water research 43 (2009) 3503–3510

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Optimization of laccase mediated biodegradation of

2,4-dichlorophenol using genetic algorithm

S.S. Bhattacharyaa, S. Karmakarb, R. Banerjeea,*

a
Microbial Biotechnology and Downstream Processing Laboratory, Department of Agricultural and Food Engineering, Indian Institute of
Technology, Kharagpur-721 302, India
b
Department of Industrial Engineering and Management, Indian Institute of Technology, Kharagpur-721 302, India

article info abstract

Article history: The present investigation focuses on the development of an effective strategy to determine
Received 8 January 2009 the optimum environmental conditions leading to the maximum rate of biodegradation of
Received in revised form 2,4-DCP by coupling response surface methodology (RSM) with a developed genetic algo-
6 May 2009 rithm (GA) thereby ensuring minimum contact time. RSM is utilized to create an efficient
Accepted 9 May 2009 analytical model for biodegradation of 2,4-DCP in terms of environmental parameters: pH,
Published online 20 May 2009 temperature, enzyme activity and time of incubation. For this purpose, a number of
degradation experiments based on statistical three-level Box Behnken design methods
Keywords: were carried out. An effective response surface (RS) model is developed by carrying out
Box Behnken design experiments designed using the Box Behnken method. The RS model thus developed is
Genetic algorithm further interfaced with the GA to optimize the degradation conditions for optimum
2,4-DCP degradation with minimum contact time. The GA increases the biodegradation conditions
Response surface methodology to >99% within a time period of 8 h within the given range of experimental conditions. The
Crossover conditions obtained from GA were verified experimentally.
Mutation ª 2009 Elsevier Ltd. All rights reserved.

1. Introduction
Incessant industrialization has resulted in the efflux of an
array of chemicals of anthropogenic origin which pose
a serious threat to the natural flora and fauna. These
compounds, popularly known as xenobiotic compounds are
generally persistent and often have carcinogenic and muta-
genic effects (Hendricks et al, 1985; Gesto et al, 2009). The great
strides made in biotechnology to solve environmentally
significant issues such as biodegradation of recalcitrant
xenobiotic compounds coupled with advances in computa-
tional techniques have enabled workers to go in for optimi-
zation using computational modeling techniques for
maximization of the processes as well as their deployment
under field conditions (Sinha and Minsker, 2007). During the
last few decades there has been a growing interest in solving
optimization problems using evolutionary and hereditary
principles; these systems maintain a potential population of
candidate solutions to the problem at hand. In recent years,
the subsurface simulation model has been combined with
techniques of optimization to address important problems of
contaminated site management (Qin et al, 2009). Modeling of
site remediation was useful for dynamic evaluation/predic-
tion and/or real-time control of the remediation systems.
Campagnolo and Akgerman (1995) proposed a model for
simulating soil vapor extraction systems that was used for
biodegradation of petroleum-contaminated soils while
Christodoulatos and Mohiuddin (1996) suggested generalized
models for prediction of pentachlorophenol adsorption by
natural soils.

* Corresponding author. Tel.: þ91 3222 283104.

E-mail address: rb@iitkgp.ac.in (R. Banerjee).
0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2009.05.012
3504 water research 43 (2009) 3503–3510

