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Bioresource Technology 101 (2010) 2884–2887

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Short Communication

Artificial neural network modeling and genetic algorithm based medium
optimization for the improved production of marine biosurfactant
C. Sivapathasekaran, Soumen Mukherjee, Arja Ray, Ashish Gupta, Ramkrishna Sen *
Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal-721302, India

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

Article history: A nonlinear model describing the relationship between the biosurfactant concentration as a process out-
Received 29 July 2009 put and the critical medium components as the independent variables was developed by artificial neural
Received in revised form 23 September network modeling. The model was optimized for the maximum biosurfactant production by using
genetic algorithm. Based on a single-factor-at-a-time optimization strategy, the critical medium compo-
Accepted 24 September 2009
Available online 14 November 2009
nents were found to be glucose, urea, SrCl2 and MgSO4. The experimental results obtained from a statis-
tical experimental design were used for the modeling and optimization by linking an artificial neural
network (ANN) model with genetic algorithm (GA) in MATLAB. Using the optimized concentration of crit-
ical elements, the biosurfactant yield showed close agreement with the model prediction. An enhance-
Design expert ment in biosurfactant production by approximately 70% was achieved by this optimization procedure.
Neural network modeling Ó 2009 Elsevier Ltd. All rights reserved.
Genetic algorithm

1. Introduction ous engineering fields, this modeling and optimization approach
has not been fully exploited to enhance the production of biomol-
Biosurfactants are extracellular surface-active molecules pro- ecules such as biosurfactants (Pal et al., 2009).
duced by various bacteria including Bacillus sp. (Desai and Banat, In this study, an attempt was made to optimize the biosurfac-
1997). The production of these molecules has gained a consider- tant production by using ANN-GA based nonlinear modeling and
able interest in recent times mainly due to their high surface activ- optimization. The critical medium components were identified by
ities, heterogeneity and great potential for therapeutic applications a single-factor-at-a-time strategy. The model was linked with GA
like antimicrobial, antifungal, antioxidant and antiviral agents to find the maximum production level and the optimum concen-
(Cameotra and Makkar, 2004). Despite their multifarious advanta- trations of the critical medium components that affect significantly
ges and diverse potential applications, their production on a com- the production process.
mercial level could not be achieved due to the low yields in
production process (Mukherjee et al., 2008). One of the primary ap-
proaches applied for obtaining increased yields in fermentative 2. Methods
production is the medium optimization. The most effective method
used for the optimization of factors is the statistical approach (Sen, 2.1. Microorganism, medium, production and quantification
1997). There is fairly good amount of available literature on med-
ium optimization using different statistical methods. These include Bacillus circulans MTCC 8281 previously isolated from Andaman
experimental designs such as Plackett–Burman (Mukherjee et al., and Nicobar Islands, India was used in this study. The medium
2008), response surface methodology (Sen, 1997; Mutalik et al., used both for inoculum preparation and biosurfactant production
2008) and factorial designs (Rodrigues et al., 2006). Other mathe- was a Modified Marine Medium (MMM) containing the following
matical approaches such as ANN coupled to GA have also been pro- components in g L 1: glucose 20, urea 3.3, K2HPO4 2.2, SrCl2 0.2,
ven to be powerful tools in optimization and superior to statistical NaCl 0.1, MgSO4 0.6, FeSO4 0.04 and microsalt solution 500 ll
approaches such as response surface methodology (Pal et al., L 1. The microsalt solution consisted of (g/L): H3BO3 0.56, Na2-
2009). Although ANN-GA has gained popularity for its use in vari- MoO42H2O 0.39, KI 0.66, CuSO45H2O 0.4, CoCl26H2O 0.42,
ZnSO47H2O 2.0, EDTA 1.0, NiCl26H2O 0.002, MnSO44H2O 0.90,
CaCl2 0.04 and KH2PO4 0.14. The fermentation (5% pre-inoculum
was used) was carried out for 28 h at 37 °C on a rotary shaker at
* Corresponding author. Tel.: +91 3222 283752; fax: +91 3222 278707. 180 rpm. The biosurfactant produced in the fermentation medium
E-mail address: (R. Sen). was quantified directly from cell-free supernatant (CFS) by a HPTLC

0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.

