Bioresource Technology 99 (2008) 8742–8747

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Bioresource Technology

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Modelling of Scenedesmus obliquus; function of nutrientswith modified Gompertz model

Abuzer Çelekli a,*, Muharrem Balcı b, Hüseyin Bozkurt c

a Department of Biology, Faculty of Art and Science, University of Gaziantep, 27310 Gaziantep, Turkiyeb Department of Biology, Faculty of Art and Science, University of Abant _Izzet Baysal, 14280 Bolu, Turkiyec Department of Food Engineering, Faculty of Engineering, University of Gaziantep, 27310 Gaziantep, Turkiye

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

Article history:Received 22 January 2008Received in revised form 9 April 2008Accepted 10 April 2008Available online 22 May 2008

Keywords:BiovolumeGompertz modelNutrientScenedesmus obliquus

0960-8524/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.biortech.2008.04.028

* Corresponding author. Tel.: +90 3423171925.E-mail addresses: celekli.a@gmail.com (A. Çelekli)

(M. Balcı), hbozkurt@gantep.edu.tr (H. Bozkurt).

This study attempted to investigate variation in biovolume of Scenedesmus obliquus, in the modified John-son medium at 20 ± 2 �C, under 16 kerg cm�2 s�1 continuous illumination. The experiments were carriedout at four nitrate (8, 12, 16, and 20 mM) and four phosphate (0.1, 0.3, 0.5 and 0.7 mM) concentrations atpH 7 and 8. The best response for algal growth was found at 0.3 mM phosphate and 12 mM nitrate at pH7, as it was obtained from weight averaging method. Besides, optimum phosphate and nitrate concentra-tions significantly distinguished (p < 0.01) from other concentrations according to Tukey’s HSD test. Keyfeatures of the growth of S. obliquus under phosphate and nitrate influenced batch culture was success-fully predicted by modified Gompertz model. Through the cultivations, specific growth rate (l) rangedfrom 0.30 to 1.02 day�1, while biovolume doubling time (td) varied from 0.68 to 2.30 days. There wereimportant differences (p < 0.05) for both l and td among response variables. Both nutrients displayednoteworthy effect (p < 0.01) on the algal biovolume.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Providing of food and feed supplies and treatment of wastewa-ter become important problems in the world with great increase inpopulation and industrial activities during recent decades. Thesesituations have prodded to seek new alternative sources and pro-vide new technologies such as algal technology to solve theseproblems. During the last decades, microalgal studies generallyhave been focused on two points of using algal biomass (Boro-witzka, 1999; Pulz and Gross, 2004) and the usages of algae biovo-lume to remove unwanted materials such as heavy metal(Rangsayatorna et al., 2004), textile dyes (Aksu and Dönmez,2006), and excess fertilizers (Voltolina et al., 2005) from wastewa-ter and its’ commercial products (Colla et al., 2007; de Morais andCosta, 2007). Furthermore, information about the morphologicaland physiological properties of algal species influenced byecological stressors (biotic and/or abiotic factors) and dynamic ofwater bodies have been sought by the researchers (Reynoldset al., 2002).

A green microalga of Scenedesmus obliquus (Chlorococcales) ischaracterized typically by two-dimensional arrangement of twoor more celled in regular aggregates called coenobia (Komárekand Fott, 1983; Soeder and Hegewald, 1992). Scenedesmus sp. is

ll rights reserved.

, balci.muharrem@gmail.com

one of the first cultured algae in vitro due to its rapid growth andability to handle (Trainor, 1998; Lürling, 2003). Additionally, spe-cies of the genus have been worldwide used for industrial purposebecause of the ease of cultivation and adaptation to the environ-mental conditions (Pulz and Gross, 2004; Li et al., 2005). S. obliquusis versatile organism for the use in domestic and industrial waste-water treatment (Voltolina et al., 2005). Thus, it has recently beenintroduced for the production of oxygen, removing of heavy metals(Martínez et al., 2000; Omar 2002) and conversion of waste prod-ucts into beneficial organic substances (Nuñez et al., 2001). The celldimension and cell number in coenobia varied in water bodieswith surrounded environmental conditions, i.e. nutrients, pH andtemperature (Trainor, 1993; Lürling, 2006; de Morais and Costa,2007). Each strain of S. obliquus with different biovolume capacitiesshows different response to combination of explanatory variables(Trainor, 1993; Lürling, 2006). Modification of the culture mediumis the fundamental factor to establish maximum biomass produc-tion and accumulation of desired products.