In the present study, an RS model using Box Behnken 2.2. Genetic algorithm
experimental design for predicting values of factors affecting
biodegradation of 2,4-DCP in wastewater was employed. 2,4- These systems have some selection process based on fitness
DCP is a potent pollutant that comes as an efflux from paper of individuals and some operators called evolutionary opera-
and textile industries. It is also used as soil fumigants and for tors. GA is one of the best known of these evolutionary
the manufacture of germicides. The regression model was programs (EP).
developed using statistical response surface methodology. In general a GA has five basic components: (a) a genetic
The accuracy of the RS model is verified with the experimental representation of solutions to the problem; (b) a way to create
measurement. The developed RS model is further coupled an initial population of solutions; (c) an evaluation function
with a developed GA to find the optimum environmental rating solutions in terms of their fitness; (d) genetic operators
conditions leading to the maximum degradation of 2,4-DCP (crossover and mutation) that alter the genetic compositions
with minimum contact time. Degradation conditions are of offspring during reproduction; (e) values of parameters of
represented with degradation parameters of enzyme GAs.
concentration, initial pH, temperature and incubation time. A GA has the following structure:
These factors may affect the efficiency of enzymatic treat-
ment, and their effects may be either independent or inter- procedure GA
active. Bhattacharya and Banerjee (2008) studied the begin
interaction of different parameters in the biodegradation of t ) 0;
2,4-DCP using RSM and got a regression coefficient of ~89%. In initialize P(t);
order to improve the efficacy of an enzymatic process, evaluate P(t);
response surface models can be coupled with modern opti- while (not termination condition) do
mization techniques to attain the optimal conditions for begin
enzymatic treatment (Liu et al., 2008). The predicted optimum t)tþ1
degradation condition by GA is validated with an experi- select P(t) from P(t - 1)
mental measurement. alter P(t)
Many global optimization methods have been proposed, evaluate P(t)
such as GA (Holland, 1975; Mitchell, 1996) and swarm intelli- end
gence (SI) which differ from other traditional search tech- end
niques in that they search among a population of points and
use probabilistic rather than deterministic transition rules. where P(t) is population of individuals in generation ‘t’.
The RS model and GA developed and utilized in this study In GA a fitness function or cost function was formed
present several advantages over other methods in the litera- depending upon the optimization of the objective function
ture. The RS model is a higher order and more sophisticated (Fig. 1). The fitness function was to be formed in such a way
polynomial model with greater accuracy (Myers and Mont- that the objective was maximized as the objective of the
gomery, 2002). The GA eliminates the difficulty of user defined present investigation was to get maximum degradation. The
parameters of the existing RS models. main objective of this study was to find the optimum condi-
The present study emphasizes the optimization of the tions which give maximum degradation which is at most
degradation of 2,4-DCP with minimum contact time by
coupling RSM and GA in the given set of experimental
parameters. The optimization of the biodegradation using
Selection of Individual
evolutionary and hereditary principles of the potent xenobi- Parameters
otic compounds is rarely reported in the literature. To the best
knowledge of the investigators, optimization of the biodeg-
radation of 2,4-DCP by coupling RSM with GA has not been
reported in the literature to date. Design of Experiments by Box
Behnken technique