70 tual experimental production values and the ANN-simulated 14 31. Imandi et al. The range and levels of the four independent in the hidden layer and one in the output layer of the network to variables along with the central values for these critical media model the dependence of the biosurfactant production on the four components are listed in Table 1.95 The training of the ANN was done for 3000 epochs after which 11 13.0 tively.00 0. Single-factor-at-a-time experiments and experimental design ANN models.25 2. Results and Discussion experiment (g L 1) 2 1 0 +1 +2 Biosurfactant production was found to be cell growth associ- Glucose 5 13.225 0. six (Mutalik et al.25 2.00 0. 2.40 2.00 3.1.e. Pal et al. 22 22.60 10 31.0 (Stat-Ease it was trained to accurately model the given phenomenon by using Inc.60 2. Imandi et al.60 2.70 from 22 g L 1 to 32 g L 1. USA) (Mutalik et al.75 4.50 3.30 21 22.0 9 13. Optimization of biosurfactant production 2.23 0.1 (Math- works Inc. 1a). average mean value of attain the maximum production.00 0.60 1.60 2..00 0. the regression correlation coefficient between the ac- 13 13.40 1. Table 2 3.80 (Fig. that optimizes a given function over a particular range. ( 2) indicates the low level. In a previous report urea was response was coded.00 0..31 0.00 0.14 0.75 4.90 evident from Table 3. 18 40.31 0.40 1.23 0.00 0.40 1.00 0.60 5 13. 2008).00 0. US)..50 5. based on the evolutionary methods of natural selection of the best individuals in a population (Goldberg.40 0..80 1. which measures the quality of individuals The boundaries of the experimental domain and the levels of the four variables.90 mined using the methods of ANN-GA. (Table 2) was obtained using Design Expert version 7. GA was used to optimize and used in modeling and simulation of various systems.50 1.50 3. The average and maximum per- 16 31.60 ously reported one (Sen.00 0.05 0. the experimental data (Table 2) in MATLAB version 7.23 0.00 0. Once the ANN model was devel- ANN is a biologically inspired computational tool that is widely oped to the desired level of precision. mean absolute error (MAE) were used as measures of closeness GA) as a modeling and optimization tool of the model to the actual system.60 optimum concentration of urea in the medium was decreased from 30 22.60 1.2. For the optimization using GA.23 0.00 0. the one of our interest was the feed forward back The critical media components that influence the biosurfactant propagation network (Pal et al. on the determine the maximum possible biosurfactant production and other hand.00 0...25 40 ated as reported earlier for other biosurfactant producers (Rodri- Urea 1 2 3 4 5 gues et al.75 2.8 1 mentation in MMM. Critical media component Range of the media component for designing 3.25 2.20 1.23 0.60 0.60 26 22. 2009). a single hidden layer comprising of six (CCD) was applied for the four factors and the experimental design neurons was used (Fig. 2008).00 0.40 outputs was found to be 0.80 1..00 0. therefore.2.. Also. 1997).31 0. Sivapathasekaran et al.23 1.70 of successive generations towards the final optimum value. This value was very close to the previ- 25 22.2%.60 2.20 components for enhancing biosurfactant production were deter- 4 31. 2008).80 After the optimization.75 2.0094 and 0.0 to 1.14 0. Zero indicates the central value.70 surfactant production (Rodrigues et al. Optimization of critical medium components using ANN-GA 8 31.0 g L 1 which meant less amount of urea was required to a Experiment performed in triplicate manner.9996.00 0.00 0. Minneapolis.00 0.00 0.75 2.00 0. the glucose concentration required for 23 22.5 31. The mean standard error (MSE) and the 2.60 cally influenced the cell growth and biosurfactant production as 2 31.2.25 4.14 0.80 2. urea. likewise (+2) indicates the in the population. C.31 0.3125 0. A central composite design media components.60 1.. 1b) maps the gradual convergence of the best fitness values 20 22.31 0. 2006.40 0.31 0...23 0. respectively.23 0.2. 2009.00 3.14 0.80 0.50 3. high level. Pal et al.00 0.75 22. Among the various 2.23 0.31 0. is a commonly used globalized optimization technique the point in the input space where this maximum was to be ob- tained.1375 0. Once the neural network was created.25 4..14 0. 2009).00 0.5% and 2.90 15 13.00 0.2 0.60 2.10 centage errors were 0.25 4.75 4.2. The best fitness plot for the GA 19 22. i.50 3.23 0.74 ter than previously reported values in a similar study (Pal et al.90 7 13. four crit- Run Glucose Urea SrCl2 MgSO4 Biosurfactant ical medium components glucose. respec- 12 31.1.80 0.31 0.25 4.14 0. Briefly.50 3. MgSO4 and SrCl2 were order (g L 1) (g L 1) (g L 1) (g L 1) concentration (g L 1 a ) identified in MMM.60 0.2.50 3. production were identified by single-factor-at-a-time experiments in the present study four neurons were used in the input layer.00 0. the ANN model was used Table 1 as the fitness function.50 3..80 2.4 0.00 0. Single-factor-at-a-time optimization strategy The 24 full factorial central composite design (CCD) consisting of 30 experiments with six central experimental runs.4 concentration of 2. 2008).20 3.23 0.1. Imandi et al.40 the maximum biosurfactant production showed a marked increase 24 22.00 0. The maximum biosurfactant SrCl2 0. / Bioresource Technology 101 (2010) 2884–2887 2885 based densitometric method (Mukherjee et al.50 3.14 0. Artificial neural network linked with genetic algorithm (ANN..40 0.2.25 2. 2006.05 0.60 2.50 3.50 3. used for the production of biosurfactant by Candida (Harrop .75 4.0066. On the basis of single variable at a time experiments. and is roborated by dry weight method (Sen.20 2009.00 0. all the experiments were performed up to 28 h.80 1.00 0. 1997) and this sharp increase clearly 27 22.80 1. The optimum concentrations of these critical 3 13. Natick.23 0. 6 31. 1989. which was bet- 17 5.00 0.60 showed that the carbon source was the key factor affecting the bio- 28 22.75 2. These four components in the medium criti- 1 13.60 2.00 0.70 3.14 0. Feed forward back propagation network. The 29 22.40 1..58 g L 1 was found to be at 28 h during the fer- MgSO4 0.00 0. n = 3. 2008).40 2.90 the MSE and the MAE were found to be 0. 2008) and also cor.6 0. GA.