Modeling the growth of microorganisms is important to under-stand the behavior of organisms under different environmentalconditions such as temperature, light intensity, pH, and nutrients(Zwietering et al., 1990; Whiting, 1995). Models allow predictionof microbial development, optimization growth conditions andalso foretelling the microbial safety and the quality in differentenvironmental conditions. All the models have been remarkablysuccessful in describing growth data and the success of these orsimilar models in describing and explaining field data (Bozkurt

A. Çelekli et al. / Bioresource Technology 99 (2008) 8742–8747 8743

and Erkmen, 2001; Costa et al., 2002). Within the last decades sev-eral growth models have been proposed to describe growth ratesand certain compositional characteristics of microalgae under lightand nutrient limited conditions (Kiefer and Mitchell, 1983). Themicroalgae development in terms of growth and content is drivenby environmental stressors such as temperature, light intensity,pH, and nutrients. The growth of Spirulina platensis under nutrientsin batch culture was modelled by using response surface method-ology (Costa et al., 2002). Investigators have applied different lin-ear or non-linear models to describe growth curves and tosimplify measured data for stressors factors. A variety of non-linearregression models such as Gompertz, Richards, Stannard, Schnute,and Logistic have been proposed to describe the whole microbialgrowth curve (Zwietering et al., 1990; Whiting, 1995). One of thewidely used models is the modified Gompertz model which giveslag time, specific growth rate and maximum biovolume (stationaryphase) (Zwietering et al., 1990; Whiting, 1995). From differences intheoretical approaches, all the models predict relationship be-tween environmental variables and growth rate.

In this study, modified Gompertz model was applied to describethe growth of a green microalga S. obliquus at four phosphate andnitrate concentrations and at two different pH values. This studywas attempted to evaluate and understand the relationship be-tween predict variable (algal biovolume) and response factors(nutrients; phosphate and nitrate concentrations) at pH 7 and 8in batch culture.

2. Methods

2.1. Microorganism and experimental design

The experiments were performed with a green microalga S. obli-quus (Çelekli and Balcı, 2006) inoculated on Johnson’s medium(Johnson et al., 1968). The culture was incubated at 20 ± 2 �C, atpH 7, and under 16 kerg cm�2 s�1 continuous illumination. ThepH (Hanna, pH 211, microprocessor pH meter) of the growth med-ium was adjusted by dilute HCl (0.01 M) and concentrated NaOH(1 M) solutions.

A series of experiments in the batch culture were carried outwith 150 ml of medium in 250 ml Erlenmeyer flasks for 30 days.Algal developments were postulated by changes in the phosphate(0.1, 0.3, 0.5 and 0.7 mM KH2PO4) and nitrate (8, 12, 16 and20 mM NaNO3) concentrations. Acclimated algal culture(5 � 105 cell mL�1) was transferred to cultivations to evaluate theinfluence of variables. Experiments were carried out in triplicatewith control Johnson’s medium without the alga.

Throughout cultivations, algal growth was monitored by count-ing cell numbers in a counting chamber (Thoma hemocytometer,0.1 mm deep) under Olympus BX51 model light microscope. Thealgal biovolume was estimated from cell density and measure-ments of cell volume taken the approximating geometric shapesof cells (Rott, 1981). Dimensions (width, length, and diameter aslm) of the least 25 individuals were measured and mean cell vol-ume was calculated. The microscopic ocular scale bar was cali-brated carefully using a standard scale bar before measuring(Rott, 1981).

2.2. Modelling

The non-linear modified Gompertz model given in Eq. (1) wasfitted to experimental data for the growth of S. obliquus (Zwieteringet al., 1990; Whiting, 1995). The fitting procedure was performedusing commercial computer software SigmaPlot version 6.0 (JandelScientific, San Francisco, USA) via the Marquardt–Levenbergalgorithm

ln ðBiovolumeÞ ¼ A � exp � expl � expð1Þ

Aðk� tÞ þ 1

� �� �ð1Þ

where l is the specific growth rate (day�1), k is the lag phase (day),A is the maximum biovolume (ln biovolume mm3 L�1) and t is thetime (day). In order to evaluate the goodness of fit, the predicteddata obtained using Eq. (1) were plotted against the experimentaldata. Regression coefficients (r2) and sum of squares (SSQ) betweenpredicted and experimental data were calculated.

