Optimization and morphology for decolorization of reactive black 5 by Funalia trogii

Enzyme and Microbial Technology 40 (2007) 1758–1764

Optimization and morphology for decolorization of reactive black 5 by Funalia trogii Chulhwan Park a,b,∗ , Jung-Soo Lim c,d , Yuri Lee e , Byunghwan Lee f , Seung-Wook Kim c , Jinwon Lee g , Sangyong Kim a,∗∗ a

Green Engineering Team, Korea Institute of Industrial Technology (KITECH), Chonan 330-825, Republic of Korea b Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, Republic of Korea c Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Republic of Korea d Digital Appliance R&D Team, Samsung Electronics Co. Ltd., Suwon 443-742, Republic of Korea e R&D Team, Jeonbuk Bioindustry Development Institute, Jeonju 561-360, Republic of Korea f Department of Chemical System Engineering, Keimyung University, Daegu 704-701, Republic of Korea g Department of Chemical and Biomolecular Engineering, Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul 121-742, Republic of Korea Received 1 June 2006; received in revised form 10 November 2006; accepted 4 December 2006

Abstract The influence of the various carbon and nitrogen sources on the decolorization of reactive black 5 (RB 5) by Funalia trogii was examined using a classical method of medium optimization. Of the carbon sources examined as effective co-substrates, fructose, glycerol, and starch were found to be satisfactory, whereas lactose and sucrose were not. When ammonium tartrate, yeast extract, and peptone were used as nitrogen sources, the decolorization rate was high. An optimal pH is important for fungal growth, and our system operated best at a pH 5–6. By combining a Latin square design with a classical method of medium optimization, the essential medium components were identified using liquid culture experiments. Morphological changes of F. trogii were found to be significantly related to decolorization, and changes in fractal dimension followed RB 5-decolorizing enzyme activity. © 2006 Elsevier Inc. All rights reserved. Keywords: Latin square method; Funalia trogii; Reactive black 5; Optimization; Morphology; Fractal dimension

1. Introduction Synthetic dyes are extensively used in the textile dyeing, paper printing, color photography, pharmaceutical, food, cosmetic, and other industries. Approximately, 10,000 different dyes and pigments are used industrially, and over 0.7 million tons of synthetic dyes are produced annually worldwide [1]. Of various contaminated industrial wastewaters, those from the textile and dyestuff industries are among the most difficult to treat. This is because the dyes usually have a synthetic origin and complex aromatic molecular structures which make them ∗ Corresponding author at: Green Engineering Team, Korea Institute of Industrial Technology (KITECH), Chonan 330-825, Republic of Korea. Tel.: +82 41 589 8426; fax: +82 41 589 8580. ∗∗ Corresponding author. Tel.: +82 41 589 8356; fax: +82 41 589 8580. E-mail addresses: [email protected] (C. Park), [email protected] (S. Kim).

0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.12.005

more stable and more difficult to biodegrade [2,3]. Of those dyes that cause pollution problems, the azo dyes represent the largest class, and have the greatest variety of colors. A necessary criterion for the use of azo dyes is that they must be highly stable to light and during washing [4]. They must also be resistant to microbial attacks, and therefore, they are not readily degradable and are typically not removed by conventional wastewater treatment systems. Ligninolytic fungi are well known as decomposers of lignin and for their ability to degrade a wide variety of organopollutants [5,6]. Several studies have shown the degradation of azo, anthraquinone, heterocyclic, triphenylmethane, and polymeric dyes by Phanerochaete chrysosporium, the most extensively studied white-rot fungus [7]. Decolorization by fungi is dependent on several factors and, it is necessary to create an optimal environment for to ensure that fungi are fully competent to decolorize dyes. Fungi are usually grown in pure nutrient, without dyes or dye wastewater, to form a biosorbent living mass