2. Materials and methods

2.1. Design of experiments

Model Generation Genetic Algorithm
An important stage of RS model generation by RSM is the
design of experiments. In the present investigation, the
degradation experiments are planned using a statistical three-
level Box Behnken experimental design. Overall 27 experi-
ments were conducted considering four parameters affecting
Validation of the model
the biodegradation of 2,4-DCP: pH, temperature, enzyme
activity and time of incubation. The range of the experimental
parameters was selected based on the single parameter Fig. 1 – Different stages of optimization of 2,4-DCP by
selection technique. coupling RSM and GA.
 water research 43 (2009) 3503–3510
100%. The parameter sets predicting degradations more than
100% were discarded by the GA program. To find the param-
eter sets, the coding was done in MATLAB.
2.3. Fungal strains
A hyperactive strain of Pleurotus sp. was isolated locally from
the forests of Kharagpur, India. It was maintained on the
slants of potato dextrose agar. Subculturing was done every
ninth day to maintain its viability.
2.4. Chemicals
All the solvents and reagents used in the present study were
purchased from Merck Chemicals, Germany.
2.5. Biodegradation conditions
A 1 mM solution of 2,4-DCP was prepared in distilled water
and the degradation was monitored at different conditions of
pH, temperature, time and enzyme concentration. The
optimal conditions for biodegradation of 2,4-DCP was estab-
lished using response surface methodology.
2.6. Laccase assay
Laccase was assayed by monitoring the oxidation of 2,20 -
azino-bis-(3)-ethylbenzothiazoline-6-sulphonic acid(ABTS) in
a reaction mixture containing 1 mM ABTS in 0.1 M sodium
acetate buffer (pH 4.5) and 5–50 ml of enzyme sample. Oxida-
tion was monitored at 436 nm, and 1 IU of activity was defined
as 1 mM of ABTS oxidized per minute (3436 ¼ 29,300 M1 cm1)
2.7. Analysis of 2,4-DCP
The degradation of 2,4-DCP was monitored using an HPLC
system (Agilent 1200 series) equipped with a Zorbax C18
reverse phase column (4.6  250 mm), Chemstation system
controller, a G1310A isopump and a UV–vis detector set at
280 nm. The solvent system used in the analyses comprised
methanol (60%), and HPLC grade water (38%) acidified with
acetic acid (2%). The flow rate was maintained at 0.75 ml
min1. The sample injection volume was 20 ml.
2.8. Response surface methodology
Response surface methodology is an empirical modeling
technique used to evaluate the relationship between a set of
controllable experimental factors and observed results. This
optimization process involves three major steps: (i) perform-
ing statistically designed experiments; (ii) estimating the
coefficients in a mathematical model; and (iii) predicting the
response and checking the adequacy of the model (Box and
Behnken, 1960). A class of three-level complete factorial
design for the estimation of the parameters in a second-order
model was developed by the Box Behnken method.
3505
3. Results and discussion
In this study, amongst several factors that affect the biodeg-
radation of 2,4-DCP, four controllable parameters were
selected to find their influence on biodegradation and opti-
mize the rate of degradation. Enzymes being proteins, they are
greatly stabilized by weak bonds such as hydrogen bonds and
hence are greatly affected by variations in their pH (Nelson
and Cox, 2000). An optimum pH is the ideal condition for the
biodegradation of a recalcitrant compound as it ensures the
minimum residence time of the compound in the environ-
ment owing to the maximum activity. The effect of pH on the
biodegradation of 2,4-DCP was studied and it was found that
a maximum degradation of 2,4-DCP was obtained at pH 7
(Fig. 2). Hence it emphasizes the efficacy of the process to be
used at neutral pH thereby negating any need for pH alteration
for the process to take place. The conferring of structural
stability by hydrogen bonds decides the selection of yet
another factor under investigation, the temperature. It was
found that the maximum rate of degradation was between 36
and 40  C. At higher temperatures, there was a sharp decline
in the activity (Fig. 3). An increase in temperature essentially
results in more kinetic energy of the reactants as well as of the
enzyme molecules, thereby increasing the frequency of colli-
sion and thus enhancing the rate of reaction. However, the
increase in temperature beyond a certain point results in the
breakage of hydrogen bonds thus resulting in a change of
structural conformation which adversely affects the rate of
enzymatic reaction. The efficacy of the process is further
dependent upon the process time. It was observed that at
a hold value of pH 7 and temperature 36  C ~96% 2,4-DCP was
degraded within a time period of ~10 h. An optimum 2%
inoculum accounting to 10 IU ml1 final enzyme activity was
taken during the investigation. The interaction of the above
mentioned parameters was studied by Bhattacharya and
Banerjee (2008), who carried out optimization of laccase
mediated biodegradation of 2,4-DCP employing the Box
Behnken design considering the four parameters, namely pH,
temperature, time and enzyme concentration. The design was
applied using Minitab to study four variables at three levels.
The boundary parameters for the above experiments were
fixed as pH (5.5 and 7), temperature (20 and 40  C), time
120
100
% Degradation of 2,4-DCP
80
60
40
20
0
4 4.5 5 5.5 6 6.5 7 7.5 8
pH
Fig. 2 – The effect of pH on the biodegradation of 2,4-DCP.
3506 water research 43 (2009) 3503–3510