Doubling time (td) was calculated by using

td ¼ ðln 2=lÞ ð2Þ

where td is the doubling time (day) and l is specific growth rate(day�1).

Non-regression analysis was carried out to include the effect ofboth time and concentration in a single equation (Tyrer et al.,2004):

ln ðBiovolumeÞ

¼ ðaþ b � CÞ � exp � expfðc þ d � CÞ � expð1Þg

ðaþ b � CÞ

��

� ½ðeþ f � CÞ � t� þ 1Þ� ð3Þ

where biovolume value is in mm3 L�1; C is the concentration (eitherphosphate or nitrate); a, b, c, e and f are the constants and t is thetime (day). Eq. (3) was fitted to the experiment data in order toevaluate the goodness of fit. The predicted data obtained using Eq.(3) were plotted against the experimental data to calculate linearregression coefficients (r2) and sum of square. The regression coef-ficient indicated that Eq. (3) was well fitted to the experimentaldata (r2 > 0.79).

2.3. Statistical analysis

Analysis of variance (ANOVA) was performed for biovolume val-ues as function of time, nutrient concentrations (either PO4 or NO3)and pH to determine significant differences (p < 0.05) by usingSPSS (version 15.0; SPSS Inc., Chicago, IL, USA). Calculated biologi-cal parameters (l, k, A, and td) from Eq. (1) among factors were alsocompared using ANOVA. Tukey’s honestly significant difference(HSD) multiple range test was also carried out. The CALIBRATE pro-gram was used for weighted average (WA) regression to estimateoptimum and tolerance of biovolume values in the algae (Jugginsand ter Braak, 1992).

3. Results and discussion

The morphological properties of isolated species addressed to S.obliquus by organization of coenobia, dimension and shape of cell(Komárek and Fott, 1983; Soeder and Hegewald, 1992). The speciesshowed 2, 4 and 8 celled colonies linearly or alternately in coeno-bia containing parietal chloroplast with pyrenoid. The cell dimen-sions varied 6–8 lm in width, 9–12 lm in length, and 2.5–3.0 lm indiameter during cultivations.

With regard to morphology of the species, the treatment popu-lation exhibited different number celled coenobia throughout thegrowth phases. Population comprised mainly unicelled individualespecially in the lag phase, while formations of four-celled coeno-bia and eight-celled coenobia were found in the exponentialgrowth and stationary phases. These findings were also confirmedin the coenobia of the same species during growth (Lürling, 2003).However, Lürling (2003) keynoted that four-celled coenobia wereespecially found throughout the entire experiment. Moreover, Ver-schoor et al. (2004) noticed that change in environmental condi-tions caused variation in the formation of coenobia of strainsbelonging to S. obliquus. In the present study, the morphological re-sponses of S. obliquus in terms of cell volume and cell number of

Fig. 1b. Variation in the biovolume of Scenedemus obliquus at pH 8 as a function ofcultivation time for phosphate gradient. Symbols represent experimental (Exp.)data and lines represent the fitted line (Predicted data; Pred.) by Eq. (1).

Fig. 2a. Influence of nitrate concentrations on the biovolume of Scenedemus obliq-uus at pH 7. Symbols represent experimental (Exp.) data and lines represent the

8744 A. Çelekli et al. / Bioresource Technology 99 (2008) 8742–8747

coenobia were closely related with both nutrient gradients andpopulation density. Plasticity of the strain changed in the treat-ment populations from initial lag phase to steady state phase ofcultivation. Investigated strain exhibited variations in the cell vol-ume values in different conditions. These variations were in goodagreement with previous studies (Agustí and Kalff, 1989; Lürling,2006). Strong linear correlation was found between density andcell volume (density = 0.242 � cell volume � 17.2 with r2 = 0.91)under optimum environmental conditions. Ecological stressors(biotic and/or abiotic factors) regulate morphological and physio-logical properties of phytoplankton species, which are importantto understand the dynamic of water bodies (Reynolds et al., 2002).

