European Journal of Scientific Research ISSN 1450-216X / 1450-202X Vol. 97 No 4 March, 2013, pp.516-526 http://www.europeanjournalofscientificresearch.

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Modelling of Nutrient Mist Reactor for Hairy Root Growth using Artificial Neural Network
Khwaja Osama Department of Biotechnology, Institute of Engineering and Technology G.B. Technical University, Sitapur Road, Lucknow-226021, India E-mail: osama.khwaja@gmail.com Pallavi Somvanshi Department of Biotechnology, TERI University 10 Institutional Area, Vasant Kunj, New Delhi-110070, India E-mail: psomvanshi@gmail.com Asheesh Kumar Pandey Department of Biotechnology, Institute of Engineering and Technology G.B. Technical University, Sitapur Road, Lucknow-226021, India E-mail: pandeyashish@gmail.com Bhartendu Nath Mishra Department of Biotechnology, Institute of Engineering and Technology G.B. Technical University, Sitapur Road, Lucknow-226021, India E-mail: profbnmishra@gmail.com Abstract Artificial neural networks are complex mathematical algorithm, which mimics the biological neural network. In this work, modelling of Nutrient Mist Reactor for hairy root growth was done using Artificial Neural Network (ANN) model. The significant features for culture parameters viz. inoculum size, mist ON time, mist OFF time, initial packing density, media volume, initial sucrose concentration in media and time of culture were considered as input of the network. The final biomass of hairy roots on dry weight basis was taken as network output. We analysed the performance of three back propagation training algorithms, namely, Feed forward back propagation, cascade forward back propagation and recurrent neural networks and were trained using gradient descent, gradient descent with adaptive learn rate and Levenberg-Marquardt training algorithms. In all, Levenberg-Marquardt training algorithm was observed to be the fastest and accurate. All the networks were found efficient, but recurrent neural network was found slightly more accurate with a correlation coefficient of 0.9679.

Keywords: Artificial Neural Network, Nutrient Mist Reactor, Levenberg-Marquardt, Network Performance.