C. Park et al. / Enzyme and Microbial Technology 40 (2007) 1758–1764

[8]. These media are predominantly composed of carbon and nitrogen sources and other nutrients. Filamentous fungi in submerged culture may grow in the form of either free mycelium or pellets [9–11]. The morphology of mycelial growth is influenced by many factors such as genetic factors [12], the size and nature of the inoculum [13], medium composition [14,15], physical culture conditions [16,17], and so on. Many studies have described the morphologies of industrially important filamentous microorganisms. Moreover, fractals also referred to as self-similarities, have been applied to describe many natural phenomena: the deposition of inorganic material, the shape of seashores, and the diffusion of liquid into a porous material [18]. Fractal geometry offers a new approach to the description of organism morphologies. In microbiological systems, fractals have previously been used to describe growth patterns and morphologies [19–21]. In this study, we investigated the effect of various environmental conditions, such as, pH, temperature, and media composition (carbon, nitrogen, and phosphate sources) and concentration on the decolorization efficiencies of reactive black 5 by Funalia trogii. In addition, the relationship between F. trogii morphology and decolorization was also investigated using fractal analysis. 2. Materials and methods 2.1. Fungal strains and culture conditions F. trogii ATCC 200800 was used in this study. Mycelia were raised and stored on 1.5% agar plates with PDA (potato dextrose agar) medium. For inoc-

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ulation, mycelia blocks were cut from a plate cultures. Briefly, 10 blocks were placed in 20 ml plates with glucose dextrin yeast peptone (GDYP) medium containing glucose, dextrin, yeast extract, peptone, KH2 PO4 , and MgSO4 , and incubated for 6 days at 28 ◦ C. After cultivation, the mycelia were harvested from plates, and homogenized at 2000 rpm for 30 s. In order to activate the strain, the mycelia were cultivated in the potato dextrose broth (PDB) for 3 days. Eight milliliters of the activated strain under suspension condition were used to inoculate each flask, which contained 80 ml of Kirk’s Basal Salts medium containing 5 g/l of glucose, 0.22 g/l of ammonium tartrate, 0.2 g/l of KH2 PO4 , 0.05 g/l of MgSO4 ·7H2 O, 0.01 g/l of CaCl2 , 0.001 g/l of thiamin·HCl, and 10 ml of trace element solution (details below), but it did not contain Tween solution or veratryl alcohol. Various carbon sources (fructose, dextrin, sucrose, glycerol, lactose, starch, and glucose) and nitrogen sources (NaNO3 , asparagines, tryptone, ammonium nitrate, peptone, yeast extract, and ammonium tartrate) were used to identify an optimal medium. Reactive black 5 (RB 5) were added to the flask, and the pH was adjusted to 4.5 using 0.1N NaOH and 0.1N HCl. The trace element solution contained (g/l of distilled water): CuSO4 ·7H2 O, 0.08; H2 MoO4 , 0.05; MnSO4 ·4H2 O, 0.07; ZnSO4 ·7H2 O, 0.043; Fe2 (SO4 )3 , 0.05. Culture flasks were shaken at 120 rpm in a shaking incubator for 3 days at 28 ◦ C under aerobic conditions. Dye concentration (from 150 mg/l to 400 mg/l) was changed to verify the change of decolorization efficiency with medium optimization because color is well removed at low dye concentration (150 mg/l).

2.2. Analytical methods Samples were withdrawn from the cultures at appropriate times and centrifuged (10,000 rpm, 15 min). Clear supernatants were analyzed for pH, glucose concentration, decolorization, and ligninolytic enzyme production. Dye decolorization was measured photometrically at the maximum absorbance wavelength for each dye using a UV/VIS spectrophotometer (Bio-Tek Instruments, Milano, Italy), and was calculated from the decreases of absorbance of maximum peak for the dye. Laccase (E.C. 1.10.3.2) activity was measured using 2,2 -azino bis(3ethylbenzthiazoline-6-sulphonic acid) (ABTS) in 0.1 M sodium acetate buffer

Fig. 1. Schematic diagram of the determination of fractal dimension using box counting method. Microscope image (a), image after processing (b), and fractal dimension determination in calculator (c).

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(pH 3.4) at 30 ◦ C. The oxidation of ABTS was used to determine enzyme activity, one unit of which was defined as the amount of enzyme require to reduce absorbance at 420 nm per min per ml of the culture filtrate [22,23].