120 and enzyme concentration respectively) are given below in

terms of coded factors:
100
% Degradation of 2,4-DCP

Y ¼ 87:73  5:5A þ 11:35B  9:17C þ 4:74D  4:19A2  0:99B2

 18:95C2 þ 1:05D2 þ 2:43AB þ 0:47AC  6:25AD þ 1:24BC
80
þ 1:38BD þ 7:10CD
60
This quadratic equation was considered as the initial function
for the genetic algorithm for further optimization of the process
40
and to eliminate the limitations of user defined boundaries of
RSM. The statistical significance of the enzymatic biodegadation
20 of 2,4-DCP was analyzed by ANOVA (Bhattacharya and Banerjee,
2008) of the quadratic equation for the enzymatic biodegrada-
0 tion of 2,4-DCP which has been summarized in Table 1. Since
25 30 35 40 45 50 55
f ¼ 2.62 at 14 and 12 degrees of freedom, it can be assumed that
Temperature (Celcius)
the regression for the quadratic equation of 2,4-DCP biodegra-
Fig. 3 – The effects of temperature on the biodegradation of dation is significant. The calculated value of ‘f’ is more than the
2,4-DCP. tabulated value ( f ¼ 3.26) at 4 and 12 degrees of freedom,
implying the significance of linear and square effect of the
quadratic equation for 2,4-DCP biodegradation. The insignifi-
cance of the interaction effect between different parameters is
interval (7 and 10 h), final enzyme concentration (5 and 10 IU indicated since f ¼ 3 at 6 and 12 degrees of freedom (Mont-
ml1). Three different pH (5.5, 6.25, 7), temperature (20, 32.5, gomery, 2004). This observation can be attributed to the fact that
40  C), different time intervals (7, 8.5 and 10 h) and final some parameters under study do not have a profound effect on
enzyme concentration (5, 7.5, 10 IU ml1) were chosen as the each other for 2,4-DCP biodegradation which is evident from
critical variables and designated as A, B, C, and D. The four their parallel mesh plot.
significant variables can be approximated by the quadratic
model equation as follows:

Y ¼ k0 þ ka A þ kb B þ kc C þ kd D þ kaa A2 þ kbb B2 þ kcc C2 þ kdd D2 3.1. Optimization of degradation using

þ kab AB þ kac AC þ kad AD þ kbc BC þ kbd BD þ kcd CD genetic algorithms

where Y is the predicted response; k0 is a constant; ka, kb, kc, kd The developed second-order RSM-based model for degrada-
are the linear coefficients; kab, kac, kad, kbc, kbd, kcd are the tion was utilized to optimize the degradation process. This
cross-coefficients; kaa, kbb, kcc, kdd are the quadratic consists of finding the combination of input variables that
coefficients. result in maximum degradation. Hence, the degradation
This response is preferred because relatively few experi- optimization can be stated as follows:
mental combinations of the variables are adequate to esti-
mate potentially complex response function. A total number Find pH, temperature, time and enzyme activity
of 27 experiments were necessarily carried out to estimate the To Maximize degradation ¼ f (pH, temperature, time and enzyme
15 coefficients for the biodegradation of 2,4-DCP. Data were activity)
analyzed using the Minitab program including ANOVA to Subjected to constraint: degradation £ 100%
find out the interaction between the variables and the Parameter ranges:
response. The quality of the fit of this model was expressed by 5.5  pH  7
the coefficient of determination (R2) in the same program. 25  temperature  40
The mathematical expression of the relationship of 2,4-DCP 7  time  10
degradation with variables A, B, C and D (pH, temperature, time 5  enzyme activity  10

Table 1 – ANOVA results for the quadratic equation of Minitab for degradation of 2,4-DCP.
Source d.f. Seq. SS Adj. SS Adj. MS F P