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1. Introduction
Around 80,000 plant species with medicinal value incorporated in them have been reported until now. These plants may find application in diverse industries viz. pharmaceutical, cosmetic, agriculture and food (Verma et al., 2008). Artemisia annua also known as sweet wormwood or Qing Hao is an aromatic plant of Chinese origin having anti-bacterial, anti-periodic, anti-septic, carminative, digestive and anti-malarial properties. Artemisinin is a secondary product in this plant which is reported to be effective anti-malarial compound and acts against multidrug resistant Plasmodium sp. (Ferreira, et al., 2005). The developmental patterns followed by biological processes are complex, time dependent, non linear and difficult in predicting which cann processes is done by genetic and environmental factors which show high degree of variability and were inconsistence (Gallego et al., 2010; Mehrotra et al., 2008). The biological process cannot be predicted by general algorithms and mathematical equations due to the noisy and incomplete dataset (Gallego et al., 2010). Plant secondary metabolites are source of various pharmaceuticals, flavours and dyes (Kim et al., 2002). Many phytochemicals are produced in small amounts and accumulate in specialized parts of the plant. These compounds are costly and difficult to synthesize chemically due to their complex structure and optical properties, hence extraction of chemicals from plants is an economical method of producing these chemicals (Kim et al., 2001). Hairy roots, result of Agrobacterium rhizogenes mediated genetic transformation are considered to produce secondary metabolites not produced by the mother plant also they are genetically stable, grow fast, do not require hormones for their growth and produce more quantities of secondary metabolites then their mother plant. Hairy roots have been cultured for large scale for production of secondary metabolites in different bioreactors. In large scale culture and production of hairy roots the cost and the time of production is very important. The conventional modelling techniques often become ineffective in monitoring and predicting the growth pattern of hairy roots. Culture of hairy roots in bioreactor is difficult due to their special morphological characteristics (Kim et al., 2002). Design of bioreactor depends upon growth behaviour, morphological changes in hairy roots during growth, rheological properties, shear stress, roots injury and callus formation, type of metabolite i.e. extracellular or intra cellular etc. A variety of reactor configurations have been used to cultivate hairy roots, including the stirred tank reactor (Hilton et al., 1990), trickle bed reactor, airlift reactor (McKelvey et al., 1993), rotating drum (Kondo et al., 1989) and nutrient mist reactors (Dilorio et al., 1992). Nutrient mist reactor is a gas phase reactor in which nutrients are delivered to immobilized root bed by in form of mist droplets using ultrasonic transducer (Wyslouzil et al., 1997). Nutrient mist reactors have some advantages for hairy root culture. It provides low shear stress environment, rapid replenishment of nutrients and removal of toxics. Due to improved gas exchange secondary metabolite production is increased in nutrient mist reactors (Liu et al. 1999). Mathematical models and other machine learning approaches have been used in monitoring the growth pattern of hairy roots. Wyslouzil et al. (1997) showed that mist deposition on roots is a function of the root diameter and packing fraction, the length of the root bed, the mist particle diameter, density, and diffusivity, and the carrier gas velocity. Ranjan et al. (2009) deduced a mathematical model describing the evolution of saturation ratio of liquid hold up in root bed. An artificial neural network is a complex mathematical model consisting of simple functions (called neurons) that mimics the structure and function of a biological neural network. The neurons in an artificial neural network are similar to the cell body, and the connections made to other nodes represent the axon and synapses and the arrangement of neurons are known as architecture of ANN. Each of these neurons follow three simple rules i.e. multiplication addition and activation (Krenker et al., 2010). ANNs can learn the complex and non-linear relationship between input and output of generalized and distorted biological dataset and detect patterns and trends which are too complex to be detected by other computational techniques (Gallego et al., 2010). Artificial neural networks have been successfully used in growth modelling, biomass estimation of cell cultures, image analysis to distinguish between embryos and non embryos and pattern recognition (Zhang et al., 1999; Prasad and

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Dutta Gupta, 2006). Sankar et al., in 2011 used Artificial Neural Network to classify respiratory signals with 99% efficiency. Estimation of hairy root biomass and culture conditions have been done by ANN (Mehrotra et al. , 2008; Prakash et al., 2010). In the present study we demonstrate the use of artificial neural network, in particular feed forward and radial basis neural network for modelling culture parameters like mist duty cycle, culture time, inoculums density, packing density and volume of nutrient mist reactor for hairy root culture.

2. Material and Method

2.1. Experimental Data Artimisia annua hairy root cultured in a nutrient mist reactor by Towler, (2005) was taken as model system. The hairy roots of A. annua were cultured in a nutrient mist reactor in different culture conditions. Initial inoculum on dry weight basis, sucrose concentration, initial packing fraction, culture time in days and media volume were the culture conditions considered in the model as the input data set. The output of model was final hairy root biomass on dry weight basis. The complete experimental data were used in the model, and was divided in two sets. First set was used for training and the second set was used as test data. The test data was used to validate the performance and robustness of the model. The training data was chosen so that it comprises of the complete range of the culture conditions used in the experiment. 2.2. Neural Network Architecture In this study we have used back propagation and radial basis neural network. Three types of back propagation networks i.e. feed forward back propagation, cascade forward back propagation and Elman recurrent network were used. (Figure 1) Feed forward is a network topology in which the neurons of same or different layers are connected in unidirectional fashion so that the information flows only in forward direction whereas in recurrent network topology both forward and backward connections are present between two neurons and in some case even with the same neuron. Recurrent artificial neural networks can use their internal memory to process any sequence of inputs. Basic topology of recurrent network is fully recurrent network in which each neuron is directly connected to every other neuron in both directions. Other recurrent artificial neural networks such as Hopfield, Elman, Jordan, bidirectional and other networks are just special cases of recurrent artificial neural networks. Elman network also referred as Simple Recurrent Network is special case of recurrent artificial neural networks. It differs from conventional two-layer networks in that the first layer has a recurrent connection. It is a simple three-layer artificial neural network that has back-loop from hidden layer to input layer trough so called context unit. Feed forward and Cascade forward neural networks are similar except that cascade forward network includes an extra weight connection from the input to each layer. Back propagations are generalization of Windrow-Hoff learning rule and multiple layer networks. The basic principle of these networks is the weights of the hidden layers are adjusted by back propagation of the error in the network output i.e. the difference between the network calculated value and the target value (Krose et al., 1996). The efficiency of feed forward, cascade forward and recurrent neural networks were checked for different learning functions.

