Research Article

Received: 6 January 2010 Revised: 9 February 2010 Accepted: 22 February 2010 Published online in Wiley Interscience: 7 April 2010

(www.interscience.wiley.com) DOI 10.1002/jctb.2391

Enzymatic hydrolysis of sugarcane bagasse

for bioethanol production: determining
optimal enzyme loading using neural networks
Elmer Ccopa Rivera,∗ Sarita Cândida Rabelo, Daniella dos Reis Garcia,
Rubens Maciel Filho and Aline Carvalho da Costa∗

Abstract
BACKGROUND: The efficient production of a fermentable hydrolyzate is an immensely important requirement in the utilization
of lignocellulosic biomass as a feedstock in bioethanol production processes. The identification of the optimal enzyme loading
is of particular importance to maximize the amount of glucose produced from lignocellulosic materials while maintaining low
costs. This requirement can only be achieved by incorporating reliable methodologies to properly address the optimization
problem.

RESULTS: In this work, a data-driven technique based on artificial neural networks and design of experiments have been
integrated in order to identify the optimal enzyme combination. The enzymatic hydrolysis of sugarcane bagasse was used as a
case study. This technique was used to build up a model of the combined effects of cellulase (FPU/L) and β-glucosidase (CBU/L)
loads on glucose yield (%) after enzymatic hydrolysis. The optimal glucose yield, above 99%, was achieved with cellulase and
β-glucosidase concentrations in the ranges of 460.0 to 580.0 FPU L−1 (15.3–19.3 FPU g−1 bagasse) and 750.0 to 1140.0 CBU L−1
(2–38 CBU g−1 bagasse), respectively.

CONCLUSIONS: The dynamic model developed can be used not only to the prediction of glucose concentration profiles for
different enzymatic loadings, but also to obtain the optimum enzymes loading that leads to high glucose yield. It can promote
both a successful hydrolysis process control and a more effective employment of enzymes.


c 2010 Society of Chemical Industry

Keywords: enzymatic hydrolysis; sugarcane bagasse; enzyme loading; optimization; modeling; artificial intelligence

NOTATION η learning rate

bk is the bias in the kth neuron in the output layer θj bias of the jth neuron in the hidden layer.
d desired output vector
ECell cellulase activity (FPU L−1 )
Eβ−glu β-glucosidase activity (CBU L−1 ) INTRODUCTION
f activation function of the jth neuron in the hidden Ethanol from lignocellulosic materials has been investigated
layer during the past few years with great attention, but its production
F activation function of the kth neuron in the output in large-scale plants has not yet become viable. Studies taking into
layer account process integration, increase of fermentation yields and
g vector of the values predicted by the ANN model integration of unit operations are still needed in order to make
G glucose concentration (g L−1 ) hydrolysis a competitive technology.1,2 Bagasse, the by-product
N agitation (rpm) of bioethanol manufacture from sugarcane fermentation, is a very
t time (h) promising raw material for bioethanol production in the large-
T temperature (◦ C) scale process perspective. It is already available on the ethanol
wji weight connecting the ith neuron in the input layer plant site, since it is produced in the mills where sugar is extracted
and the jth neuron in hidden layer
Wkj weight connecting the jth neuron in the hidden layer
and the kth neuron in the output layer ∗ Correspondence to: Elmer Ccopa Rivera and Aline Carvalho da Costa, Labo-
x1 coded values of cellulase concentration ratory of Optimization, Design and Advanced Control, School of Chemical
x2 coded values of β-glucosidase concentration Engineering, State University of Campinas, P.O. Box 6066, 13083-970, Camp-
inas, SP, Brazil. E-mail: elmer@feq.unicamp.br, accosta@feq.unicamp.br
Yield glucose yield (%)
Laboratory of Optimization, Design and Advanced Control, School of Chemical
Greek letters Engineering,StateUniversityofCampinas,P.O.Box6066,13083-970,Campinas,
983