Regression 14 5946.89 5946.89 424.778 6.22 0.002

Linear 4 3188.93 3188.93 797.231 11.68 0.000
Square 4 2320.17 2320.17 580.042 00 0.002
Interaction 6 437.80 437.80 72.966 1.07 0.432
Residual error 12 819.1 819.1 68.258
Lack-of-fit þ1 818.67 818.67 81.867 383.75 0.003
Pure error 2 0.43 0.43 0.213
Total 26 6765.99
 water research 43 (2009) 3503–3510
As seen from the response surface analysis, there exists
a non-linear relationship between the degradation and
process parameters. Further, the combinations of process
parameters required to maximize degradation are different.
Thus, it requires an efficient optimization tool to determine
the combination of process parameters which maximize
degradation and hence GA is employed.
The GAs are more likely to converge to global optimum
than conventional optimization techniques, since they search
from a population of points, and are based on probabilistic
rules. The conventional optimization techniques are ordi-
narily based on deterministic hill-climbing methods, which
may find local optima. The GAs can also tolerate discontinu-
ities and noisy function evaluations. In each cycle of genetic
operation, termed an evolution process, a subsequent gener-
ation is created from the chromosomes in the current pop-
ulation. This consists of manipulation of genes, where genes
of parents are mixed and recombined for the production of
offspring in the next generation. This evolution process
consists of selection or reproduction, crossover and mutation.
3.1.1. Genes, chromosomes and fitness function
The genes are the searching parameters for the optimization
problem. In the simple GA, the genes are represented with
finite length of binary codes, 0 and 1. The chromosomes are
the strings containing defining genes. Thus, the chromosome
for the optimization consisted of four genes corresponding to
four searching parameters: pH, temperature, time and
enzyme activity. Each gene was represented by six bits of
binary codes and hence the chromosome constructed was of
24 bits length as shown below:
Sample chromosome ¼ [110001111001011001100100]
The genes of each chromosome were decoded as:
 
X ¼ DðZmax  Zmin Þ= 2nb  1 þ Zmin ;
Z ¼ pH; temperature; time and enzyme activity
where D is the decimal equivalent of binary, Zmax the
maximum limit for Z, Zmin the minimum limit for Z, and nb is
the number of binary bits for genes (i.e. 6).
3.1.2. Fitness function
The uncoded response surface found earlier was used as the
objective function which is to be maximized. A penalty func-
tion was introduced in the fitness function to discard the 100%
or more degradation values. The fitness function F(x) was of
the following structure:
1
Fitness function ¼ FðxÞ ¼ þ PðxÞ
Degradation
where P(x) represents the penalty function.
3.1.3. Selection
In the present study, the tournament selection with replace-
ment and selection size 4 (called tournament size) is applied
for the selection of chromosomes. This method randomly
chooses four chromosomes and picks out the best chromo-
some for reproduction based on fitness value. Randomization
3507
is indispensable in genetic algorithms as it helps to push out
a trapped solution from a local optima to a global optima.
Hence the probability of reaching the global optima increases
with the induction of randomization (Mitchell, 1996).
3.1.4. Crossover and mutation
Crossover is a recombination operator where gene informa-
tion is exchanged at random locations between two parent
chromosomes, which are randomly selected high fitness
valued chromosomes from the population (Gen and Cheng,
1999). The crossover fraction ( pc) is also randomly selected.
For instance, if crossover fraction ( pc) is 0.5 then the first 30
higher fitness valued chromosomes out of 60 chromosomes
are to be selected for crossover when the chromosomes are
sorted according to the decreasing fitness values.
For constrained optimization the central problem is to
handle the constraints as the manipulation of chromosomes in
the process of crossover and mutation often yield infeasible
chromosomes which may give better results but violating the
constraints. Four techniques can be used for handling the
constraints (Michalewicz, 1996):
 Rejecting strategy: Simply discards the infeasible
chromosomes.
 Repairing strategy: Repairs the infeasible chromosomes.
 Modifying genetic operators strategy: Modifies the problem
representation and genetic operators (Crossover, Mutation
and Selection).
 Penalty strategy: Penalizing the infeasible solutions.
Penalty strategy is the most popular and common
strategy amongst the above mentioned methods. Except
penalty strategy, others never take infeasible solution at all
into consideration. As in the constrained optimization,
infeasible solutions generally take a bigger portion than
feasible solutions and in such cases feasible solutions
are really difficult to find out if genetic search is confined to
feasible regions only. Penalty strategy allows the movement
of genetic search in infeasible regions too to yield more rapid
optimization and produce better results than do approaches
limiting search trajectories only to feasible regions of search
space. Hence it provides greater adaptability to biological
systems which in general have lower consistency. This
technique transforms the constrained optimization problem
into a unconstrained one, in which a penalty function is
added to the objective function for any violation of
constraints (Michalewicz, 1996).
In the present investigation, the penalty function P(x) is
defined as:
PðxÞ ¼ maximumf0; Mðf ðxÞ  100Þg
where x is a solution to the problem, M is a large number such
as 10,000,000, and f(x) is the degradation function.
This depicts that, whenever the degradation value is more
than 100 then a large positive quantity will be added to the
fitness value and as it is a minimization problem, it is auto-
matically discarded otherwise it would take a zero value when
the degradation value is less than 100, hence proving its
feasibility.
3508 water research 43 (2009) 3503–3510

