Artificial neural network to optimise parameters for enhancing xylitol production in bioreactor

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Artificial neural network to optimise parameters for enhancing xylitol production in bioreactor

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

Regular article

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.

Candida [6] and Debaryomyces [7,8] are the best known yeast

Xylitol is a naturally occurring sugar alcohol with one third species for xylitol production. Biotechnological production of xyl-

calories lesser than sucrose [1]. In recent years, interest in xyli- itol is limited by several factors which includes age and inoculum

tol has increased considerably, mainly due to many commercial concentration, initial substrate concentration [8], pH, temperature

applications in several industrial sectors like food, dental and [7], aeration and agitation conditions of the fermentation process

pharmaceuticals. Industrially, xylitol is currently manufactured by [9,10].

chemical hydrogenation of pure xylose in the presence of metal Debaryomyces nepalensis NCYC 3413, a halotolerant yeast strain

catalysts like nickel, palladium and ruthium [2] at elevated tem- was isolated from rotten apple, which is capable of efficiently utiliz-

perature (80140 C) and pressure (50 atm) [3]. Chemical method ing hemicellulose-derived sugars to produce industrially important

of xylitol manufacturing is laborious, energy and cost intensive. metabolites like xylitol, arabitol and ethanol [11]. Being halotol-

Furthermore, it also requires pure substrate (xylose) for hydro- erant, D. nepalensis can be considered an ideal strain for xylitol

genation, thus adding the refining cost to the total production production using lignocellulose as substrate, since pretreatment

cost. Microbial or enzymatic production of xylitol is becoming a of lignocellulose releases large amounts of inorganic salts that are

more sustainable alternative. Biotechnological production of xyli- inhibitory to microbes [12]. However, the large scale microbial pro-

tol is gaining more interest as it takes place under mild operating duction is rendered by physical and operational parameters such as

conditions, ease in purification and relatively economical and safe pH, temperature, initial xylose concentration and oxygen transfer

process [4]. rate [7,8,10,13,14]. It is necessary to optimize various parameters to

Microbial process uses bacteria, yeast and fungi for production make a process more cost effective and efficient. Statistical method-

of xylitol from xylose or hemicellulose hydrolysate derived from 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].

Corresponding author.

E-mail address: gummadi@iitm.ac.in (S.N. Gummadi).

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 137

Mathematical models could be used for prediction of micro- pH probe (Mettler Toledo, Switzaerland) and a temperature sen-

bial development, production of metabolites and optimization of sor. The dimensions of the bioreactor and the impeller were as

growth conditions in fermentation process. Information gained follows: Total volume: 2.5 L, working volume 1 L, tank height

from modelling might direct the adjustment of process parame- 270 mm, diameter of the tank 110 mm, Number of impeller

ters and model-based simulations proposes regulation of process 2, Type of impeller Rushton flat blade, Number of blades 6,

parameters, prediction of substrate and product concentrations impeller diameter 46 mm, height of the impeller blade 11 mm,

[16]. Regression analysis is a statistical technique for investigating width of the impeller blade 11 mm, Number of baffles 3. Sam-

and modelling the relationship between variables. In the recent ples were collected at regular time intervals and centrifuged at

years, complex biotechnological process has been modelled and 10,000 rpm for 10 min. The supernatant was used for substrate and

optimized using artificial intelligence techniques to attain high metabolite concentrations and the cell pellet was used to measure

operational performance [17,18]. Artifical neural network (ANN) biomass concentration. The aeration and agitation rates were var-

and genetic algorithm (GA) are more suitable for analyzing data in ied to obtain different kL a values using dynamic gassing out method

which the process variables project non-linear relation to output [9]. Prediction of rate of absorption of gaseous materials in a stirred

response or when it is too difficult to develop deterministic models. tank are usually based on correlations of overall volumetric mass

ANNs offer many advantages such as adaptive solutions and re- transfer coefficient (kL a) with mechanical agitation power per unit

estimation of model parameters in relatively simpler forms when volume (Pg /VL ) and gas sparging rate expressed as the superficial

compared to other modelling approaches. ANN model predicts the gas velocity (Vg ).

