response surface optimization for efficient dye removal by isolated strain pseudomonas sp

Cent. Eur. J. Eng. • 2(3) • 2012 • 425-434DOI: 10.2478/s13531-012-0001-9

Central European Journal of Engineering

Response surface optimization for efficient dyeremoval by isolated strain Pseudomonas sp.

Research article

Shanmugam Senthilkumar1∗, Muthiah Perumalsamy1† Harinarayan Janardhana Prabhuy, ChiyaAhmedBasha2, Narayan Anantharaman1

1 Department of Chemical Engineering, National Institute of Technology, Trichy-620015, Tamilnadu, India

2 Department of Chemical Engineering, Adhiyamaan College of Engineering, Hosur 635109, Tamilnadu, India

Received 31 December 2011; accepted 15 February 2012

Abstract: Response surface methodology (RSM) involving the central composite design (CCD) was employed to optimizethree important process variables for the decolourization of synthetic dye solutions containing Remazol TurquoiseBlue (RTB) and Reactive Black 5 (RB5) with isolated bacterial strain Pseudomonas sp. The interaction betweenthree variables i.e. Initial concentration of dye, carbon source and nitrogen source were studied and modeled.According to the Analysis of variance (ANOVA) results the predicted results were found to be in good agreement withexperimental results (R2: 0.9726; Adj R2: 0.9480 for RTB and R2: 0.9789; Adj R2: 0.9750 for RB5) which indicatedexcellent evaluation of experimental data from the second order polynomial regression model. Mathematicalmodels were developed by the proposed system, for each process variable showed the effect of each factor andtheir interactions on biodecolourization process. The optimum concentrations of Dye, Carbon source, and Nitrogensource were found to be 20 mgL−1, 1.5 g/L and 1.5 g/L, respectively for RTB and RB5 to obtain maximum dyeremoving capacity. Predicted values were validated with experimental results, which indicated appropriateness ofthe employed model and the success of RSM.

Keywords: Bacteria • Carbon source • Dye • Nitrogen source • RSM© Versita sp. z o.o.

1. Introduction

Dyes and dyestuffs are the colorants which are extensivelyused in the food, pharmaceutical, cosmetic, textile andleather industries. Over 100,000 commercially availabledyes exist and more than 7 × 105 tones of dyestuff areproduced annually [1]. These dyestuffs which are presentin textile effluent causes significant problems in treat-ment plants since these compounds are hard to degrade∗E-mail: [email protected]†E-mail: [email protected]

by biological means. Color can be removed from effluentby many methods such as physical, chemical and advanceoxidation methods such as adsorption, coagulation floccula-tion, oxidation, filtration and electrochemical methods [2–4].However, these methods are not cost effective and havesome other practical difficulties. Moreover, these dyestuffscannot be completely mineralized by conventional treat-ment methods. Complete mineralization of dyestuff can bedone by chemical or biological oxidation. On the Basis ofthe degree of fixation, of dye to fiber, more than 10–15%dye does not bind to the fiber during color processingand release into water bodies as effluent causing serious425

Response surface optimization for efficient dye removal by isolated strain Pseudomonas sp.