pool for crossover. In this way when 100 chromosomes were

A B C D E F G H I J K L selected creating a mating pool, the chromosomes were ready
Parent Chromosome1
for crossover. The chromosomes were then arranged in
Parent Chromosome2 1 0 1 0 1 1 1 0 1 0 1 1
increasing order of fitness F0 1, F0 2 F0 3 .. F0 100. The cumulative
After two point Crossover
fitness values were calculated as:

Q1 ¼ F01
1 0 1 D E F G H I 0 1 1 Q2 ¼ F01 þ F02
Progeny Chromosome1
..;
Progeny Chromosome2 A B C 0 1 1 1 0 1 J K L Q100 ¼ F01 þ F02 þ ..F0100

Selection of a population of chromosomes to undergo the

Fig. 4 – Two point crossover.
genetic operators was performed in the following way. For
applying crossover operator, 100 suitable random numbers (ri)
were generated. The crossover was started with a crossover
The genetic algorithm was initialized with a population of fraction or probability pc ¼ 0.9. From that population, chro-
chromosomes randomly generated assumed as x1,x2, x3 mosomes, which satisfied Qi < ri were checked. The chromo-
..x100 with the initial population size 100. For each indi- somes for which this relation held true were selected for
vidual xi, where i varies from 1 to 100, the fitness function (F ) crossover and placed in a mating pool of chromosomes. The
values were calculated and obtained as F1, F2 F3 .. F100. To reason behind choosing a higher crossover fraction was to
select the chromosomes for crossover, tournament selection allow almost all the chromosomes to undergo crossover.
(tournament size 4) with replacement was applied, in which In the present investigation, a two point crossover was
100 times four chromosomes were selected at random and the used. Two point crossover was carried out between two
chromosome with best fitness value was kept to fill the mating chromosomes from the chromosome pool as shown in Fig. 4.

a Mutation Probability v/s Fitness

11.10
Fitness(10e-3)

10.80 Crossover probability=0.5

Population size=1000
10.50
Maximum no. of generations=100
10.20 Mutation probability varied from 0.001 to 0.02
9.90
9.60
0.001

0.003

0.004

0.006

0.007

0.009

0.010

Mutation Probability

b 11.1 Population size v/s Fitness

Crossover probability=0.5
10.8
Fitness(10e-3)

Mutation probability=0.008
Maximum no. of generations=100
10.5 Population size varied from 100 to 1000
10.2

9.9

9.6
100

250

400

550

700

850

1000

Population size

c 11
Maximum Generation No. v/s Fitness
Fitness(10e-3)

10.8 Crossover probability=0.5

10.6 Mutation probability=0.008
10.4 Population size =450
10.2 Maximum no. of generations varied from 50 to 1000
10
9.8
9.6
50
150

250
350
450

550
650
750

850

950

Maximum Generation No.