α momentum coefficient SP, Brazil

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from sugarcane, and better technologies of cogeneration are
expected to result in an increased surplus of bagasse at the
plant site. Bioethanol from sugarcane bagasse may share the
infrastructure where conventional bioethanol is produced, such as
fermentation and distillation units, which diminishes equipment
costs. The product obtained after hydrolysis may be diluted in
the sugarcane juice, thus decreasing the impacts of potential
fermentation inhibitors, such as furfural and its derivatives formed
during cellulose hydrolysis.
Of the two possible hydrolysis methods, acid and enzymatic
hydrolysis, acid hydrolysis is relatively inexpensive, but it forms
compounds that might seriously inhibit the subsequent fermen-
tation. On the other hand, enzymatic hydrolysis takes place under
milder process conditions than that of dilute acid hydrolysis,
thus leading to a decreased formation of by-products. Techni-
cal feasibility, however, is not sufficient: economic potential is
the driving force. Thus, there is a great interest in the modeling
and optimization of all second-generation bioethanol production
steps.
The present market offers many cellulase preparations (includ-
ing those obtained from Trichoderma reesei) containing low levels
of β-glucosidase, which leads to an increased accumulation of cel-
lobiose in the enzymatic hydrolyzates of cellulose. The inability of
industrial glucose-fermenting yeasts to ferment cellobiose results
in incomplete conversion of the hydrolyzate to ethanol, signifi-
cantly diminishing final yield. These drawbacks may be overcome
by supplementation of the cellulase complex with β-glucosidase
from other sources. Thus, the task of identifying an optimal en-
zyme combination is of extreme importance in obtaining a high
glucose yield for the overall economy of the process.3 – 6 However,
successful optimization of the enzymatic hydrolysis step can only
be achieved by incorporating methodologies for rapid develop-
ment of reliable mathematical models. These models can be used
to facilitate the implementation of suitable operating strategies to
achieve high operational performance.
Design of experiments (DOEs) have successfully been used to
investigate the influence of physical parameters (temperature, pH,
substrate concentration, agitation, among others) in enzymatic
hydrolysis.7 – 10 This approach has been widely used in various ap-
plications due to its well-established methodology.11 Considering
the difficulties involved in biotechnological processes, the main
difficulty in model-based techniques for definition of operational
strategies and optimization in the enzymatic hydrolysis of sug-
arcane bagasse is the problem of obtaining an accurate model
to aid in the decision making process. Although DOEs provide
understanding about the process and a reliable measurement of
its parameters,12 practical experience has shown that the behavior
of enzymatic reactions with varying enzymes loading is extremely
complex and hence difficult to handle statistically.13,14
Artificial intelligence, such as artificial neural networks (ANN),
has been used successfully for solving biotechnological complex
problems related to the field of modeling and optimization in
order to achieve high operational performance. This technique
is required to efficiently combine all available knowledge and to
direct the development towards an improved process operation
strategy. ANNs can be used to offer adaptive solutions, since the
reestimation of their parameters is a straightforward procedure.15
These characteristics are suitable for analyzing data from more
complex processes such as the influence of enzyme loading on
the enzymatic hydrolysis of sugarcane bagasse. At this point, it
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is worthwhile mentioning that in many studies the combination
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c 2010 Society of Chemical Industry
E. Ccopa Rivera et al.
of modeling and optimization techniques enhances the overall
prediction accuracy.14,16
The application presented in this study illustrates the efficiency
and usefulness of a model-based approach for the identification
of the optimum ratio of cellulase and β-glucosidase on the
enzymatic hydrolysis of sugarcane bagasse with regard to cellulose
conversion to glucose. DOE was used to obtain statistically well
distributed data of cellulase and β-glucosidase concentrations in
the input domain for the training and validation of the ANN model,
and glucose concentration (g L−1 ) was used as the model output.
This is a very important issue to be addressed, as neural networks
have very limited extrapolation properties. As a consequence,
an accurate nonlinear model was obtained, which provided an
optimal region of glucose yield (%) during the enzymatic hydrolysis
of sugarcane bagasse.
MATERIAL AND METHODS
Substrate
Sugarcane (Saccharum officinarum) was grown and mechanically
harvested in 2006, and bagasse was obtained from the sugar plant
Usina da Pedra, located in Serrana, São Paulo, Brazil. This material
was dried outdoors for 72 h and, to obtain more uniform particles,
was milled in a knives mill (Wiley Mill, Philadelphia, PA, USA, model
3) and in a hammer mill (General Electric, Plainville, CT, USA), for
10 min at each mill, and went through a process of screening using
a Tyler 35 sieve (Paulinia, SP, Brazil). The dry matter (DM) content
was approximately 95% (w/w).
Pretreatment
Bagasse was pretreated with alkaline hydrogen peroxide. The
pretreatment was performed in the conditions determined as
optimal for this process, with bagasse concentrations of 8% (w/w),
11% (v/v) of hydrogen peroxide and pH adjusted to 11.5 with
sodium hydroxide. The pretreatment solution was incubated in an
orbital shaker (Marconi, Piracicaba, SP, Brazil, MA-832), agitated at
150 rpm, 25 ◦ C for 1 h.17,18 The pretreated biomass was washed
until pH 7.0 and dried at 50 ◦ C for 24 h.