Modelling of Nutrient Mist Reactor for Hairy Root Growth using Artificial Neural Network
Figure 1: Architecture of cascade forward, feed forward and recurrent neural network.

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2.3. Training and Simulating Network The Neural Network Toolbox of MatLab 8.0 was used to design, train and simulate networks. Different combinations of network parameters, i.e. number of neurons in each hidden layer, transfer function, error goal etc. was considered. Different variants of back propagation algorithm like gradient decent algorithm, gradient descent algorithm with adaptive learn rate and Levenberg-Marquardt algorithm were explored for training. Each network had tansig and purelin as activation function for hidden layer and output layer respectively. The training data set contains 50 records comprising of different culture conditions. The test data contained 31 records which were not present in the training data set. The input data contained various culture conditions i.e. mist on cycle, mist off cycle, reactor volume, culture time, initial packing density and initial inoculums dry weight. The network was trained for different training parameters to check best result. The weights of input layer were initialized using mapstd weight initialization function of neural network toolbox of MatLab. The trained network was tested with validation data and outputs were compared with experimental results. The performance of networks was evaluated using Mean Square Error (MSE) and Correlation Coefficient. The network with the best performance were simulated to predict biomass productivity for increasing reactor volume with constant initial packing fraction and initial inoculums, time and mist on cycle.

3. Results and Discussion

3.1. Comparative Study of Training Functions Three training algorithms i.e. gradient descent algorithm, gradient descent algorithm with adaptive learn rate and Levenberg-Marquardt were used in this study for all the three networks. The training parameters for these algorithms were optimized by considering a range of values. The optimized training parameters for different training algorithms are given in Table 3. The networks were tested by 31 records of validation data. The performance of feed forward, cascade forward and recurrent neural networks with different training algorithms for both training and validation data is shown in Fig 4-6. The mean square error and correlation coefficient for both training and validation data are given in Table 1 and a comparison of time taken by networks to converge is given in Table 2.

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Table 1:

Khwaja Osama, Pallavi Somvanshi, Asheesh Kumar Pandey and Bhartendu Nath Mishra
Comparison of cascade and feed forward back propagation neural network for different training functions
Training Data Mean Square Correlation Error (MSE) Coefficient 0.0427 0.9074 0.0214 0.0527 0.0368 0.0631 0.0484 0.0284 0.0469 0.0067 0.9555 0.9028 0.9248 0.8667 0.9150 0.9402 0.8988 0.9445 Validation Data Mean Square Correlation Error (MSE) Coefficient 0.0539 0. 9492 0.01 0.0214 0.0260 0.0145 0.0149 0.0142 0.0161 0.0097 0. 9568 0.9624 0.9414 0.9488 0.9553 0.9446 0.9457 0.9679

Network Architecture

Training Function Gradient decent Gradient decent with adaptive learn rate Levenberg-Marquardt Gradient decent Gradient decent with adaptive learn rate Levenberg-Marquardt Gradient decent Gradient decent with adaptive learn rate Levenberg-Marquardt

Feed Forward

Cascade Forward

Layer Recurrent

Table 2:

Comparison of time taken in training by different networks.