environmental pollution [5–8]. These dyestuffs enter intothe ground water and indirectly affect our food chain.Several bacterial strains have been investigated, by a num-ber of researchers, having the ability to decolorize azodyes aerobically and they have been isolated during thepast few years to decolorize various textile azo dyes [9–13].Several microorganisms are capable of decolorizing the azodyes, including Gram-positive and Gram-negative bacteria[14]. These are known not only for degradation but alsofor detoxification. Dye degradation using isolated Pseu-domonas species as biocatalyst is a cost effective systemand it involves the elimination of the reactive dyes [15, 16].The bacterial isolate Pseudomonas aeruginosa NBAR12was capable of decolorizing 12 different dyes with varyingdecolorization efficiency. Among them, diazo Reactive Blue172 was found to be decolorized when glucose and yeastextract was supplied in the medium [17]. P. aeruginosadecolorized a commercial tannery and textile dye, NavitanFast blue S5R, over 90% with in 24 hours in the presence ofglucose under aerobic conditions. This organism was alsoable to decolorize various other azo dyes [18].There are onlyvery few bacteria that are able to grow on azo compoundswhich utilizes the dye stuff as the sole carbon and energysource. These bacteria have the capability to cleave theazo −N=N− bonds and utilizes the aromatic amines asthe source of carbon and energy for their growth [19].Numerous statistically designed experimental models havebeen applied to optimize the process variables in biolog-ical processes. In conventional optimization process, Inorder to determine the influence of process variables, theexperiments were carried out varying systematically theparticular process variable by keeping the other parameteras constant. In order to optimize the effective parameterswith the minimum number of experiments, application ofthe experimental design methodologies can be useful. Re-sponse surface methodology (RSM) is a good alternatewhich provides elaborative vision over various combinationsof parameters with minimum number of experimental runs.RSM is essentially a particular set of mathematical modeland statistical methods for designing experiments, buildingmodels, evaluating the effects of variables, and searchingoptimum conditions of process variables to predict targetedresponses [20–22].The main advantage of RSM is the re-duced number of experimental trials needed to evaluatemultiple parameters and their interactions [20–23].Recently several studies have described the use of RSMfor optimization of process parameters (dye concentration,pH, temperature and inoculum size) for the decolouriza-tion/degradation of dyes from synthetic solutions. However,to the best of our knowledge until now no study used CCDto analyze the optimal media condition for the decolour-ization of RTB and RB5 using isolated bacterial strainPseudomonas sp.

Hence the objective of the present study is to identifyoptimal condition that would efficiently decolorize thesynthetic dye solutions containing RTB and RB5 usingisolated strain Pseudomonas sp. based on CCD and toevaluate the role of the key parameters and their interac-tions.2. Materials and methods

2.1. Microorganism and dyePseudomonas sp. bacterial culture has been used to studythe degradation of synthetic dye solutions containing RTBand RB5. This Organism is well known for its potential todegrade textile dye effluents. It was isolated from textiledisposal site soil and the pure culture of this bacterium wasobtained by culture enrichment technique. The structureand properties of RTB and RB5 were presented in Table 1.2.2. Maintenance of CultureStock cultures were stored at −20°C in 20% glycerol.Periodical subculture has been done and the organismsfrom stock cultures were used for biodegradation studiesafter preculturing in nutrient broth. 5 days old culture hasbeen used for this study.2.3. Culture media and dyeMineral salts medium (MSM) of following composition(g/L): K2HPO4, 1.73; KH2PO4, 0.68; MgSO4 · 7H2O, 0.1;NaCl, 0.1; FeSO4 ·7H2O, 0.03; NH4NO3, 1.0; CaCl2 ·2H2O,0.02 and Glucose, 1. The pH of the medium was adjusted to7.5. The medium without glucose was sterilized at 121°Cfor 20 min. Glucose was sterilized separately and addedto the medium. Wherever the effects of nitrogen sourceswere studied, NH4NO3 in the medium was replaced withinorganic nitrogen source such as (NH4)2SO4. The effectof carbon sources was studied by replacing glucose withsucrose [18]. Dye solutions were filter sterilized as stocksolution and added aseptically to the mineral salt mediumto the desired concentration of dye. Remazol turquoiseand Reactive black 5 were bought from Sigma Aldrich andwere used without any purification.2.4. Decolourization studies Reactive black5 and Remazol turquoise blue by Pseudo-monas sp.

Experiments carried out for 3 days in a 250 mL Erlenmeyerflask containing Reactive black 5 and Remazol turquoise426

S. Senthilkumar, et al.

Table 1. Structure and properties of RTB and RB5.

S No Dye Molecular structure Molecularweight Absorptionmaxima(λmax)

1 Remazolturquoiseblue576.10 628 nm

2 Reactiveblack 5 991.82 597 nm

blue dyes were mixed with 100 mL of nutrients solutionseparately and covered with sterilized cotton. The conicalflask was kept in a sterilizer at 121°C for 15 minutes. Af-ter this solution was allowed to cool to room temperature,the culture was added to the dye solution. The conicalflask was kept in an orbital shaking incubator and shakencontinuously for aeration. 10 mL of the solution was peri-odically taken (for every 24 h) and centrifuged to obtaina clear solution. The optical density of the solution wasmeasured in the UV visible spectrophotometer at 628 nmfor remazol turquoise blue and 595 nm for Reactive black 5.% decolourization is calculated as shown in Equation 1:%Decolourization = C0 − Ci