Chemical analysis of bagasse samples
Samples of raw bagasse and bagasse pretreated with alkaline
hydrogen peroxide were analyzed to determine chemical com-
position. Samples of approximately 4 g were extracted with 95%
ethanol for 12 h in a Soxhlet apparatus.19 For determination of ash
content, samples of 1 g were burned in a muffle furnace at 575 ◦ C
for 4 h.20
Extracted bagasse samples were hydrolyzed with 72% sulfuric
acid at 30 ◦ C for 1 h (300 mg of sample and 3 mL of sulfuric acid).
The acid was diluted to a final concentration of 4% (addition of
84 mL of water) and the mixture heated at 125 ◦ C, 1 atm for 1 h.21
The residual material was cooled and filtered through filter paper
previously dried.
The acid-insoluble lignin was measured as the weight of
insoluble residue remaining at 105 ◦ C. The acid-soluble lignin
was measured by UV–Vis spectroscopy (Mini-1240 Shimadzu) at
205 nm.22 A solution of 4% H2 SO4 was utilized as blank. The total
lignin content was the sum of acid-insoluble and acid-soluble
lignin.
Carbohydrates in biomass were determined using the soluble
fraction. Glucose, xylose and arabinose were determined by high
performance liquid chromatography (HPLC; Waters Corporation,
J Chem Technol Biotechnol 2010; 85: 983–992
Optimization of enzymatic hydrolysis of sugarcane bagasse using ANN
Table 1. Chemical composition of untreated bagasse and bagasse
pretreated with alkaline hydrogen peroxide
Untreated bagasse Bagasse pretreated
Components (%) with H2 O2 (%)
Ash 1.79 ± 0.02 – a Sugar-Pak I column (Waters Corporation) at 70 ◦ C with deionized
Extractives 3.25 ± 0.2 – water as eluent at a flow rate of 0.5 mL min−1 .
Total lignin 25.10 ± 0.5 9.87 ± 0.4
Glucan 37.35 ± 0.5 60.09 ± 0.9
Xylan 23.66 ± 0.9 16.58 ± 0.6
Massachusetts, USA) equipped with a refractive index detector.
The separation was performed in a Sugar-Pak I column (Waters
Corporation) at 70 ◦ C with a flow rate of 0.5 mL min−1 , using
filtered deionized water as the mobile phase. The sample was
centrifuged and then, filtered through 0.2 µm (Acrodisc) and a
volume of 10 µL was injected.
Acetyl content was determined using a Biorad HPX87H column
at 45 ◦ C, eluted at 0.55 mL min−1 with 0.01 mol L−1 sulfuric acid.
Acetyl groups were detected in a 65 ◦ C temperature-controlled RI
detector (Knauer, Berlin, Germany, HPLC pump and detector).
Glucose, xylose, arabinose, and acetic acid were used as
external calibration standards. Sugar loss by acid degradation was
considered using the Sugar Recovery Standards as suggested by
the NREL method.21 The factors used to convert sugar monomers
to anhydromonomers were 0.90 for glucose and 0.88 for xylose
and arabinose. Acetyl content was calculated as the acetic acid
content multiplied by 0.7. These factors were calculated based on
water addition to polysaccharides during acid hydrolysis. Table 1
shows the composition of the bagasse untreated and pretreated
with alkaline hydrogen peroxide.
Enzymatic hydrolysis performed with variation on enzymes
loading
The enzymatic hydrolysis of pretreated bagasse was performed in
250 mL erlenmeyer flasks, containing a 100 mL mixture of citrate
buffer and solid substrate with pH adjusted to 4.8. The substrate
concentration in the hydrolysis assays was 3% (w/v).
The effect of enzymes loading was evaluated using
cellulase from Trichoderma reesei (Sigma-Aldrich, Steinheim,
Germany, ATCC 26 921) and β-glucosidase from Aspergillus niger
(Novozym 188). The values of enzymes concentrations were si-
multaneously varied based on a 22 + central composite design.
The flasks were incubated in an orbital shaker (Marconi MA-832)
agitated at 100 rpm at 50 ◦ C. The reaction time was set at 72 h and
periodically, aliquots were taken, boiled to deactivate the enzymes
and evaluated for sugar content.
The concentrations of cellulase and β-glucosidase given in FPU
L−1 and CBU L−1 , respectively, were varied according to the design
matrix described in Table 2.
Enzymatic activities
Cellulase activity was determined as filter paper units per milliliter
(FPU mL−1) , as recommended by the International Union of Pure
and Applied Chemistry.23,24 β-glucosidase activity was determined
through a solution of cellobiose 15 mmol L−1 and expressed in
units per milliliter (CBU mL−1 )25 . Enzyme activity was 64.11 FPU
mL−1 for cellulase and 308.37 CBU mL for β-glucosidase.
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Sugar analysis by HPLC
Samples were filtered through a 0.45 µm filter and the content
of monosaccharides (D-glucose, D-xylose and L-arabinose) was
quantified using a HPLC system (Waters Corporation) equipped
with a refractive index detector. The separation was performed in
The maximum yield of glucose was calculated using
Glucose yield (% theoretical maximum)
g of glucose by HPLC
= × 0.90 × 100 (1)
g of glucan
Where 0.90 is the factor used to convert sugar monomers to
anhydromonomers.
ANN MODEL DEVELOPMENT
This section presents the considerations required to develop
a modeling technique based on ANN. A multilayer perceptron
neural network (MLP) was used in this work, mainly for its easily
understandable architecture and simple mathematical form, which
results in a simple tool for modeling and optimization.
Important elements of ANNs are model structure (architecture)
and ANN training, as will be discussed below.
ANN structure selection
An MLP with one hidden layer of sigmoidal neurons and a layer of
linear output neurons was employed. This structure has nonlinear
processing capabilities and universal approximation property,26
and has already been used successfully to describe the dynamic
behavior of biotechnological processes.15,27
A MLP consists of three types of layers: an input layer, an output
layer and one or more hidden layers, whose numbers of neurons
are N, M and K, respectively. Each layer may have a different number
of neurons, which are interconnected by adjustable parameters
(weights and biases) associated with them. The relationship is
given mathematically as:
  N  