Effects of phosphate concentrations on the algal biovolume areshown in Figs. 1a and 1b. It was found that the effect of phosphateconcentration on the biovolume was found to be significant(p < 0.01, F = 14.33). Tukey’s HSD test showed that biovolume ob-tained from S. obliquus was the highest (p < 0.01) at 0.3 mM phos-phate concentration. This result was in agreement with Sanchoet al. (1997). Besides, WA regression indicated that the species pre-ferred relatively low phosphate concentration such as0.32 ± 0.1 mM. Remaining three phosphate (0.1, 0.5, and 0.7 mM)groups obviously had biovolume (p > 0.05) according to Tukey’sHSD test. Despite the difference in biovolume value between bothpH values, there was no significant difference (p > 0.05) in the algaldevelopment. Having wide tolerance range for phosphate gradienton reaching considerable algal biovolume provides advantages tothis species which can be used to remove excess nutrients, heavymetals, and textile dyes from wastewaters is and also importantin natural ecological system (Reynolds et al., 2002; Rangsayatornaet al., 2004; Voltolina et al., 2005; Aksu and Dönmez, 2006).

Effect of nitrate concentrations on the biovolume is shown inFigs. 2a and 2b. The species was able to display remarkableimprovement at the worked nitrate gradients. Nitrate valuesplayed a noteworthy role (p < 0.01) on the variation of biovolume.However, the species displayed similar growth curves (p > 0.05)between two pH values. Indeed, Tukey’s HSD test significantly dis-tinguished (p < 0.01) optimum group containing 12 mM nitratefrom remaining three nitrate groups, whereas no remarkable dif-ferences were observed for biovolume values between two pHs.Differed from S. platensis (Colla et al., 2007), the green alga requiredlower nitrate value to reach maximum biovolume level. As growthpattern, different from Dunaliella viridis (Jiménez and Niell, 1991),S. obliquus preferred higher nitrate value, which also caused to ap-

Fig. 1a. Variation in the biovolume of Scenedemus obliquus at pH 7 as a function ofcultivation time for phosphate gradient. Symbols represent experimental (Exp.)data and lines represent the fitted line (Predicted data; Pred.) by Eq. (1).

fitted line (Predicted data; Pred.) by Eq. (1).

pear discernable growth of Haematococcus pluvialis (Fàbregas et al.,2000) and a marine alga Chroomonas sp. (Bermúdez et al., 2004).

With regard to the consequence of the study, the major biovo-lume as 2208.9 mm3 L�1 was obtained in the Johnson medium con-taining 0.3 mM phosphate and 12 mM nitrate concentrations at pH7. Substantial cell number of S. obliquus achieved at high nitrateand low phosphate concentrations in semi-continuous culture(Nuñez et al., 2001; Voltolina et al., 2005). Plasticity of the algawas affected by temperature (Martínez et al., 2000), nutrient avail-ability (Holtmann and Hegewald, 1986) and illuminance (Sanchoet al., 1997). Previous studies (Martínez et al., 2000; Nuñez et al.,2001; Hodaifa et al., 2008) also noticed that S. obliquus had poten-tial to nitrogen and phosphorous removal from artificial wastewa-ter. High biovolume value can be used to the treatment ofindustry’s pollutants in terms of removal of heavy metals, textiledyes, pesticides.

The Gompertz model was used to describe biovolume value ofthe strain in the modified Johnson’s medium including the afore-mentioned nutrient conditions. Validation of the model waschecked by using the linear regression coefficient (r2) between

Table 2Predicted parameters (a, b, c, d, e and f) obtained from secondary model (Eq. (3)),linear regression coefficient (r2) and sum of square values (SSQ) for Scenedesmusobliquus

Parameters PO4 NO3

pH 7 pH 8 pH 7 pH 8

a 7.5033 7.2133 9.1819 10.3123b �3.8497 �1.4788 �0.1764 �0.2405c 0.1824 0.1783 0.0187 �0.1056d 1.2113 0.6650 0.0336 0.0386e �7.0557 �9.6988 �11.6538 �14.9745f 9.6219 12.8317 0.5344 0.7056r2 0.79 0.90 0.87 0.91SSQ 23.39 12.83 16.59 10.95

Fig. 2b. Influence of nitrate concentrations on the biovolume of Scenedemus obli-quus at pH 8. Symbols represent experimental (Exp.) data and lines represent thefitted line (Predicted data; Pred.) by Eq. (1).