Table 1 The effect of temperature on the decolorization of RB 5 (150 mg/l) at 96 h Temperature (◦ C)

Decolorization (%)

2.3. Image analysis for cell morphology

23 28 33 38

92 95 88 80

Cell morphology was studied on photomicrographs (Samwonscientific Ind. Co. Ltd., Korea) using Image Pro 3.0 software (Media Cybernetics, Silver Spring, MD, USA). Mean areas of pellets were measured automatically after being sorted and classified by the image analysis software.

2.4. Determination of fractal dimension The fractal dimensions of cells were determined using a box counting method, which was derived using the method described by Obert et al. [24]. Binary images of mycelium were edited to remove foreign particles and to correct optical errors. Mycelium images were covered by a square grid of length (L), and the number of boxes (N) containing mycelium were counted. The number of boxes overlapped by a mycelium image grows as the side length (L) of box is increased. For well-defined fractal subjects, the following equation should be satisfied: N(L) = αLD

(1)

where α and D are the proportionality constant and the fractal value of the subject, respectively. Eq. (1) is expressed in logarithmic form as: log N(L) = D log L + log α

(2)

For a well-defined fractal subject, logarithmic number of the overlapped boxes is linearly related to the logarithmic value of the side length of the box with a slope of fractal D. The fractal Dimension calculator (ver. 1.1, Bar-Ilan University, Israel) automatically counts the number of overlapped boxes and calculates fractal dimensions by linear regression. In the present study, more than 30 images were processed, and average fractal dimensions were calculated at different culture times. A schematic diagram of the process used to determine fractal dimension is shown in Fig. 1.

3. Results and discussion 3.1. The effect of temperature and initial pH on decolorization Different fungi have different optimal growth temperatures. The decolorization of RB 5 (150 mg/l) was conducted at different

temperatures (23 ◦ C, 28 ◦ C, 33 ◦ C, and 38 ◦ C). Decolorization activity was high at 28 ◦ C (95% color removal in 4 days) and was relatively lower at 33 ◦ C and 38 ◦ C (88% and 80% color removal in 4 days, respectively) suggesting that the optimum decolorization temperature is about 28 ◦ C (Table 1). The effect of pH on the decolorization process is important, since it was noted that some compounds responsible for the wastewater color are soluble over a certain basic pH ranges and insoluble at acid pHs [8]. In order to quantify these relations, experiments were carried out at different initial pHs in the range of 2.5–8.5, each value being adjusted using 0.1N HCl and 0.1N NaOH before autoclaving. The optimum pH for decolorization of RB 5 (150 mg/l) by F. trogii was pH 4.5–7.5 (Table 2). Best color elimination (96%) was achieved at an initial pH of 6.5, whereas cultures incubated at pH 2.5 and 3.5 showed only 60% and 80% decolorization, respectively. Fu and Viraraghavan [25] reported that the initial pH of the dye solution significantly influences the chemistry of both dye molecules and the fungal biomass. 3.2. The effect of carbon and nitrogen sources on decolorization The effects of various carbon sources on the decolorization of RB 5 (150 mg/l) by F. trogii were examined by substituting glucose in the basal medium with other carbon sources. As shown in Table 3, decolorizing activity was high when fructose, glycerol, starch, and glucose were used as carbon sources. Maximum decolorizing activity (over 90%) after 4 days was achieved using fructose, glycerol, and glucose. Zhang et al. [26] studied car-

Table 2 The effect of pH on the decolorization of RB 5 (150 mg/l) at 96 h Initial pH

Decolorization (%) Final pH

2.5

3.5

4.5

5.5

6.5

7.5

8.5

60 2.64

80 3.36

91 3.38

95 4.21

96 4.37

92 4.91

88 4.39

Table 3 The effect of various carbon and nitrogen sources on the decolorization of RB 5 (150 mg/l) at 96 h Carbon source

Decolorization (%)

Nitrogen source

Decolorization (%)