M 
gk = F  Wkj f wji xi + θj + bk 
j=1 i=1
(j = 1, . . . , M); (k = 1, . . . , K) (2)
where wji is the weight connecting the ith neuron in the input
layer and the jth neuron in the hidden layer; θj is the bias of the jth
neuron in the hidden layer; Wkj is the weight connecting the jth
neuron in the hidden layer and the kth neuron in the output layer;
bk is the bias in the kth neuron in the output layer; f (·) and F(·) are
the activation functions of the jth neuron in the hidden layer and
of the kth neuron in the output layer, respectively.
Selection of the input domain using DOE and training
The available process information that can be used for building
ANN models consists of the measurable process output and the
process inputs. A problem which arises is to select from the amount
of information available the appropriate information for the ANN
inputs. When ANNs are used to build inferential models, a study of
their input domain is crucial in order to provide a reduction in the
dimension of the input space, which can remarkably reduce the
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time needed for training.28
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Table 2. Experimental design (coded levels in parentheses) and results of 22 plus star configuration central composite design, including three
replicates at the center point

Trial Cellulase ECell (FPU L−1 ) β-glucosidase Eβ−glu (CBU L−1 ) Glucose G (g L−1 ) Glucose yield Yield (%) Production rate (g L−1 h−1 )

1 174.0 220.0 16.59 82.81 0.230

(−1) (−1)
2 174.0 1280.0 15.73 78.52 0.218
(−1) (+1)
3 775.0 220.0 16.26 81.19 0.226
(1) (−1)
4 775.0 1280.0 17.10 85.42 0.238
(1) (+1)
5 50.0 750.0 8.57 42.76 0.119
(−1.4142) (0)
6 900.0 750.0 19.66 98.15 0.273
(+1.4142) (0)
7 475.0 0.0 12.72 63.51 0.177
(0) (−1.4142)
8 475.0 1500.0 18.65 93.08 0.259
(0) (+1.4142)
9 (C) 475.0 750.0 19.67 98.21 0.273
(0) (0)
10 (C) 475.0 750.0 19.66 99.39 0.273
(0) (0)
11 (C) 475.0 750.0 19.91 99.43 0.277
(0) (0)

Design of experiments (DOE) allows one to optimize the input 22

domain by reducing the number of experiments and ensuring
that the dataset is statistically well distributed. Thus, this dataset 18
Glucose (g/L)

should be sufficient to build effective ANN models.29 This approach 13

has been used recently in areas of biotechnological processes
optimization,30 where statistical techniques such as multivariate
regression models have traditionally been used.
In this work, in order to obtain data for the training of the neural
network, the inputs are distributed according to a DOE within
the enzyme concentration intervals. These intervals were defined
based on prior knowledge of enzymatic hydrolysis of sugarcane
bagasse.17,18 The aim was to include as learning patterns those data
that contain most of the important information about enzymatic
hydrolysis.
The DOE was carried out using a central composite design
(CCD), consisting of two factors (concentrations of cellulase (ECell )
and β-glucosidase (Eβ−glu )) at three levels with three replicates
at the center point. The DOE matrix is shown in Table 2. The
response variable (glucose concentration) chosen as the reaction
time after which no significant changes in these variables were
detected (72 h) is also shown in Table 2, where the corresponding
glucose yield, Yield (%) and production rate (g L−1 h−1 )) are also
reported. Nevertheless, to include the kinetic behavior in the data-
driven identification of the process, ANN training was processed
using glucose concentration profiles, G (g L−1 ), for each trial in
the DOE matrix (Fig. 1). A PCHIP routine (piecewise cubic hermite
interpolating polynomial) implemented in Matlab (MathWorks,
Natick, MA, USA) was used to fit the experimental data for G with
the purpose of increasing the dataset by interpolation. Thus, each
trial used for training provides 60 interpolated data points. Tests
have shown that this procedure led to good results when dynamic
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9
4
0
0 20 40 60 80
Time (h)
Figure 1. Experimental glucose concentration values for each trial in the
DOE matrix (Table 2). Trial 1 ( – × – ), Trial 2 ( –
– ), Trial 3 ( –  – ), Trial
4 ( – ◦ – ), Trial 5 ( – • – ), Trial 6 ( –  – ), Trial 7 ( – ♦ – ), Trial 8 ( –  – ),
Trial 9 (average value of the three replicates at the center point) ( –  – ).
Table 3 details the input (ECell , Eβ−glu and t) and the output (G)
vectors used to carry out the ANN training. In this table, the dataset
from trial 9 is an average value of the three replicates at the center
point of the DOE matrix (Table 2).
A representative dataset containing 480 input/output patterns
was presented in a randomized sequence to the neural network for
the estimation of its parameters (weight and bias). The predictive
capability of neural networks or validation was assessed on a
different sequence of experimental observations, comprising the
experimental data from trial 2, which was randomly chosen from
the DOE matrix.
In this study, both input and output data to the ANN were
scaled in the interval [0.1, 0.9]. Small random values were
used for the initialization of weights and biases. Subsequently,
the standard backpropagation learning algorithm,31 based on

processes were identified using ANN.15 a gradient descendent method implemented in FORTRAN was