model output of the process using the same input as the process The following correlation is frequently found in the literature

after training and no additional measurements are needed during [24]:

the prediction phase. An artificial intelligence based stochastic non-

linear optimization technique, genetic algorithm (GA) is used to

kL a = ! (Pg /VL )" (Vg )c , (1)

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 where P is the mechanical agitation power in gas liquid dispersion

proved to be an ideal technique for solving non-linear optimization (W), VL is the working volume of the reactor (m3 ), Vg is the super-

problems [20]. ficial gas velocity (m/s) which was calculated by dividing the gas

No modelling studies have been published to date that address flow rate (m3 /s) by internal tank area (m2 ), ! is the constant, " and

the complete interaction of fermentation parameters accounting c are exponents.

biomass and by-product concentration as variables influencing Since accurate measurement of power input is difficult, power

production of metabolites. Indeed, many existing models have been number (NP = P3 5 ) was used to calculate the ungassed power

!N D

i i

developed using less number of input variables and trained with (Ni Stirrer speed, Di Impeller diameter). As the six blade rushton

reduced number of experimental data points [21,22]. However, turbine is employed for agitation, Np = 6 was used in the calculation

inclusion of biomass and byproduct concentration of previous data as reported in the literature [25]. An expression for the ratio of

point could improve the prediction capability of the model. In this gassed to ungassed power as a function of operating conditions as

study, regression models and ANN were evaluated as predictive given in Eq. (2) was used to calculate the power consumption with

tools for production of xylitol by Debaryomyces nepalensis in biore- sparging [26].

actor and to compare the performances of statistical and artificial

intelligence based optimization techniques.

Pg Fg

! "0.25 # N 2 D4 $0.20

i i

= 0.10 (2)

2. Materials and methods P0 Ni V gWi V 2/3

where Pg is power consumption with sparging, P0 is power con-

Debaryomyces nepalensis NCYC 3413, isolated from rotten apple, sumption without sparging, Fg is volumetric gas flow rate, Ni is

was maintained on a solid YEPP medium containing yeast extract stirrer speed, V is liquid volume, Di is impeller diameter, g is gravita-

10 g L1 , peptone 20 g L1 and pectin 5 g L1 at pH 7.0 and incubated tional acceleration and Wi is impeller blade width. Experimentally

at 30 C for 24 h and stored at 4 C. A single colony was transferred calculated kL a for varying agitation speed and aeration rate was

from an overnight-grown culture plate into the YEPD medium correlated to Pg /VL and Vs according to Eq. (1). Parameter values of

(50 mL) containing yeast extract 10 g L1 , peptone 20 g L1 and dex- ! = 0.34, " = 0.12 and c = 0.52 was estimated using feval and fmin-

trose 20 g L1 and incubated for 12 h at 30 C at 180 rpm 8% (v/v) search inbuilt functions of MATLAB. (Supplementary file: Estimated

seed culture was used to inoculate the fermentation medium in KL a values for different values of agitation speed and aeration rate).

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

2.3. Analytical methods

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 ;

Cell pellet was resuspended in water and A600 was measured.

3N H3 PO4 and 3N NaOH were used to adjust pH. All the components

The cell concentration was calculated from pre-calibrated graph of

were autoclaved separately and mixed subsequently as described

A600 vs cell dry weight (experimentally determined). A600 of 1.0

earlier [23].

corresponds to 0.34 g cell dry weight per liter culture as mentioned

earlier [27]. The concentration of xylose and metabolites (xylitol

2.2. Batch fermentation and glycerol) were analyzed by High Performance Liquid Chro-

matography (HPLC, Jasco, Japan) equipped with refractive index

The batch fermentation was carried out under varying opera- detector and Aminex HPX-87H column (Bio-Rad, Richmond, USA).

tional conditions as indicated in Table 1, in 2.5 L round bottomed The mobile phase used for the analysis was 0.01N H2 SO4 at a

cylindrical glass vessel bioreactor (Minifors, Infors HT, Switzerland) flow rate of 0.6 mL min1 and oven temperature was maintained

equipped with a dissolved oxygen probe (Hamilton, Switzerland), 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