C0 × 100, (1)where C0 and Ci are Initial and Final concentration re-spectively.2.4.1. Effect of Carbon sourceThe rate of decolourisation for various amount of carbonsource (sucrose) was studied. RTB and Reactive black 5dye solutions were mixed with 100 mL nutrient medium.To this 0.5, 1, 1.5 and 2 g of sucrose were added. Experi-ments carried out in a 250 mL Erlenmeyer flask containingReactive black 5 dye solution and Pseudomonas sp. agi-tated using a orbital shaking incubator. The samples werewithdrawn at regular intervals centrifuged and analyzedin UV spectrophotometer.

2.4.2. Effect of Nitrogen source

The rates of decolourization for various amounts of Nitro-gen source in the form of Diammonium sulfate salts werestudied. RTB and RB5 dye solutions were mixed with100 mL nutrient medium. To this 0.5, 1, 1.5 and 2 g ofDiammonium sulfate were added. The same procedure wasfollowed as that of carbon source.2.4.3. Effect of agitated and still condition

The effects of agitated and still conditions on the decolour-ization were studied. Two Erlenmeyer conical flasks weretaken with 100 mgL−1 of RB5 dye solution added to 100 mLof nutrient medium. One conical flask was kept in a orbitalshaking incubator and another flask in still condition. Therate of decolourization was studied and compared with andwithout agitated dye solutions.2.5. Analytical procedure

The pure dye and Metabolites formed after decolorizationof RB5 with Pseudomonas sp. were characterized byusing the Fourier Transform Infrared Spectroscopy (FTIR).FTIR analysis was done in the mid IR region of 400–4,000 cm−1 with 16 scan speed. The pellets preparedusing spectroscopic pure KBr (5:95), were fixed in sampleholder and analyses were carried out.427


Table 2. Experimental range and level of the input variables.Factor Process Variable Unit Range and levels of coded variables−1 (Min) 0 (Mean) +1 (Max)A Initial dye Concentration mgL−1 20 60 100B Carbon Source gL−1 0 1 2C Nitrogen source gL−1 0 1 2

2.6. Experimental designOnce we had determined the optimal pH value and tem-perature, we adopted a 20 run Central composite designwas adopted to optimize the process inducers, becausethe predicted and actual responses can be simply relatedto the chosen factor [23–25]. The following second orderpolynomial regression equation was used to correlate theboth variables.

Xi = (Xi − X0∆Xi

), (2)

where Xi is the coded value, X0 is the actual value on thecenter point and ∆Xi is step interval.The following parameters and their range are consideredfor the experimental design method: (i) Initial concentrationof the dye [20, 40, 60, 80 & 100 mg/L]; (ii) Carbon source [0,0.5, 1, 1.5, 2 g/L]; (iii) nitrogen source [0, 0.5, 1, 1.5, 2 g/L]at pH 7.0 and Temperature 30°C [24, 25]. The followingresponse equation was used to correlate the dependentand independent variables:Y = b0 + b1x1 + b2x2 + b3x3 + b11x21 + b22x22+ b33x33 + b12x1x2 + b13x1x3 + b23x2x3 (3)

where Y is predicted response to the bio decolorizationprocess, bo is a constant coefficient, b1, b2 and b3 are thelinear regression coefficients, x1 is the Initial concentrationof the dye, x2 is carbon source, x3 is Nitrogen source, b11,b22 and b33 are the quadratic coefficients and b12, b13 andb23 are the interaction coefficients and b1, b2 and b3 arethe coded factors. The low middle and high levels of eachindependent variable were designated as –1, 0, and +1respectively, and the corresponding actual values for eachvariable shown in Table 2.Fig. 1(a) & (b) demonstrates the normal probability andstudentized residual plot for RTB and RB5 decolourization.Residuals are indication of difference between predictedversus actual values which validates the Analysis of vari-ance model.3. Results and discussionCentral composite statistical experiment design and theresponse surface methodology were employed to inves-

(a)

(b)Figure 1. The studentized residuals and normal % probability plot of

bio decolourization of RTB (a) and RB5 (b) dyes.

tigate the effects of the three independent variables onthe response functions. The independent variables wereInitial concentration of dye (A), concentration of sucrose(B), and concentration of ammonium nitrate (C). The low,center and high levels of each variable are designated as–1, 0, and +1, respectively as illustrated in Table 2. Theseexperimental levels for each variable were selected basedon the literature values, available resources and resultsfrom preliminary experiments.428


Table 3. Biochemical characterization of isolated strain.S. No. Name of the test Observationa Pseudomonas sp.1 Colony morphology Complex irregular2 Shape Rod3 Grams stain –4 Spore staining +5 Methyl red +6 Voges proskauer –7 catalase +a+ Positive; – Negative.