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Table 3. Input and output vectors used for the ANN training and validation

Input vectors Output vector

ECell (FPU L−1 ) Eβ−glu (CBU L−1 ) t (h) G (g L−1 )

Trial {ECell i , i = 1 . . . 60} {Eβ−glu i , i = 1 . . . 60} [60 discrete data] [60 discrete data]

1 ECell i = 174.0 Eβ−glu i = 220.0 [0.2 . . . 72.0] [0.299 . . . 16.587]

2∗ ECell i = 174.0∗ Eβ−glu i = 1280.0∗ [0.2 . . . 72.0]∗ [0.358 . . . 15.728]∗
3 ECell i = 775.0 Eβ−glu i = 220.0 [0.2 . . . 72.0] [1.134 . . . 16.260]
4 ECell i = 775.0 Eβ−glu i = 1280.0 [0.2 . . . 72.0] [0.879 . . . 17.104]
5 ECell i = 50.0 Eβ−glu i = 750.0 [0.2 . . . 72.0] [0.040 . . . 8.570]
6 ECell i = 900.0 Eβ−glu i = 750.0 [0.2 . . . 72.0] [0.808 . . . 19.659]
7 ECell i = 475.0 Eβ−glu i = 0.0 [0.2 . . . 72.0] [0.151 . . . 12.722]
8 ECell i = 475.0 Eβ−glu i = 1500.0 [0.2 . . . 72.0] [0.680 . . . 18.647]
9 (C) ECell i = 475.0 Eβ−glu i = 750.0 [0.2 . . . 72.0] [0.763 . . . 19.747]
∗ Trial 2 is used to validate the ANN model. Here the vectors ECell , Eβ−glu , t and G contain 14 experimental points (i = 1 . . . 14)

and glucose concentration, G (g L−1 ), was the model output.

ENZYMATIC HYDROLYSIS The purpose of this approach was to obtain an optimal region
of glucose yield, Yield (%), through the determination of the
optimum ratio of ECell (FPU L−1 ) and Eβ−glu (CBU L−1 ). In the current
Eβ-glu optimization problem of determining the optimum enzyme
{E1 … En}
Enzymes

concentrations, the effectiveness of the pretreatment and the

G optimal physical parameters of hydrolysis (temperature, T, pH and
ANN (g/L)
ECell agitation, N) are known. Furthermore, it is worthwhile mentioning
that the systematic approach developed can also be used to build
models with more than two inputs (in the case of this work, enzyme
t concentrations {E1 . . . En }). In order to accomplish this, the data-
driven identification procedure must be carried out adequately
Optimal pretreatment conditions with a suitable set of data representative of the process to be
Optimal T, pH and N studied. This requirement was fulfilled in the present case.

Figure 2. General framework of the model-based approach used to

optimize the enzymatic hydrolysis of sugarcane bagasse.
employed to train the ANN. This algorithm makes use of the two
adjustable terms; learning rate, η and momentum coefficient, α, in
order to stabilize convergence and accelerate the optimization of
the ANN parameters.32
The appropriate number of neurons in the hidden layer was
found by the cross-validation technique in order to avoid model
over-fitting and to achieve good generalization from the training
dataset. This technique splits the data sample into a training
dataset and a validation dataset. Then neural networks with
different numbers of hidden nodes are trained with the training
dataset, and their performances evaluated on the ability to make
correct predictions of the validation dataset in terms of mean
square error (MSE):

K
MSE = (dk − gk )2
k=1
In Equation (3), gk is the prediction of the neural networks and dk
is the desired output, which in this study is glucose concentration
G (g L−1 ).
Figure 2 illustrates the general framework of the model-based
RESULTS AND DISCUSSION
Results of the DOE
Development of the data-driven technique based on ANN and
DOE started with selection of the appropriate information to build
the inferential model.
Initially, the results of experiments obtained utilizing a central
composite design (CCD) with three replicates at the center point
were analyzed by considering glucose yield after hydrolysis of
pretreated bagasse as response variable. The CCD matrix is seen
in Table 2. The ranges of the input variables (ECell and Eβ−glu ) were
selected based on the results of preliminary studies described
elsewhere.17,18
It can be seen from Table 2 that, in the operational conditions
used in this work, maximum glucose release was obtained
with ECell = 475.0 FPU L−1 and Eβ−glu = 750.0 CBU L−1 ,
corresponding to the center point. Trial 6 (ECell = 900.0 FPU
L−1 and Eβ−glu = 750.0 CBU L−1 ) also led to high values of glucose
yield.
(3)
Statistical analysis of the data obtained in the CDC was
performed using the software Statistica 7.0 (Statsoft, Inc., Tulsa,
OK). A second-order statistical model was obtained:
Glucose yield = 99.01 + 10.45 × x1 − 12.38 × x 1 2 (4)
+ 5.22 × x2 − 8.46 × x 2 + 2.13 × x1 × x2
2

approach proposed in this work. The concentrations of cellulase,

ECell (FPU L−1 ), β-glucosidase, Eβ−glu (CBU L−1 ), and the hydrolysis where x1 and x2 are the coded values of cellulase and β-glucosidase
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time, t (h), were the inputs of the MLP neural network model, concentrations.