( 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)

XNorm = + 0.05 (3)

Xmax "0 is intercept coefficient, "ii is the coefficient of squared terms,

"ij is the coefficient of interaction terms and xi represents inde-

where XNorm represents normalized value of the variables, Xori pendent variables considered for modelling (x1 temperature

denotes the original value of the variables before normalization, ( C), x2 time (h), x3 pH, x4 kL a, x5 biomass(i) , x6

Xmin and Xmax represents the minimum and maximum value of glycerol(i) , x7 xylose(i) ). Subscript i represents values of vari-

the variable considered for analysis [28]. The experimental data ables at ith h (where i = 0,6,. . .114) and i+1 represents data at

obtained was fitted to the quadratic polynomial model equation (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 139

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- where n is the number of experimental data points used for test-

ing significance tests and analysis of variance (ANOVA). Coefficient ing; YExp,i is the experimental value; YPred,i is the predicted values

of determination (R2 ) values was used to statistically assess the obtained from the model. The lower values of NRMSE means a

adequacy of polynomial model and significance of the regression better performance of the developed ANN [22].

coefficient was tested by using Students t-test. Regression analysis

of the experimental data and statistical analysis of the model were 2.5. Optimization by genetic algorithm (GA)

carried out using MINITAB 16 software.

Regression equation and trained neural network model were

used as the fitness function of GA for optimization of fermenta-

2.4.2. Artificial neural network (ANN) model

tion parameters to enhance the production of xylitol. GAs operate

Artificial neural network methodology employed in this study

simultaneously on a population of potential individuals employ-

was a multilayer perceptron model. Parameters (time, pH, temper-

ing the principle of natural evolution i.e., survival of the fittest

ature, kL a, biomass(i) , xylose(i) , glycerol(i) ) were used to construct

strategy, to produce better and better approximation to a solu-

ANN topology to identify the non-linear relationship between input

tion of the problem. The major operations of GA is summarized as

variables and output response. Transfer functions tan sigmoid

follows: (i) Initialization: initial population of a set of individuals

(f1: tansig) and pure linear (f2: purelin) were used in the hidden

(Input variables) was generated randomly, (ii) Evaluation: using the

and output layer respectively. Predicted output response can be

predefined objective function (Regression equation/ANN weights

described as follows:

and biases), the fitness of individuals were evaluated, (iii) Selec-

Response = f 2[wo f 1 (wH Inputvector + bH ) + bo ] (5) tion: ranking method was used in this study to select individuals

based upon its fitness such that the better individuals have an

where wH , bH and wo , bo are weights and biases of hidden and increased chance of being selected, (iv) Genetic operators: new

output layer respectively. LevenbergMarquardt back propagation set of individuals were generated from the existing individuals in

(BP) algorithm was used to train the network. In this training algo- the populations by using genetic operators such as cross-over

rithm, the error between the predicted and observed values was with a probability of 0.8, (v) Termination: GA is terminated after

calculated and propagated backward through the network to the a pre-specified number of generations and the resulted solution is

weights of each layer. The algorithm autonomously adjusted the tested against objective function. Overall schematic representation

connection weights in the direction of reducing errors between pre- of regression and ANN coupled GA is shown in Fig. 1.

dicted and observed until the errors met the requirement. Trained The objective function of hybrid regression GA (Eq. (8)) and

ANN must be evaluated by testing samples to determine the pre- hybrid ANN-GA (Eq. (9)) can be defined as follows:

diction capability. Adequacy of the developed model was estimated Maximize y = f(x, regression model);

using mean squared error (MSE) and normalized root mean squared xiL xi xiU , i = 1, 2, . . ..n (8)

error (NRMSE) as represented in Eqs. (6) and (7).