40 ± 0. Best and average fitness values with successive generations showed gradual convergence to the optimum value. (a) Schematic representation of a (4–6–1) neural network (having four neurons in the input layer. respectively. The results were factant production. The model was validated by running the GA several times using Acknowledgements different randomly selected initial populations. 1989. R. Optimization and Machine a Experiments were performed in triplicate (mean ± standard deviation)..38 g L 1 could be achieved the desired functional behavior more accurately than previously by using the aforementioned optimized concentrations of the vari. Biol. SMST. Control (including all nutrients) 4.03 2. which was also validated by experimental study. cation of GA predicted an increase in biosurfactant production by approximately 70% as compared to the un-optimized parent 3.06 2.10 2. circulans MTCC 8281 in MMM. The model predicted that tion by B.03 Cameotra. 1. MgSO4.1.21 ± 0.02 and immunological molecules.S.. Sivapathasekaran et al.20 ± 0.04 Desai. Microbiol. Opin. 2003). however. biosurfactant yield of 4.. 262–266. IIT-Kharagpur for his valuable Urea ND ND MgSO4 ND ND support. 1997). The present study showed the ANN-GA based optimization of tion. Makkar. respectively.. However.47 ± 0. I. components as well as biosurfactant production.388 Mean: -4. Results of single factor at a time experiments – effect of medium components on Government of India for the project grant (BT/PR-6827/AAQ/03/ biomass and biosurfactant production. Model validation and confirmative studies MMM.5 g L 1 (HPTLC method) and was also used in the medium to enrich the growth of the marine 4. Mol. ND – not detected.06 2..M. Addison-Wesley.84 g the optimized media. has been reported as a trace element in the biosynthesis of biosurfactant and a key factor 4.89 ± 0. Recent application of biosurfactant as biological Microsalts 4.2886 C.01 ± 0.24 ± 0. Goldberg. Rev. SM acknowledges CSIR. For SrCl2 and MgSO4. for biosur. There are several reports describing the optimization study in biosurfactant produc. this is the first report showing the use of successive runs showed very slight variation in the values of media urea as nitrogen source for the cultivation of Bacillus sp.08 g L 1 and 0. this is the first report showing the optimization of critical media components for the enhanced biosurfactant produc- critical parameters like urea and SrCl2. 1941). Genetic Algorithms in Search. Conclusion required for the sporulation of cells (Sen. 47–64.D.jj X1 Output layer X2 Results X3 X4 Best: -4.06 1.58 ± 0. / Bioresource Technology 101 (2010) 2884–2887 (a) hidden layer Input layer Wii. Authors acknowl- Glucose ND ND edge Roshan Joy Martis. S. SrCl2 1. J. (b) Representative plots generated from the optimization by GA using MATLAB 7.56 ± 0. a rare salt present in marine environments.3.07 NaCl 3. 61.3281 3 (b) 2 Best fitness Mean fitness 1 Fitness value 0 -1 -2 -3 -4 -5 0 20 40 60 80 100 120 140 160 180 200 Generation Fig. the confirmed by conducting sets of shake flask experiments using optimum concentrations were found to be 0. Learning. SrCl2. Banat. This is mental results were significantly close to the value predicted by the first report to indicate its importance in the biosurfactant pro. The results of 10 Table 3 RS and CS acknowledge the Department of Biotechnology (DBT). D.96 ± 0. on the other hand. 263/2005) in marine biotechnology. and its optimization.20 0. These experi- strain and it is used as a growth stimulant (Zobell. the ANN-GA model.34 ± 0.05 References K2HPO4 3. 7. six in the hidden layer and one in the output layer).35 ± 0. duction process. 2004.6 g L 1 (dry weight method). Microbial production of surfactants and their commercial potential. reported studies and the optimized values obtained from the appli- ables in the medium.. 1997..S. Authors acknowledge Palashpriya Das for strain isolation and her support in this study. BS – biosurfactant.71 ± 0. ANN was able to model the maximum biosurfactant yield of 4. . The experimental results showed the higher L 1. Curr.04 FeSO4 2. Microbiol. New Nutrients absent in MMM Biomass (g L 1 a ) BS (g L 1 a ) Delhi for his fellowship.98 ± 0. et al.

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