Training Function Gradient decent Gradient decent with adaptive learn rate Levenberg-Marquardt Gradient decent Gradient decent with adaptive learn rate Levenberg-Marquardt Gradient decent Gradient decent with adaptive learn rate Levenberg-Marquardt Training Time (Epochs) 1000 29 13 1000 26 8 1000 85 16

Network Architecture Feed forward

Cascade forward

Layer Recurrent

Levenberg-Marquardt training algorithm was found to be most efficient and fast. The Levenberg-Marquardt training algorithm is a damped least square technique which is a combination of steepest descent and the Gauss-Newton method. It uses the advantages of the two methods. It uses positive constant (damping) which is added to the diagonal of Jacobian matrix to reduce error. It solves t the equation (Jt update the network weights. Levenberg-Marquardt is very popular algorithm used for non linear data set. It is used for faster training as it provides rapid convergence and stability to the system (Sankar et al., 2011). Gradient descent algorithm was found to be most slow and was less efficient. Gradient descent also known as steepest descent is one of the most important breakthroughs in neural network training. It is simple easy to use and it guarantees to find a minimum of a function if present. However, it is slow converging at small step size while, convergence speed could be increased with increasing step size, larger step size results in large error. Gradient descent with adaptive learn rate is an improvement on gradient descent in which the step size (learn rate) is adjusted during the training to produce an optimum convergence rate and error. In our study gradient descent with adaptive learning algorithm converged faster than gradient. A comparative analysis of convergence of training parameters is given in figure 2.

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Figure 2: Comparison of convergence of gradient descent (a), gradient descent with adaptive learning (b) and Levenberg-Marquardt training algorithms (c) for recurrent neural network.

3.2. Comparative Study of Networks The comparative study of the network performances showed that all the networks were efficient in predicting the hairy root biomass productivity of nutrient mist reactor while recurrent neural network being most accurate and produced highest correlation coefficient of 0.9679 (Figure 3) and lowest Mean Square Error of 0.0097 for validation data (Table 1). Recurrent neural networks are also called feedback neural network due to both forward and backward flow of information in the network. Due to this complex structure recurrent neural networks are more efficient than feed forward neural network in analysing noisy data, (Salmasi et al., 2011) time series prediction (Connor et al., 1994) estimation of bioprocess variables (Karim and Rivera 1992) and other applications (Saha and Raghava 2006; Welch et al.,2009). However, cascade forward network learned faster than other networks. The fast learning of cascade forward neural networks is due to the extra connection between the input layer and all other layers.
Table 3: Optimum training parameters for different training functions
Learn rate / Momentum 0.285 0.01 0.001 0.31 0.01 0.001 0.71 0.01 0.001 1.05 23 1.05 15 1.05 14 0.65 0.7 0.7 0.4 0.8 0.4 Learn rate / Momentum Increase Learn rate / Momentum Decrease No. of nodes in Hidden layer 9 6 7 14 2 6 16 12 6

Network Architecture

Training Function Gradient decent Gradient decent with adaptive learning Levenberg-Marquardt Gradient decent Gradient decent with adaptive learning Levenberg-Marquardt Gradient decent Gradient decent with adaptive learning Levenberg-Marquardt

Feed forward

Cascade forward

Layer Recurrent

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Figure 3: Linear regression of validation data and network output obtained from recurrent neural network with Levenberg-Marquardt training algorithm.

The optimized training parameters of feed forward network with highest correlation coefficient are given in the table no. 2. The simulation result of the networks i.e. hairy root biomass on dry weight basis was compared with the experimental data to calculate correlation coefficient, mean square error, root mean square error, covariance coefficient, slope and intercept.
Figure 4: Prediction of hairy root biomass on dry weight basis by feed forward neural network for training data (a) and validation data (b)

Figure 5: Prediction of hairy root biomass on dry weight basis by cascade forward neural network for training data (a) and validation data (b)

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Figure 6: Prediction of hairy root biomass on dry weight basis by layer recurrent neural network for training data (a) and validation data (b)