Maximize y = f(x, W);

n

1 %& '2 xiL xi xiU , i = 1, 2, . . ..n (9)

MSE = YExp,i YPred,i (6)

n

i=1 where f represents the objective function (regression model); W

( represents the weight vectors; x denotes the input vector, the

1

)n & '2

fermentation parameters to be optimized; y refers to the xylitol

n i=1

YExp,i YPred,i

NRMSE = & ' (7) concentration; n denotes number of input variables; and xiL and

YExp,min YExp,max 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

function: where x1 temperature ( C), x2 time (h), x3 pH, x4 kL a, x5

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

data at (i+6)th h (i+1 = 6,12,. . .120).

where Errorj denotes the fitness value of the candidate solution

j

Positive and negative signs of the coefficients in the regression

and ypred denoted the model predicted xylitol concentration for model equations indicate that any increase in that particular factor

particular individual. A constrained based GA optimization was leads to a statistically-significant decrease or increase in p-value

implemented, which searches for the optimal only in the feasible [30]. Adequacy and statistical significance of the regression models

region and the optimization is subjected to the constraint that tem- thus obtained was evaluated through analysis of variance (ANOVA).

perature of fermentation process is maintained at 30 C. Training Table 2 depicts analysis of variance of xylitol regression model, in

of artificial neural network and optimization by genetic algorithm which the F and p values of the model were observed to be 78.06 and

was performed using MATLAB 2013. <0.0001 respectively, which implied that the model was significant

at 95% confidence level (p < 0.05). Terms in the model equation with

3. Results and discussion p < 0.05 exhibits a significant contribution to the output response.

In the regression model for xylitol, linear terms of factors (tem-

Xylitol production in bioreactors was carried out at different

perature, time, pH, kL a, biomass and glycerol) were found to be

physiological conditions of pH, temperature, aeration and agita-

significant. Interactions terms involving pH and kL a, pH and xylose

tion rates as shown in Table 1. The samples were collected at every

and interaction of kL a with biomass and glycerol were found to

6 h interval resulted in 340 data points (27 runs). The fermenta-

have p < 0.05 which implies that pH, agitation and aeration affects

tion conditions, xylitol concentration, productivity, time at which

the production of xylitol. Lack of fit with high t-value and low

maximum xylitol concentration was produced, product yield coef-

p-value denoted that the model is significant relative to the pure

ficient (YP/S ) and the number of data points for each run that were

error. Further to test the model adequacy, determination of coef-

considered for ANN model training were tabulated in Table 1. All

ficient (R2 ) was evaluated which was observed to be 86.56% that

340 data points from Table 1 were used to train ANN model (Sec-

indicated the model could explain 14% of the variability. Regres-

tion 3.2). Experiments were performed at various combinations of

sion models for biomass prediction exhibits high adequacy with

pH (4.0, 5.0 and 6.0), temperature (25 C, 30 C and 35 C) and kL a

R2 value of 96.43% and F and p values of the model were observed

(0.14 h1 , 0.28 h1 and 0.56 h1 ) were considered as test data. To

to be 327.8 and <0.0001 respectively (Table 3), which implied that

check the robustness of the model, these set of experiments were

the model was significant at 95% confidence level (p < 0.05). It has

not included in the training data. Fermentation profiles for fermen-

been reported that xylose to xylitol conversion by microorganisms

tation run# (1,7 and 13) were provided as Supplementary figures

is strongly affected by oxygen supply. In the presence of excess aer-

(Supplementary Fig. S1ac).

ation, NADH is reoxidized by respiratory chain, catalyzed by NAD+

dependent xylitol dehydrogenase (XDH) and xylitol is consumed

3.1. Regression model for prediction of xylitol production by

for growth [31]. pH of the medium also plays a vital role in the

Debaryomyces nepalensis NCYC 3413 in batch reactor

enhanced production of xylitol as pH affects the transport of xylose

across the cell membrane [32]. In addition to aeration, agitation and

The experimental conditions and experimental values (Table 1)

pH, the activities of enzyme responsible for microbial production

were used to build a second order polynomial model by a multi-

of xylitol are very sensitive to the substrate concentration. since

ple regression technique to examine the individual and combined

the real substrates for xylitol producing microorganisms are ligno-

effect of input variables on xylitol and biomass production. The

cellulosic hydrolysate which often contain a variety of sugars [33].

coefficients of each term (normalized) of the second order polyno-

These results suggest that pH of the medium, aeration and agita-

mial equation for biomass and xylitol were given in Eqs. (11) and

tion rates are very much crucial for enhanced xylitol production in

(12).

bioreactor.

