Ni catalyst in the hydrogenation
of xylose to xylitol, Appl. Catal. Gen. 196 (2000) 143155, http://dx.doi.org/
10.1016/S0926-860X(99)00453-6.
[3] J.C. Paraj, H. Domnguez, J.M. Domnguez, Production of xylitol from raw
wood hydrolysates by Debaryomyces hansenii NRRL Y-7426, Bioprocess. Eng.
13 (1995) 125131, http://dx.doi.org/10.1007/BF00369695.
[4] R.C.L.B. Rodrigues, W.R. Kenealy, T.W. Jeffries, Xylitol production from DEO
hydrolysate of corn stover by Pichia stipitis YS-30, J. Ind. Microbiol.
Biotechnol. 38 (2011) 16491655, http://dx.doi.org/10.1007/s10295-011-
0953-4.
[5] K.-K. Cheng, H.-Z. Ling, J.-A. Zhang, W.-X. Ping, W. Huang, J.-P. Ge, J.-M. Xu,
Strain isolation and study on process parameters for xylose-to-xylitol
bioconversion, Biotechnol. Biotechnol. Equip. 24 (2010) 16061611, http://dx.
doi.org/10.2478/V10133-010-0013-7.
[6] M.F.S. Barbosa, M.B. Medeiros, I.M. Mancilha, H. Schneider, H. Lee, Screening
of yeasts for production of xylitol fromd-xylose and some factors which affect
xylitol yield in Candida guilliermondii, J. Ind. Microbiol. 3 (1988) 241251,
http://dx.doi.org/10.1007/BF01569582.
[7] A. Converti, J.M. Domnguez, Influence of temperature and pH on xylitol
production from xylose by Debaryomyces hansenii, Biotechnol. Bioeng. 75
(2001) 3945.
[8] A. Converti, P. Perego, A. Sordi, P. Torre, Effect of starting xylose concentration
on the microaerobic metabolism of Debaryomyces hansenii: the use of carbon
material balances, Appl. Biochem. Biotechnol. 101 (2002) 1530, http://dx.
doi.org/10.1385/ABAB:101:1:15.
[9] K. Himabindu, S. Gummadi, Effect of kLa and fed-batch strategies for
enhanced production of xylitol by Debaryomyces nepalensis NCYC 3413, Br.
Biotechnol. J. 5 (2015) 2436, http://dx.doi.org/10.9734/BBJ/2015/14273.
[10] S.S. Suva, M.G.A. Felipe, I.M. Mancilha, Factors that affect the biosynthesis of
xylitol by xylose-fermenting yeasts: a review, Appl. Biochem. Biotechnol.
7072 (1998) 331339, http://dx.doi.org/10.1007/BF02920149.
[11] S. Kumar, S.N. Gummadi, Purification and biochemical characterization of a
moderately halotolerant NADPH dependent xylose reductase from
Debaryomyces nepalensis NCYC 3413, Bioresour. Technol. 102 (2011)
97109717, http://dx.doi.org/10.1016/j.biortech.2011.07.030.
[12] H.B. Klinke, A.B. Thomsen, B.K. Ahring, Inhibition of ethanol-producing yeast
and bacteria by degradation products produced during pre-treatment of
biomass, Appl. Microbiol. Biotechnol. 66 (2004) 1026, http://dx.doi.org/10.
1007/s00253-004-1642-2.
[13] J.C. Paraj, H. Domnguez, J. Domnguez, Biotechnological production of
xylitol. Part 2: Operation in culture media made with commercial sugars,
Bioresour. Technol. 65 (1998) 203212, http://dx.doi.org/10.1016/S0960-
8524(98)00036-4.
[14] F.C. Sampaio, V.M. Chaves-Alves, A. Converti, F.M. Lopes Passos, J.L. Cavalcante
Coelho, Influence of cultivation conditions on xylose-to-xylitol bioconversion
by a new isolate of Debaryomyces hansenii, Bioresour. Technol. 99 (2008)
502508, http://dx.doi.org/10.1016/j.biortech.2007.01.017.
[15] L.V.A. Reddy, Y.-J. Wee, J.-S. Yun, H.-W. Ryu, Optimization of alkaline protease
production by batch culture of Bacillus sp. RKY3 through Plackett-Burman
and response surface methodological approaches, Bioresour. Technol. 99
(2008) 22422249, http://dx.doi.org/10.1016/j.biortech.2007.05.006.
[16] H. Abu Qdais, K. Bani Hani, N. Shatnawi, Modeling and optimization of biogas
production from a waste digester using artificial neural network and genetic
algorithm, Resour. Conserv. Recycl. 54 (2010) 359363, http://dx.doi.org/10.
1016/j.resconrec.2009.08.012.
[17] E.C. Rivera, S.C. Rabelo, D. dos Reis Garcia, R.M. Filho, A.C. da Costa, Enzymatic
hydrolysis of sugarcane bagasse for bioethanol production: determining
optimal enzyme loading using neural networks, J. Chem. Technol. Biotechnol.
85 (2010) 983992, http://dx.doi.org/10.1002/jctb.2391.
[18] S. Vats, S. Negi, Use of artificial neural network (ANN) for the development of
bioprocess using Pinus roxburghii fallen foliages for the release of
polyphenols and reducing sugars, Bioresour. Technol. 140 (2013) 392398,
http://dx.doi.org/10.1016/j.biortech.2013.04.106.