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the predicted data from the modified Gompertz model and theexperimental data. The high correlation coefficients (r2 > 0.92)indicated that modified Gompertz model fitted well to the experi-ment data and it could be regarded as sufficient to describe thegrowth of S. obliquus in the medium.

Biological parameters, i.e specific growth rate (l), lag time (k),and maximum biovolume value (A) were calculated by using themodified Gompertz model from Eq. (1) and their results are givenin Table 1. The maximum biovolume (A) varied from 4.90–7.21 to5.83–7.21 ln biovolume (mm3 L�1) for phosphate and nitrate,respectively. The best result for A levels were obtained with con-centrations of 0.3 mM phosphate and 12 mM nitrate at pH 7. Sta-tistical analysis indicated that changing pH of the medium didnot change maximum biovolume (p > 0.05). The steady state timefor S. obliquus was reached around 30 days, in agreement with pre-vious study (Kim et al., 2007). The findings indicated that the mod-ified model was good enough (r2 > 0.92) to describe relationshipbetween predict variables (A, l, k, and td) and response factors(phosphate, nitrate, and pH).

Through the cultivations, the l and td for phosphate concentra-tion ranged 0.37–1.02 day�1 and 0.68–1.87 days�1, respectively.The l with 0.32–0.81 day�1 and td with 0.85–2.3 days were foundfrom the growth curve at nitrate value. Both nutrient concentra-tions and pH value of the medium significantly affected (p < 0.05)

Table 1Predicted specific growth rate (l, day-1), lag phase (k, day), maximum biovolume (A, ln mmprimary model (Eq. (1)) of biovolume for Scenedesmus obliquus

(mM) A l k

pH 7 pH 8 pH 7 pH 8 pH 7

PO4

0.1 5.42a, A 5.78a, A 1.02a, A 0.41a,b, B �1.09a

0.3 7.21b, A 7.04a, A 0.81a,b, A 0.58c, B �1.84b

0.5 5.07a, A 6.39a, A 0.69b, A 0.37b, B �1.83b

0.7 4.90a, A 6.34a, A 0.88a,b, A 0.54a,c, B �1.21a

NO3

8 6.27a, A 6.30a, A 0.52a, A 0.30a, B �2.77a

12 7.21a, A 7.04a, A 0.81b, A 0.58b, B �1.84b

16 6.58a, A 6.43a, A 0.39a, A 0.59b, B �3.96c

20 5.83a, A 6.02a, A 0.45a, A 0.32a, B �3.42a

Different lower-case letters indicate statistical difference at a = 0.05 level in each columDifferent capital letters indicate a statistical difference at a = 0.05 level among pH valueValues with the same letters in the same parameters indicate that the values did not di

l and td values. Moreover, Tukey’s HSD test importantly distin-guished optimum phosphate value (0.3 mM) from other threephosphate concentrations at pH 8. Slightly higher l levels of S. obli-quus were obtained from the cultivations than in previous studiesfor same species (Sancho et al., 1997; Martínez et al., 2000). Be-sides, the species exhibited higher specific growth rate and lowerdoubling time compared with other algae such as H. pluvialis(Kaewpintong et al., 2007) and S. platensis (Radmann et al.,2007). Moreover, monitored alga exhibited lower growth rate thanthat in the previous study concerning same species cultured in Chu10 medium (Lürling, 2006), whereas it is higher than that in MCmedium (de Morais and Costa, 2007). This could be due to conse-quence of species having different response to different environ-mental conditions. Algal biovolume was closely associated andsurrounded by a variety of environmental variables such as tem-perature (Trainor, 1993; Martínez et al., 2000), light (Sanchoet al., 1997; Çelekli and Dönmez, 2006), nutrient availability (Lür-ling, 2006; de Morais and Costa, 2007), and zooplankton grazing(Trainor, 1998; Lürling 2003).