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 3.2. Development of ANN model for prediction of xylitol

x5 6.278 x 1 x6 + 1.651 x 1 x7 + 0.593 x 2 x3 + 1.171 x 2 production in batch reactor

x4 0.282 x 2 x5 0.586 x 2 x6 0.188 x 2 x7 0.455 x 3 Experimental data from 27 fermentation batches (Table 1) were

integrated (Total number of data points: 340) and then divided

x4 0.494 x 3 x5 1.476 x 3 x6 1.053 x 3 x7 2.024 x 4

randomly into two divisions: training data (75%) and validation

x5 + 1.246 x 4 x6 0.256 x 4 x7 1.533 x 5 x6 0.322 x 5 data (10%). The mean square error (MSE) between predicted and

experimental values was set at 0.005 and the maximum number of

x7 0.301 x 6 x7 (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

Biomass(i+1) = 0.345 1.264 x 1 0.599 x 2 + 0.0402 x 3

the validation set begins to increase typically. When the validation

+ 0.096 x 4 + 2.106 x 5 0.825 x 6 0.386 x 7 error increases for a specified number of iterations (set n = 6), the

network training is stopped and the weights and biases of the net-

+ 3.10346 x 1 x2 6.433 x 1 x5 + 4.741 1 x6 + 1.551 x work at the minimum of the validation error were obtained. The test

0.399 x 2 x3 + 0.201 x 2 x4 0.021 x 2 x5 0.023 x 2 data set, which are not used for training and validation were used

1 x7

to examine the trained network. ANN topology was identified by

x6 + 0.0351 x 2 x7 0.029 x 3 x4 + 0.377 x 3 x5 + 0.197 x 3 training with different neural networks varying the number of neu-

rons in the input and hidden layer. The NRMSE values of the neural

x6 0.0423 x 3 x7 0.311 x 4 x5 0.215 x 4 x6 0.009 x 4

network N/w I (6-10-3), N/w II (4-10-3) and N/w III (5-10-2) for

x7 0.0593 x 5 x6 + 0.177 x 5 x7 0.104 x 6 x7 (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.

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.

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.

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

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

predictability, the model was trained at fermentation temperature work. Since, glycerol is a by-product in the fermentation of xylose,

of 30 C (Table 1, Run # 126, 325 data points) and one single run of the concentration of biomass(i) and glycerol(i) of the previous data

fermentation temperature 25 C (Table 1 run # 27, 15 data points). point aids in better prediction of xylitol production. Network with

To check this, test experiments were performed at the following 6-10-3 topology shows equal contribution of temperature (21%),

conditions: varying pH (4.0, 5.0, 6.0) but at constant temperature kL a (22%), biomass(i) (22%), glycerol(i) (20%) and time (6%) and pH

of 30 C and kL a 0.28 h1 (Run# 13); varying temperature (25, 30 (9%). Network II (N/w II 4-10-3) was trained by excluding input

and 35 C) but at constant pH 4.0 and kL a 0.28 h1 (Run# 1, 4 and variables time and pH and shows increased contribution of

5); and varying kL a (0.14, 0.28 and 0.56 h1 ) but at constant pH 4.0 biomass(i) (31%). Network III (N/w III 5-10-2) was trained by

and temperature 30 C (Run# 1, 6 and 7) (Table 4). The experimen- including xylose concentration of previous data point as input vari-

tal conditions and data considered for testing the model (Table 4) able and exhibits contribution of temperature as 21%, kL a 23%,

were not included in the training data set (Table 1). biomass(i) 26%, glycerol(i) 13% and xylose(i) 17%. The pre-

The effective network topology for xylitol prediction was 5- diction capability of N/w I 6-10-3, N/w 4-10-3 and N/w