The bacterial strain which is used in this study was iso-lated from textile contaminated site soil from real textile

effluent outlet. The results of Chemical and Biochemicalcharacterization of isolated strain, Pseudomonas sp. waspresented in Table 3.Table 4 depicts the experimental results for dye removal %(RTB and RB5) and using three factor 20 run CCD experi-mental designs. Each run was performed in triplicate andmean values for % removal of RTB and RB5 were presentedin Table 4, while the predicted values of responses wereobtained from quadratic model fitting techniques using thedesign expert (trial version) software.The experimental results were evaluated and approximatedthe function of biodecolourization in terms of percentagedye removal was obtained from the following equation: interms of coded factors:for RTB

%Removal(RTB) = + 58.67625− 0.15788 ∗ Co + 35.1825 ∗ CS + 9.97 ∗NS − 0.099 ∗ Co ∗ CS − 0.097∗ Co ∗NS − 6.25 ∗ CS ∗NS − 3.53125E − 04 ∗ C 2

o − 4.705 ∗ CS2 + 2.94 ∗NS2 (4)and for RB5:%Removal(RB5) = + 37.53182 + 0.69574 ∗ Co + 33.24568 ∗ CS + 26.43818 ∗NS − 0.071250 ∗ Co ∗ CS

− 0.05475 ∗ Co ∗NS − 2.25 ∗ CS ∗NS − 0.011146 ∗ C 2o − 6.19409 ∗ CS2 − 7.60909 ∗NS2 (5)

The results of analysis of variance (ANOVA) for Eqs. 4& 5 were shown in Table 5. The model of the equationwas significant at 1% level and each term was significantat 5% level. A coefficient of determination (R2) value of(R2: 0.9726; Adj R2: 0.9480 for RTB and R2: 0.9789;Adj R2: 0.9750 for RB5) showed that the equation washighly reliable, the model also revealed a statisticallyinsignificant lack of fit. The models were found to beadequate for prediction within the ranges of variables.The results showed that the experimental values weresignificantly in agreement with the calculated values andalso suggested that the models (Eqs. 4–5) were satisfactoryand accurate (Fig. 2).The three-dimensional response surface plots are thegraphical representations of the regression equation.These plots are presented in Figs. 3–5. Fig. 3(a) & (b)represents the response surface for RTB and RB5 deco-lorization respectively. In Fig. 3(a) & (b), the responsesurface and contour plots were developed as a functionof initial dye concentration and carbon source while thenitrogen source being kept constant at 1 gL−1 (centrallevel). The most effective decolorization of RTB and RB5were observed with 20 mgL−1 of initial dye concentration.

The contour plots demonstrate maximum percentage re-moval of RTB (Fig. 3(a)) and RB5 (Fig. 3(b)). Increasein dye concentration suppresses the growth of cells anddecolourization rate. Higher concentration may be toxic tobacterial growth [14, 15, 26, 27]. As shown in Figs. 3(a) &(b), the maximum decolorization of 96.88% (predicted valueof 98%) was observed in 20 mg/L concentration for RTBand maximum decolorization of 96.2% (predicted value of97%) was observed in 20 mg/L concentration for RB5. Thepercentage removal was increased with increase in glucoseconcentration by the keeping nitrogen source as optimalvalue. As it can be seen from plots 3(a)– 5(b), the initialconcentration slightly influenced the decolourization.It has been inferred that the dye alone could not be usedas the sole carbon and nitrogen source by the organism.Since the organism requires additional carbon and nitrogensources to co-metabolize the dye [14, 16, 18]. Surface plotsshow the increase on dye decolourization with increasingon carbon and nitrogen source. Plots 3(a)–5(b) clearlyshow that the decolourization by Pseudomonas sp. wassensitive to carbon and nitrogen sources.Many research attempts were made to replace glucose andyeast extract with other carbon sources (sucrose, peptone,429