Negative lag phases, k (varied 1.09–5.12), were obtained for thealga in modified Gompertz model. It was concluded that accli-mated algal culture was transferred to the medium, which causedeasy adaptation to environmental conditions. Indeed, Hodaifa et al.(2008) denoted that the growth curve of S. obliquus showed no lagphases for all the experiments. The first phase was exponentialgrowth during cultivations. Besides, Masson et al. (2002) noticedthat the negative signs of the lag time refer to no adaptation formicrobial growth at environmental conditions. Both nutrientsattributed significant differences k among response variables re-vealed by statistical analysis (see Table 1). Moreover, there was

3/L), doubling time (td, day) and linear regression coefficient (r2) obtained from the

td r2

pH 8 pH 7 pH 8 pH 7 pH 8

, A �4.15a,b,B 0.68a, A 1.71a,b, B 0.95 0.95, A �3.08a,B 0.85a,b, A 1.19c, B 0.94 0.92, A �4.59b,B 1.00b, A 1.87b, B 0.93 0.94, A �1.83c,B 0.78a,b, A 1.28a,c, B 0.98 0.99

, A �5.01a, B 1.34a, A 2.30a, B 0.97 0.93, A �3.08b, B 0.85a,b, A 1.19b, B 0.94 0.92, A �2.08b, B 1.79c, A 1.17b, B 0.94 0.98,c, A �5.12a, B 1.55a,c, A 2.16a, B 0.92 0.94

n.s at each concentration.ffer by the Tukey test at 0.95 confidence interval.

8746 A. Çelekli et al. / Bioresource Technology 99 (2008) 8742–8747

remarkable differences k between pH 7 and 8 within each nutrientconcentration.

The regression coefficients (r2) and sum of square generated byusing Eq. (3) are given in Table 2. The model of fitting is indicatedin Figs. 3a, 3b, 4a and 4b, which revealed the growth curves of thespecies during cultivations. The model successfully described theeffects of phosphate and nitrate concentrations on the growthcurve of the microalga at both optimum pH. The model expressedthe sigmoidal phosphate and nitrate values dependent of specificgrowth rate at relatively low phosphate concentration (0.3 mM)

Fig. 3a. Changes in the biovolume of Scenedemus obliquus at pH 7 as a function ofcultivation time for phosphate gradient [mesh plot represents the fitted line by Eq.(3)].

Fig. 3b. Changes in the biovolume of Scenedemus obliquus at pH 8 as a function ofcultivation time for phosphate gradient [mesh plot represents the fitted line by Eq.(3)].

and high nitrate level (12 mM) at pH 7. Besides, the Gompetz mod-els usually give good fits data for the growth curve of organisms(Zwietering et al., 1990) such as growth of Pseudomonas fluorescens(Masson et al., 2002) and inactivation of Yersinia enterocolitica(Bozkurt and Erkmen, 2001).

The results obtained for constants in Eq. (3) are given in Table 2.Relatively high correlation coefficients (r2 > 0.79) show that thegrowth of S. obliquus can be modeled using Eq. (3) within the lim-its of this study (0.1–0.7 mM phosphate, 8.0–20.0 mM nitrate, andpH 7 and 8).

Fig. 4a. Changes in the biovolume of Scenedemus obliquus at pH 7 as a function ofcultivation time for nitrate gradient [mesh plot represents the fitted line by Eq. (3)].

Fig. 4b. Changes in the biovolume of Scenedemus obliquus at pH 8 as a function ofcultivation time for nitrate gradient [mesh plot represents the fitted line by Eq. (3)].

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4. Conclusion

S. obliquus was adaptable to low level of phosphate and high ni-trate value at circum-neutral pH. Both nutrients showed significanteffect on the species biovolume. Cell volume and cell number ofcoenobia changed from lag phase to stationary phase. Growthcurve of S. obliquus at both optimum pH was successfully describedby using the modified Gompertz model under the effects of phos-phate and nitrate concentrations. This species has a potential to bebiomass source for different biotechnological aims. Understandingof the optimum conditions and unique culture combination to ob-tain highest production capacity of algae comes into questionincreasingly.

Acknowledgements

We wish to thank Aziz Deveci (University of Abant _Izzet Baysal)for his help in laboratory studies. Also thanks to University ofAbant Izzet Baysal for financial supports and Scientific ResearchProjects Executive Council of University of Gaziantep.

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