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 www.soci.org
(1)ECell(L) 42.65096
ECell(Q) -42.4282
Eβ-glu(Q) -28.9883
(2)Eβ-glu(L) 21.30082
1Lby2L 6.14622
p = .05
Standardized Effect Estimate (Absolute Value)
Figure 3. Pareto chart of standardized effects for the glucose yield (%).
The Pareto chart (Fig. 3) shows the magnitude of the effects
on glucose yield. In this chart, the effect estimates divided by
their standard errors are sorted from the largest absolute value
to the smallest absolute value. The magnitude of each effect is
represented by a column, and a line going across the columns
indicates how large an effect must be to be considered statistically
significant. In this work the vertical line corresponds to a p-value
of 0.05, which implies a 95% level of significance. It can be seen
from Fig. 3 that all the effects are statistically significant and the
largest effects on glucose yield are the linear and quadratic effects
of cellulase concentration.
The ANOVA is shown in Table 4. From this table it can be
concluded that the model does not fit the experimental data
well. The calculated F value (lack of fit/pure error) is higher than
the critical F value at 95% confidence (i.e. at this confidence
level, there is evidence of lack of fit for the model, Equation (4)).
Furthermore, the results show that the model accounted for a
low percentage of the explained variance (R2 = 68.0%), and
the calculated regression F value (regression/residual) is lower
than the critical F value at 95% confidence, indicating that the
regression is statistically not significant.
The statistical model obtained for the data considered in this
work was not significant due to the high non-linearity of the
enzymatic hydrolysis process. Moreover, as the glucose yield has
been determined over a wide range of enzyme concentrations,
a second-degree polynomial was not capable of describing the
glucose yield in the range considered. As an alternative to the
statistical techniques, an ANN model was implemented.
Table 4. ANOVA for the model describing glucose yield
Source of variation Sum of squares
Regression 2119.1
Residual 1003.1
Lack of fit 1002.1
Pure error 0.961
Total 3121.3
F listed values
(95% of confidence)
Percentage of explained variance (R2 ) = 68.0; percentage of explicable variance = 99.9.
∗ F test for statistical significance of the regression = regresion/residual.
∗∗ F test for lack of fit = lack of fit/pure error.
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E. Ccopa Rivera et al.
Results of ANN training and validation
The potential of different ANN structures with a single hidden layer
for estimation of glucose concentration, G (g/L) was assessed. The
validation dataset was assembled to evaluate the performance
of the trained ANN. ECell (FPU L−1 ) and Eβ−gluc (CBU L−1 ) and t
(h) vectors corresponding to Trial 2 in Table 3 were used in the
validation set. During the validation process, the number of hidden
neurons was varied from 5 to 20, and the optimal number chosen
by the cross-validation criterion with the number of epochs fixed
at 2000 for all the structures studied. The neural network with
11 hidden neurons for G (g L−1 ) was found to give the lowest
MSE for the validation sample. The glucose concentration was
then modeled using an ANN with 56 scalar parameters (weights
and bias). The learning rate, η, and the momentum coefficient, α,
used in this work were optimized to 0.95 in the backpropagation
learning.
The ANN model selected for this work is illustrated in Fig. 4
(using the notation from Equation (2)). Table 5 shows the final
optimized parameters (weights and bias) of the model. This table
contains the additional information required to reproduce the
behavior of glucose concentration using the ANN model from a
broad range of enzyme loadings (see Table 3).
The performance of the optimal ANN model in describing the
experimental glucose concentration (g L−1 ) for the training and
validation data sets are shown in Fig. 5. As shown in Fig. 5(a)
and (b) (dataset corresponding to Trials 5 and 8 in Table 3), the
model effectively tracks the desired trajectory of experimental
observations. For the validation data set (Trial 2), it can be seen
from Fig. 5(c) that the model also described the experimental
observations accurately.
The quest for a more rigorous evaluation of a good modeling
tool leads to the use of additional performance measures (criteria).
RSD (residual standard deviation), written as a percentage of the
average of the experimental values, dk , defined by Equation (5),
was used to evaluate the quality prediction of the ANN model.
  0.5
1 n
(dk − gk )2
n k=1
RSD(%) = × 100 (5)
dk
where dk is the experimental observation, gk is the value predicted
by the ANN model and n is the number of points.
In this work, the RSD(%), the correlation coefficient (R2 ) and the
mean square error (MSE) were used to evaluate the performance
of the ANN model, as can be seen in Table 6. From these criteria,
Degrees of freedom Mean square F-ratio
5 423.0 2.11∗
5 200.6
3 334.1 695.4∗∗
2 0.480
10
∗
F5,5 = 5.5
∗∗ F3,2 = 19.16
J Chem Technol Biotechnol 2010; 85: 983–992
Optimization of enzymatic hydrolysis of sugarcane bagasse using ANN www.soci.org

θ1

w1,1

w1,2 + f1(•)

w1,3

x1 = ECell θ2

w2,1 θ1

W1,1
x2 = Eβ-gluc
w2,2 + f2(•) W1,2 + F1(•) g1 = G
•
•
x3 = t w2,3 •
W1,11
•
θ11 •
•
w11,1

w11,2 + f11(•)

w11,3

Input layer Hidden layer Output layer

Figure 4. Optimal ANN structure used for prediction of glucose concentration.