Table 4. Experimental design and results of the Central composite design forthe decolourization of RTB and RB5.Factor 1 Factor 2 Factor 3 Response 1 Response 2Run A:InitialConcentration[mgL−1]

B:Carbonsource[gL−1]C:Nitrogensource[gL−1]

% RemovalRTB % RemovalRB51 100.00 1.00 1.00 59.92 36.382 60.00 1.00 1.00 73.86 70.493 60.00 1.00 1.00 73.86 70.494 60.00 1.00 1.00 73.86 70.495 60.00 0.00 1.00 56.65 52.586 80.00 1.50 1.50 66.65 62.587 80.00 1.50 0.50 69.86 55.718 20.00 1.00 1.00 96.88 96.29 40.00 0.50 1.50 75.38 75.0910 80.00 0.50 1.50 59.86 45.8811 40.00 0.50 0.50 68.46 68.7812 40.00 1.50 1.50 89.46 94.7813 60.00 2.00 1.00 83.86 83.1814 60.00 1.00 1.00 73.86 70.4915 80.00 0.50 0.50 53.49 37.6216 40.00 1.50 0.50 85.46 84.5817 60.00 1.00 1.00 70.36 70.4918 60.00 1.00 0.00 73.86 63.2119 60.00 1.00 1.00 73.86 70.4920 60.00 1.00 2.00 81.94 76.63

Table 5. Analysis of variance (ANOVA), results for decolourization of Congo red and Reactive Red195 by Pseudomonas sp.

Source Sum ofsquares Degrees ofFreedom Meansquare F Value P-value Prob>FModel 1 (RTB) 1895.45 9 210.61 39.51 < 0.0001 significantA-Initial Concentration 1004.89 1 1004.89 188.50 < 0.0001 significantB-Carbon source 737.94 1 737.94 138.43 < 0.0001 significantC-Nitrogen source 57.15 1 57.15 10.72 < 0.0004Residual 294.95 10 29.49 residualLack of Fit 294.95 5 58.99 significantPure Error 0.00 5 0.00Total 5556.29 19

R2: 0.9469; Adj R2: 0. 8991 Adeq Precision 21.259Model 2 (RB5) 5261.34 9 584.59 19.82 < 0.0001 significantInitial Concentration 3773.03 1 3773.03 127.92 < 0.0001 significantCarbon source 821.68 1 821.68 27.86 < 0.0004Nitrogen source 129.28 1 129.28 4.38 0.0628Residual 149.48 10 14.95Lack of Fit 145.50 5 29.10 36.59 0.0006 significantPure Error 3.98 5 0.80Total 4901.31 19

R2: 0.9695; Adj R2: 0. 9421 Adeq Precision 17.369lactose) and nitrogen sources (urea, ammonium chloride,ammonium nitrate, sodium nitrate). However, it was ob-served that none of the sources at tested concentrationscould match high decolorization rates displayed in thepresence of sucrose and ammonium nitrate. Hence, itwas envisaged that optimization of a suitable combination

and concentration of carbon and nitrogen sources couldprovide results at par with the sucrose and ammoniumnitrate. Therefore In the present study, the combination ofsucrose and ammonium nitrate has been used as carbonand nitrogen sources respectively. The preliminary studiessuggested that 0–2 gL−1 of sucrose and ammonium nitrate430


(a)

(b)Figure 2. The actual and predicted values for the biodecolourization

of RTB (a) and RB5 (b) dyes.

concentrations were the optimal value for complete removalof RTB and RB5.Sucrose was used as the carbon source and it has givenbest result of 96.88% at 1.0 gL−1 sucrose concentrationwas obtained for congo red at a concentration of 20 gL−1(Shown in Fig. 4(a)) where as 96.2% removal obtained forRB5 at 1.0 gL−1 sucrose concentration (Shown in Fig. 4(b)).Absence of carbon source has given the least amount ofremoval. Increasing concentration of sucrose increasesthe percentage decolourization (Table 3). From this ob-servation it has been inferred that the dye alone can’tbe used as a sole carbon source finding suggests thatdecolourization of RTB and RB5 by Pseudomonas requirescarbohydrate metabolism and its dependence. Dye removalincreased from 52.58% to 83.18% as the amount of sucrosewas increased from 0 g/L to 2 gL−1 at 1 gL−1 ammoniumnitrate and 20 gL−1 of RTB where as 52.58% to 83.18%