[19] D.E. Goldberg, Genetic Algorithms in Search, Optimization, and Machine
Learning, Addison-Wesley Pub. Co., Reading, Mass, 1989.
 S.M.J. Pappu, S.N. Gummadi / Biochemical Engineering Journal 120 (2017) 136145
[20] D. Sarkar, J.M. Modak, Optimisation of fed-batch bioreactors using genetic
algorithms, Chem. Eng. Sci. 58 (2003) 22832296, http://dx.doi.org/10.1016/
S0009-2509(03)00095-2.
[21] V.V. Nair, H. Dhar, S. Kumar, A.K. Thalla, S. Mukherjee, J.W.C. Wong, Artificial
neural network based modeling to evaluate methane yield from biogas in a
laboratory-scale anaerobic bioreactor, Bioresour. Technol. 217 (2016) 9099,
http://dx.doi.org/10.1016/j.biortech.2016.03.046.
[22] R.C. Deo, M. Sahin, Application of the Artificial Neural Network model for
prediction of monthly Standardized Precipitation and Evapotranspiration
Index using hydrometeorological parameters and climate indices in eastern
Australia, Atmospheric Res. 161162 (2015) 6581, http://dx.doi.org/10.
1016/j.atmosres.2015.03.018.
[23] H. Kumdam, S. Narayana Murthy, S.N. Gummadi, Production of ethanol and
arabitol by Debaryomyces nepalensis: influence of process parameters, AMB
Express 3 (2013) 23, http://dx.doi.org/10.1186/2191-0855-3-23.
[24] W. Klckner, R. Gacem, T. Anderlei, N. Raven, S. Schillberg, C. Lattermann, J.
Bchs, Correlation between mass transfer coefficient kLa and relevant
operating parameters in cylindrical disposable shaken bioreactors on a
bench-to-pilot scale, J. Biol. Eng. 7 (2013) 28, http://dx.doi.org/10.1186/1754-
1611-7-28.
[25] P.M. Doran, Bioprocess Engineering Principles, Reprint Elsevier/Acad. Press,
Amsterdam, 2004.
[26] G.A. Hughmark, Power requirements and interfacial area in gas-liquid turbine
agitated systems, Ind. Eng. Chem. Process Des. Dev. 19 (1980) 638641,
http://dx.doi.org/10.1021/i260076a023.
[27] S. Kumar, S.N. Gummadi, Metabolism of glucose and xylose as single and
mixed feed in Debaryomyces nepalensis NCYC 3413: production of
industrially important metabolites, Appl. Microbiol. Biotechnol. 89 (2011)
14051415, http://dx.doi.org/10.1007/s00253-010-2997-1.
[28] F. Yazdandoost, J. Attari, International conference on hydraulics of dams and
river structures, in: Hydraulics of Dams and River Structures: Proceedings of
the International Conference on Hydraulics of Dams and River Structures,
2628 April 2004, Tehran, Iran, Balkema, Leiden, New York, 2004, http://dx.
doi.org/10.1201/b16994 (Accessed December 21, 2016).
145
[29] G. Dobrev, I. Pishtiyski, V. Stanchev, R. Mircheva, Optimization of nutrient
medium containing agricultural wastes for xylanase production by
Aspergillus niger B03 using optimal composite experimental design,
Bioresour. Technol. 98 (2007) 26712678, http://dx.doi.org/10.1016/j.
biortech.2006.09.022.
[30] F. Coelho Sampaio, T.L. da Conceico Saraiva, G. Dumont de Lima e Silva, J.
Teles de Faria, C. Grij Pitangui, B. Aliakbarian, P. Perego, A. Converti, Batch
growth of Kluyveromyces lactis cells from deproteinized whey: response
surface methodology versus artificial neural networkgenetic algorithm
approach, Biochem. Eng. J. 109 (2016) 305311, http://dx.doi.org/10.1016/j.
bej.2016.01.026.
[31] F.M. Grio, J.C. Roseiro, P. S-Machado, A.R. Duarte-Reis, M.T. Amaral-Collaco,
Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism
in Debaryomyces hansenii, Enzyme Microb. Technol. 16 (1994) 10741078,
http://dx.doi.org/10.1016/0141-0229(94)90145-7.
[32] A.K. Chandel, S.S. da Silva, W. Carvalho, O.V. Singh, Sugarcane bagasse and
leaves: foreseeable biomass of biofuel and bio-products, J. Chem. Technol.
Biotechnol. 87 (2012) 1120, http://dx.doi.org/10.1002/jctb.2742.
[33] E. Winkelhausen, S. Kuzmanova, Microbial conversion of d-xylose to xylitol, J.
Ferment. Bioeng. 86 (1998) 114, http://dx.doi.org/10.1016/S0922-
338X(98)80026-3.
[34] J.S.M. Pappu, S.N. Gummadi, Multi response optimization for enhanced xylitol
production by Debaryomyces nepalensis in bioreactor, 3 Biotech 6 (2016),
http://dx.doi.org/10.1007/s13205-016-0467-x.
[35] K. Desai, S.L. Sansom, M.L. Ackers, S.R. Stewart, H.I. Hall, D.J. Hu, R. Sanders,
C.R. Scotton, S. Soorapanth, M.-C. Boily, G.P. Garnett, P.D. McElroy, Modeling
the impact of HIV chemoprophylaxis strategies among men who have sex
with men in the United States: HIV infections prevented and
cost-effectiveness, AIDS 22 (2008) 18291839, http://dx.doi.org/10.1097/
QAD.0b013e32830e00f5.