Table 5. Optimized parameters (weights and bias) of the ANN model

Parameters connecting the input and hidden neurons Parameters connecting the hidden and output neurons

wj1 wj2 wj3 θj W1j b1 = 2.076

j=1 −1.827 −0.7160 −1.072 −0.3798 8.284 × 10−2

j=2 2.613 −4.558 −6.729 0.9368 −2.254
j=3 −2.752 −0.2179 4.301 × 10−2 −0.2609 0.1154
j=4 −3.072 −4.270 16.92 −6.356 −2.222
j=5 −1.849 −1.653 −0.9896 0.2454 −0.5380
j=6 −1.929 −0.9366 −0.6441 −1.608 0.8346
j=7 −0.2829 −22.82 1.053 0.5961 −10.46
j=8 −2.072 −4.927 2.043 3.595 −1.395
j=9 −1.784 −1.903 −1.898 0.5293 −0.8095
j = 10 −4.009 3.465 −4.913 6.030 −2.993
j = 11 −5.663 −4.807 × 10−2 5.377 7.509 2.676

by the MSE and RSD(%). Furthermore, in all cases R2 was close to

Table 6. Statistical criteria used to characterize the prediction quality
of the ANN model unity, indicating a good fit of the model to the experimental data.
Finally, it is worth mentioning that these results have shown
Trial MSE RSD(%) R2 that the dynamic model developed in this work can infer directly
the glucose release during enzymatic hydrolysis from a knowledge
1 26.8 17.0 0.98
of enzymatic loadings.
2 (Valid.) 16.8 14.4 0.97
3 13.9 10.4 0.97
4 13.4 10.0 0.98 Prediction of optimal enzyme loading using the ANN model
5 8.0 18.7 0.99 Prediction of the optimal region of glucose yield is one of the
6 49.9 17.4 0.98 main objectives in this study. Thus, after ANN model validation
7 3.2 8.8 0.99 is completed, it can be used to find processing inputs, i.e. the
8 36.4 15.8 0.98 optimum ratio of Ecell and Eβ−glu that gives the optimal glucose
9 (C) 19.6 11.6 0.99 yield. The conversion of glucose concentration, G (g L−1 ) to glucose
yield, Yield (%) is straightforward using Equation (1).
A three-dimensional response surface of glucose yield, was
obtained by keeping the time variable, t (h) constant at 72 h
(at which time no significant changes in yield were detected).
it was concluded that for the training and validation (marked in The response surface is shown in Fig. 6(a). It can be seen that
989

bold) datasets, the model showed similar performance, as judged the surface is strongly nonlinear and reveals a single maximum,

J Chem Technol Biotechnol 2010; 85: 983–992

c 2010 Society of Chemical Industry www.interscience.wiley.com/jctb
 www.soci.org E. Ccopa Rivera et al.

(a) 10 (a)
8
Glucose (g/L)

6
4
2
0
0 20 40 60 80

(b) 22
18
Glucose (g/L)

13
9
4 (b)
0
0 20 40 60 80

(c) 18
14
Glucose (g/L)

11
7
4
0
0 20 40 60 80
Time (h)

Figure 5. Experimental data (filled triangles) and performance of the ANN

model for the glucose concentration using: (a) and (b) training dataset
(corresponding to Trial 5 and 8 in Table 3) and (c) validation dataset Figure 6. (a) Response surface and (b) contour plot generated by ANN
(corresponding to Trial 2 in Table 3). model showing the effect of cellulase and β-glucosidase activities on
glucose yield at 72 h.