10-2, where 5 represents the input variables (temperature, kL a, 5-10-2 were compared using normalized root mean square error

biomass(i) , glycerol(i) , xylose(i) ), 10 as hidden neurons and 2 as out- (NRMSE). Network II and III were also trained with same number

put responses (biomass(i+1) , xylitol(i+1) ). The performance of the of data points with reduced number of input variables and tested

models was depicted in Fig. 2, which presents the prediction val- for different fermentation conditions. NRMSE values for test data

ues versus experimental values for training, validation and test data of seven fermentation runs were tabulated in Table 4 and no sig-

for network III run# 1. Figures representing the regression values nificant difference was observed in NRMSE values of network IIII.

of training, validation and test data of network III (Run# 27) as This result suggests principal components to be included in the

tabulated in Table. 4 were provided as supplementary material in prediction network were temperature, kL a, biomass(i) , xylose(i) and

MATLAB.FIG format. Even though ANN model was trained with only glycerol(i) .

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). 3.3. Optimization of fermentation parameters for enhanced

The weight matrix of each network can be employed to deter- production of xylitol by Debaryomyces nepalensis NCYC 3413 in

mine the relative importance of input variables on the output batch reactor using genetic algorithm (GA)

response. For this purpose, Eq. (13) is used in the partitioning of

connection weights. Experimental runs were carried under fermentation conditions

*# $ + (time (h), pH, agitation (rpm), aeration (vvm), biomass(i-1) (g L1 )

)Nh |W ih |

jm ho |

and glycerol(i-1) (g L1 )) optimized based on hybrid regression GA

m=1 ) Ni |W mn and hybrid ANN GA and the profiles of xylose consumption, biomass

|W ih |

Ij = , *#k=1 kmih $ +- (13) and xylitol production were shown in Fig. 3a and b respectively.

)Ni )Nh |W |

jm ho | Regression equation and weights and biases of ANN were coupled

k=1 m=1 ) Ni ih

|W mn

k=1

|W

km

| with artificial intelligence technique GA for optimizing the fer-

mentation operational conditions to enhance xylitol production.

where Ij is the relative importance of the jth input variable on the The comparison of xylitol production at optimized fermentation

output response, Ni and Nh are the number of neurons in the input conditions using different approaches (Hybrid regression-GA and

and hidden layer respectively, W is the connection weights, the Hybrid ANN-GA) are given in Table 6. Optimized fermentation

superscripts i, h and o represents input, hidden and output lay- conditions pH 5.2, agitation speed 400 rpm, aeration rate

ers respectively, and k, m and n refers to input, hidden and output 1.0 vvm by hybrid regression GA approach predicts 58.70 g L1 of

layer neuron number respectively. Relative importance of different xylitol at 78 h of fermentation, however optimized fermentation

ANN was calculated and tabulated in Table 5. Agitation and aeration conditions pH 4.9, agitation speed 500 rpm, aeration rate

shows equal percentage of contribution in predicting the output 0.5 vvm by hybrid ANN GA approach predicts xylitol concentra-

response. When temperature was included as an input parame- tion to be 65.73 g L1 at a fermentation time of 82 h. Experiments

ter, the contribution of agitation and aeration were halved which at the optimized fermentation conditions of regression coupled GA

indicates that aeration and temperature are inter dependent. Con- results in higher biomass yield (28.38 g L1 ) and low xylitol produc-

tribution of fermentation time is reduced to 15% when biomass(i) is tion (52.88 g L1 ) whereas experiments at fermentation conditions

included as an input variable in prediction of xylitol which signifies optimized by ANN coupled GA results in high xylitol production

the fact that the variation in biomass concentration as fermentation (63.42 g L1 ).

progresses accounts for the fermentation time. The contribution Regression model has predicted xylitol concentration of

of fermentation time is further reduced to 8% when both biomass 58.70 g L1 at optimized conditions. Experimental validation has

and glycerol were considered as input variables in training the net- 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.

structure of ANN responses

(Ni -Nh -No )

(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

Table 6

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

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 ).

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