Fig. 5 – Study of parameters for selection of factors for genetic algorithm: (a) mutation probability versus fitness; (b)
population size versus fitness; (c) maximum generation number versus fitness.
 water research 43 (2009) 3503–3510 3509

given by 24  100  0.05 ¼ 120, as the population size was 100

Table 2 – Regression statistics of genetic algorithm.
and each chromosome had 24 bits To select the bits to
Regression statistics undergo mutation 2400 random numbers were generated in
Multiple R 0.973028261 the range of 0,.,1. Whenever the generated random number
R square 0.946783998 was less than pm i.e. 0.05 the corresponding bit was selected
Adjusted R square 0.945486046 for mutation. The randomness was introduced here as the
Standard error 0.624902745 main objective of mutation is to force a trapped solution out of
Observations 43
local optima (Gen and Cheng, 1999). The parametric study of
GA is depicted in Fig. 5a,b,c.
After mutation a GA cycle is completed i.e. one generation
Mutation causes individual genetic representations to be is over. After crossover and mutation the generated chromo-
changed according to some probabilistic rule so as to over- somes were combined and were ready for the selection of the
come destructive crossover. The mutation operation con- next generation. In this way, the GA was run for 100
sisted of complementing the bits (replacing 0 by 1 and vice generations.
versa) at random locations. Assuming the mutation proba- It was observed that after running the GA for a set of
bility ( pm) as 0.05, the number of bits to undergo mutation are optimum parameters even >99% degradation is possible with

Table 3 – Residual analysis of the results obtained from genetic algorithm.

Observation Experimental Y (% degradation) Predicted Y (% degradation) Residuals Standardized residuals