which represents the optimal ratio of Ecell to Eβ−glu for maximum
glucose yield. This behavior, confirmed by the validation results
of the ANN-based predictive model, has shown that the addition
of Eβ−glu to all concentrations of Ecell tested does not necessarily
ensure gradual increase in Yield.
Figure 6(b) show the optimal processing conditions to maxi-
mum glucose yield. This figure shows a contour plot of Yield as
a function of Ecell and Eβ−glu . It can be seen that the maximum
yield is not obtained for the maximum enzymes loading. Analysis
of the contours indicated that glucose yield above 99.0% could
be obtained in the ECell range 460.0–580.0 FPU L−1 (15.3–19.3
FPU g−1 bagasse) with supplementation of Eβ−glu in the range
750.0–1140.0 CBU L−1 (25–38 CBU g−1 bagasse). Concentrations
of enzyme below and above these ranges led to reduced yields.
The amount of enzymes required for high yields in enzymatic
hydrolysis is directly related to the efficiency and type of
pretreatment. Thus, different pretreatments lead to different
changes in the factors that cause resistance of lignocellulosic
materials to enzymatic hydrolysis, such as the content of lignin, the
presence of acetyl groups, cellulose crystallinity, polymerization
degree, surface area, volume and particle size.33
Comparison of the results obtained in this work with those
obtained by other authors show that combining an adequate
pretreatment agent with optimization tools is a successful method
to achieve high yields with minimum enzyme loading.
Krishna34 pre-treated sugarcane bagasse with 1% alkaline
hydrogen peroxide, resulting in glucose yields of 70%, using
990
Studies carried out by Martin35 have shown that bagasse
pretreated with steam at 250 ◦ C for 10 min, after impregnation
with sulfuric acid 1% (w/w), can provide a glucose yield of 35.9%
using an enzyme loading of 41.4 FPU g−1 dry pre-treated biomass
of cellulase and 39 IU g−1 dry pre-treated biomass of β-glucosidase.
According to Zhao,36 pretreating bagasse with 10% NaOH at
90 ◦ C for 1.5 h and further delignifying with 10% peracetic acid at
75 ◦ C for 2.5 h, led to a yield of reducing sugars of 92.04% and a
glucose yield of 56.23% after enzymatic hydrolysis for 120 h with
cellulase loading of 15 FPU g−1 solid.
Mesa37 studied the organosolv pretreatment of sugarcane
bagasse. The best result in terms of glucose yield was 20.9 g
glucose per 100 g sugarcane bagasse, obtained after enzymatic
hydrolysis of biomass pretreated with 1.25% sulfuric acid as a
catalyst for 60 min. The enzymatic loading was 15.0 FPU g−1 dry
biomass pre-treated of cellulase and 15.0 IU g−1 dry biomass of
pre-treated β-glucosidase.
It is worthwhile mentioning that the optimal processing
condition obtained in this work is certainly specific for the raw
material pretreatment method and conditions used. However,
it should be emphasized that application of the model-based
approach steps presented in this work to other enzymatic
hydrolysis processes is straightforward.
CONCLUDING REMARKS
This work presents results from the development and testing of

an enzyme loading of 40 FPU g−1 biomass. a modeling approach for the optimization of enzymes loading

www.interscience.wiley.com/jctb

c 2010 Society of Chemical Industry J Chem Technol Biotechnol 2010; 85: 983–992
Optimization of enzymatic hydrolysis of sugarcane bagasse using ANN
in enzymatic hydrolysis of sugarcane bagasse. In this process,
besides considering the optimal pretreatment conditions prior
to enzymatic hydrolysis, it is also important to define an optimum
ratio of enzymes that makes it possible to reduce enzyme loading,
which is often an important requirement to provide cost-efficient
second generation ethanol processes. Thus, for reliable per-
formance prediction and optimization through modeling, a
systematic model-based approach should be implemented. In this
work, a methodology to fully optimize enzyme loading has been
developed using artificial neural networks and design of experi-
ments techniques that have been widely used for prediction and
optimization purposes in engineering applications. Several recent
works points out the potential of combining the features of both
techniques to enhance prediction and optimization performance.
The complex relationship between the enzymatic activities
and the glucose released in a hydrolysis reaction is not easy to
identify. However, the data-driven identification methodology
developed in this work can convert the correlation of these
variables into a predictive mathematical model, while maximizing
the amount of experimental information that can be obtained
about the enzymatic process using an experimental design. Based
on this methodology, an optimal neural network structure was
obtained and its performance in describing the dynamic behavior
of glucose release during hydrolysis was assessed. Prediction by the
model using the validation dataset gave acceptable performance
measures (MSE, RSE and R2 ), equivalent to those obtained for the
training dataset.
The dynamic model developed for the enzymatic hydrolysis of
sugarcane bagasse can be used not only for the prediction of
glucose concentration profiles for different enzymatic loadings,
but also to obtain the optimum enzymes loading that leads to
optimal glucose yield. It can promote both successful hydrolysis
process control and a more effective employment of enzymes.
It can be seen from the graphical plots that the effectiveness of
enzymatic hydrolysis, as evaluated from the glucose released,
was strongly dependent on the cellulase and β-glucosidase
loadings. The recommended enzyme activities from the study
were; cellulase concentration in the range 460.0–580.0 FPU L−1
(which corresponds to the range 15.3–19.5 FPU g−1 bagasse)
and β-glucosidase in the range 750.0–1140.0 CBU L−1 (which
corresponds to 25–38 CBU g−1 bagasse). In these conditions the
glucose yield was above 99%. Calculation of this optimum region
may have practical consequences for improving the hydrolytic
efficiency of the enzymes that may be independently controlled
to accomplish a desired enzyme constraint regarding glucose yield.
Although the optimal enzymatic load is highly dependent on
the pretreatment method chosen and its operational conditions,
as well as on the quality of the raw biomass used, the methodology
developed in this work is straightforward and can be easily
applied to any combination of pretreatment/biomass employed
in enzymatic hydrolysis.
ACKNOWLEDGEMENTS
The authors thank Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento
Cientı́fico e Tecnológico (CNPq) for financial support.
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