3.3. Prediction of Biomass Based on the result of comparative study of the networks cascade forward network with three nodes in hidden layer trained with Levenberg-Marquardt algorithm was simulated for prediction of hairy root biomass for an increase of time and mist on time. A graph of variation of hairy root biomass on dry weight basis is given in figure 7. 3.4. Biomass Productivity with Increase in Time The hairy root growth kinetics have been studied by various workers, models of growth of a single hairy root ( Silk et al. 1989; Morris & Silk 1992; Chavarra-Krauser & Schurr 2004; Chavarra-Krauser et al. 2005) as well that for dense clump of roots (Bastian et al. 2008 a) are also present. The growth of single roots in a root system can be assumed to be purely apical similar to that of fungi mycelia and blood vessels. Although in a dense cluster of roots the growth is influenced by nutritional heterogeneity, mechanical stresses, hydrotropism, gravitropism and growth towards the direction of more space (Bastian et al. 2008 b). Hence in a nutrient mist reactor given liquid and gaseous nutrients are not limiting, hairy roots follows a lag phase (given time required for adjustment to environmental conditions), an exponential phase, stationary phase due to the increasing density of root bed followed by a death phase due to accumulation of secondary metabolites, lack of nutrients in the inner parts of the root clump due to high packing density. The present model predicts a sigmoidal curve showing a lag, exponential and stationary phase of A. annua roots in a 0.75 liters nutrient mist reactor from zero to fifteen days. (Figure 7a)
Figure 7: Prediction of biomass productivity for an increase in time (a), mist duty cycle (b).

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3.5. Biomass Productivity with Mist Duty Cycle Increasing the duration of mist duty cycle increases the mist deposition on root beds increasing liquid hold up in the root bed, resulting in more nutrients for growth. But with increase of liquid hold up mass transfer coefficient for gas increases resulting in oxygen depletion in the root bed, which causes a decrease of growth rate of root. (Ranjan et al., 2009) In the present study final hairy root biomass was predicted on increasing the mist duty cycle from 3 minute ON / 15 minute OFF to 15 minute ON / 3 minute OFF. The final biomass initially increased with the duty cycle showing a maximum final biomass at 15 minute ON / 15 minute OFF, while on increasing the mist duty cycle future the biomass productivity decreased considerably. (Figure 7b) This result was in concurrence with earlier studies where biomass productivity was found highest at an optimum mist ON/OFF cycle. (Dilorio et al., 1992; Chatterjee et al., 1997; Liu et al., 1999; Wyslouzil et al., 2000; Kim 2001; Towler et al., 2005) Optimization of culture conditions for best productivity is essential for tissue culture studies. Conventional methods for optimization of these conditions are time-consuming and expensive, in addition optimization for large-scale bioreactor cultures requires several scale-up studies. Hairy roots are easily available and cost-effective for secondary metabolite production. Secondary metabolite production and growth of hairy roots in a nutrient mist reactor depends upon culture conditions like media flow rate, mist duty cycle, packing fraction of roots media composition, liquid hold up, saturation ratio etc. Along with these other environmental conditions like temperature, light intensity also influence secondary metabolite production and hairy root growth rate. Therefore, a prior optimization of such physical and chemical conditions for optimal growth and productivity is important obtaining maximum yields in bioreactor. The application of artificial neural networks has been proven a time, money and labour efficient method for optimization of culture conditions of hairy roots at small scale in earlier studies (Mehrotra et al., 2008; Prakash et al., 2010). In earlier studies the applicability of various neural network models in plant tissue culture studies have been shown (Albiol et al., 1995; Honda et al., 1997; Suroso Murase et al., 1996).

4. Conclusion
In present study, the efficiency of neural network models to predict final biomass of hairy roots at different culture conditions in nutrient mist reactor has been proven, also neural network models was found to be efficient in modelling the growth of hairy roots in a nutrient mist bioreactor. The present neural network model not only was efficient in optimizing the culture conditions of hairy root culture in a nutrient mist bioreactor but also it efficiently predicted the biomass productivity for different culture conditions.

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