1 98.97419397 102.3404898 3.366295881 5.452209938

2 99.67031839 99.57997089 0.090347495 0.14633102
3 99.5676115 99.47260267 0.095008838 0.153880749
4 99.71596589 99.28221539 0.433750493 0.702522545
5 99.96702715 99.14648318 0.820543969 1.328991313
6 98.74595645 98.76783378 0.021877323 0.035433533
7 98.88289896 98.61308998 0.26980898 0.436995218
8 98.74595645 98.49079654 0.255159916 0.413268911
9 98.73454458 98.2866891 0.447855484 0.725367646
10 98.96278209 98.24592621 0.716855879 1.161053242
11 97.61618076 97.96083681 0.344656045 0.558221019
12 98.03842016 97.4956993 0.542720861 0.879016039
13 97.92430141 97.46153063 0.462770776 0.74952515
14 96.69181883 97.43243009 0.740611261 1.199528569
15 96.69181883 97.28026795 0.588449118 0.953079658
16 96.46358132 96.88593476 0.422353438 0.684063343
17 96.70323071 96.7911788 0.087948094 0.142444838
18 96.82876134 96.73277627 0.095985074 0.155461907
19 96.44075757 96.72810053 0.287342961 0.465394072
20 96.07557755 96.43889403 0.363316482 0.588444332
21 96.22393193 96.34379368 0.119861752 0.194133689
22 96.5662882 96.1977389 0.368549304 0.596919655
23 96.26957943 96.17463725 0.094942185 0.153772794
24 96.20110818 95.86932737 0.331780812 0.537367689
25 95.88157566 95.47422558 0.407350076 0.659763196
26 95.40227688 95.31261039 0.089666494 0.145228039
27 95.23109874 95.13914503 0.09195371 0.14893252
28 95.18545124 95.10116639 0.084284851 0.136511679
29 95.12839186 95.07250968 0.055882187 0.090509399
30 95.03709686 94.93158614 0.105510717 0.170890083
31 94.84309497 94.76572197 0.077372997 0.125316917
32 94.51215058 94.39709022 0.115060359 0.186357128
33 94.26108931 94.04451377 0.216575539 0.350775852
34 93.74755491 93.19963284 0.547922062 0.887440148
35 93.00578299 93.07901695 0.073233968 0.118613153
36 92.83460485 92.89719828 0.062593426 0.101379234
37 92.77754547 92.83596172 0.058416252 0.094613688
38 92.60636734 92.82903517 0.222667835 0.36064322
39 92.59495546 92.82791982 0.232964355 0.377319944
40 92.56071983 92.60925986 0.04854003 0.078617698
41 92.33248232 92.0946788 0.237803526 0.38515769
42 92.03577355 92.04955397 0.013780419 0.022319411
43 88.32691396 88.3274679 0.000553944 0.000897193
3510 water research 43 (2009) 3503–3510
parameter values of 6.10021, 36.85669  C, 7.99644 h and
9.92093 IU ml1 as pH, temperature, time and enzyme activity
respectively. In the real experiment, these values of the
parameters gave a degradation of 98.91% which was very
similar to the data obtained. Bhattacharya and Banerjee (2008)
reported a maximum degradation efficiency of ~98% at a pH 6,
temperature 40  C, time 9 h and an enzyme concentration of
8 IU ml1. Zhang et al. (2008) reported 94% removal efficiency
of 2,4-DCP within 10 h at pH 5.5 while exploring the idea of
a promotion in the rate of degradation with the increase in
laccase concentration or an increase in temperature. Using
GA, >99% degradation is possible by varying the conditions.
The increase in the degradation rate can be attributed to the
greater stability of laccase at slightly higher pH. The greater
degradation efficiency at elevated temperature might be due
to the higher collision rate between the molecules of the
enzyme and 2,4-DCP. From this study, it is evident that
a balance has to be struck between temperatures of maximum
degradation of 2,4-DCP and laccase denaturation. This high-
lights the efficacy of the GA to overcome the limitations of
user defined boundary parameters. Furthermore, while
varying the concentration of 2,4-DCP ranging from 50m to
1000 mg ml1 to check the feasibility of the process across
a concentration range in addition to the concentration of
1 mM (163 mg l1) at which the optimum conditions of
biodegradation were established, it was observed that the Km
value obtained from the experiments is 350 mg ml1. The levels
of 2,4-DCP in the environment range from ~150 mg l1(Valo
et al., 1990) to 150–200 mg l1 (Etalla et al., 1992). This rein-
forces the efficacy of laccase for the biodegradation of 2,4-DCP
in field conditions. Hence, the present investigation highlights
the fastest biodegradation of 2,4-DCP which can be applied at
a concentration where whole cells fail to thrive (Bhattacharya
and Banerjee, 2008).
3.2. Validation of the model
For a model to be declared industrially fit, it is important to
validate the model. A regression analysis was carried out
(Table 2) and it was found that the model developed by GA has
an accuracy of 94.67% which is fairly good for deployment in
field conditions. Residual analysis was further carried out
(Table 3) for the validation of the model and it was found that
all the standardized residuals fell within the range of 1 to þ2
barring one observation which was 5.45. This is a peculiar case
of an outlier because the predicted value exceeded 100%
degradation which is not practically feasible. This is a fairly
good result as the acceptable range of residuals is between 3
to þ3.
4. Conclusions
In the present investigation, RSM was effectively coupled with
the GA to develop a model and optimize the experimental
conditions to effect ~99% biodegradation of 2,4-DCP within
a time period of 8 h. This was an improvement compared to
the regression model which showed ~98% degradation within
9 h. This highlights the efficacy of the methodology to over-
come the limitations of user defined parameters thereby
adding more flexibility in the model. The model was further
validated and the accuracy was found to be 94.67%. This
reinforces the deployment of the methodology in field condi-
tions for 2,4-DCP biodegradation at a concentration at which
the growth of whole cell organisms is not possible.
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