(a)

(b)Figure 3. Biodecolourization of dyes on 3-D graphics for response

surface optimization Initial dye concentration versus Car-bon source (a) RTB and (b) RB5.

was observed for RB5 concentration as is evident from therising ridge of the response surface curve along the axis forcarbon source. The similar kind of behavior was observedby many researchers [18, 26, 27].Effect of nitrogen source has been studied on decolour-ization of RTB and RB5. In this study the ammoniumsulfate was replaced by ammonium nitrate in the mineralsalt medium. This study reveals that among the differentinorganic nitrogen sources, ammonium salts were the bet-ter nitrogen sources for dye decolourization. Pseudomonassp. showed a maximum decolourization of 96.88% at anammonium salt concentration of 1.0 g/L (Fig. 5a) for RTBand for 96.2% decolourization for RB5 at an ammoniumsalt concentration of 1 g/L (Shown in Fig. 5b). Dye re-moval increased from 73.86% to 81.94% as the amount ofsucrose was increased from 0 g/L to 2 gL−1 of ammoniumnitrate at 1 gL−1 sucrose and 20 gL−1 of RTB where as70.49% to 76.63% was observed for RB5 concentration asan evident from the rising ridge of the response surface

431


(a)


surface optimization Initial dye concentration versus Nitro-gen source (a) RTB and (b) RB5.

curve along the axis for nitrogen source. Nachiyar andRajkumar [18] observed the similar finding of ammoniumsalts supporting decolourization of Navitan Fast Blue S5Rby Pseudomonas aeruginosa [11, 13–16, 18, 27, 28].The FTIR spectrum of the control dye compared with ex-tracted metabolites confirmed the degradation of Reactiveblack 5 and is shown in Fig. 6. As shown in Fig. 6,the difference in the FTIR spectrum of Reactive black5 and metabolites obtained after its decolorization re-sulted in biodegradation. The FTIR spectrum of Reactiveblack 5 showed specific peaks. This was supported by thepeaks at 672.91 cm−1 for –C=C–H: C–H bending alkynes,1637.38 cm−1 for –C=C– stretch alkenes and 3431.87 cm−1for O–H stretch, H-bonded alcohols, phenols. The FTIRspectrum of metabolites obtained after decolorization ofCongo red showed peaks at 674.84 cm−1 for C–Br stretchalkyl halides, 1228.79 cm−1 for C–N stretch aliphaticamines, 1366.49 cm−1 for C–H rock alkanes, 1421.64 cm−1for C–C stretch (in-ring) aromatics, 1637.52 cm−1 for C=O

(a)


surface optimization Carbon source versus Nitrogensource (a) RTB and (b) RB5.

Figure 6. FTIR analysis of Decolorization of RB5 by Pseudomonassp. before treatment (Black line) and its metabolites (Redline).

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stretch carboxylic acids, 3431.15 cm−1 for O–H stretch,H-bonded alcohols, and phenols O–H stretching vibrationsof primary amide [14, 16].4. ConclusionIsolated strain Pseudomonas sp. was able to decolorizethe Remazol turquoise blue and Reactive black 5. RSMstudies were showed that in the range studied, processof dye removal is affected significantly by the amount ofammonium nitrate which is a nitrogen source followed bysucrose, a carbon source. This study showed that responsesurface methodology was a suitable approach to optimizethe best growth medium conditions for achieving maximumdecolorization of the dye. Under optimized conditions,the experimental values agreed with the predicted ones,indicating the suitability of the model and the success ofRSM. UV-VIS and FT-IR spectra of RB5 solution duringbiological decolorization confirmed the biodegradation ofdye into intermediate products. The results indicate thatthis is a possible and potential application in the bioreme-diation of textile effluents contaminated with azo dyes andother toxic compounds.References

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response surface optimization for efficient dye removal by isolated strain pseudomonas sp

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