Fructose Dextrin Sucrose Glycerol Lactose Starch Glucose

93 82 84 93 82 89 91

NaNO3 Asparagine Tryptone Ammonium nitrate Peptone Yeast extract Ammonium tartrate

86 74 93 92 95 94 94

C. Park et al. / Enzyme and Microbial Technology 40 (2007) 1758–1764

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Table 4 Optimization of carbon, nitrogen, and phosphate sources for the decolorization of RB 5 (250 mg/l) using the Latin square method Ingredient type Fructose Glycerol Starch Ammonium tartrate Yeast extract Peptone KH2 PO4 K2 HPO4 Na2 HPO4 Decolorization (%)

M1 (%) 0.5

M2 (%) 0.5

M3 (%)

M4 (%)

M5 (%)

0.022

0.5

M8 (%)

M9 (%)

0.5 0.5 0.022

0.022 0.022

0.022 0.022

0.02

0.5

0.022 0.02

0.02 0.02 93

0.02 0.02

88

0.5

0.022 0.022 0.02

0.02

85

M7 (%)

0.5 0.5

90

M6 (%)

90

0.02 87

83

86

89

M8 (%)

M9 (%)

Table 5 Optimization of the concentrations of selected medium ingredients for the decolorization RB 5 (350 mg/l) using the Latin square method Ingredient type

M1 (%)

Fructose 0.5% Fructose 2.5% Fructose 1.5%

0.5

Peptone 0.088% Peptone 0.044% Peptone 0.022%

0.088

Na2 HPO4 0.03% Na2 HPO4 0.02% Na2 HPO4 0.04%

0.03

Decolorization (%)

M2 (%) 0.5

M3 (%)

M4 (%)

M5 (%)

M6 (%)

M7 (%)

0.5 2.5

2.5

2.5 1.5

0.088 0.044 0.022

96

0.022 0.022

0.022

0.03

93

0.03

0.02 0.04 93

0.02 0.04

94

bon sources as co-substrates during the decolorization of cotton bleaching effluent by an unidentified white-rot fungus and found that glucose, starch, maltose and cellobiose facilitated decolorization, whereas, sucrose, lactose, xylan, xylose, methanol, and glyoxal did not. In the present study, as a result of this experimentation and of medium optimization using Latin-square method, we selected three types of carbon sources (fructose, glycerol, and glucose). Nitrogen source in media is another essential factor for efficient decolorization by fungi. Both the nature and the con-

1.5

0.088 0.044

0.02

1.5

93

0.04 90

92

88

91

centration of the nitrogen source employed have been reported to be of considerable importance [13,27]. Thus, we assessed the effects of different nitrogen sources (NaNO3 , asparagines, tryptone, ammonium nitrate, peptone, yeast extract, and ammonium tartrate) on the decolorization of RB 5 (150 mg/l) by F. trogii. Most nitrogen sources, expect NaNO3 and asparagines, enabled over 90% of RB 5 decolorization. Eventually, we selected three types of nitrogen sources (peptone, yeast extract, and ammonium tartrate) for medium optimization using the Latin-square method.

Fig. 2. Decolorization (a) and relative enzyme activity (b) of RB 5 (400 mg/l) in basal medium and optimized medium by F. trogii ATCC 200800.

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3.3. Medium optimization for decolorization using the Latin-square method Conventional methods of medium optimization are timeconsuming, expensive and inaccurate, when interactions between different components are present. Therefore, statistical experimental designs were used to optimize the medium, and in particular, the Latin square method was used to determine carbon, nitrogen, and phosphate sources. The effects of the three different carbon sources (fructose, glycerol, and glucose), the three nitrogen sources (peptone, yeast extract, and ammonium tartrate), and the three phosphate salts (KH2 PO4 , K2 HPO4 ,

and NaHPO4 ) on the decolorization of RB 5 (250 mg/l) were studied in order to obtain optimal medium components. The decolorization of RB 5 in nine different mediums is shown in Table 4. Of the medium components tested, M3 (fructose, peptone, and NaHPO4 ) were found to be the best carbon, nitrogen, and phosphate salts sources, respectively. Maximum decolorization activity (93%) was achieved by M3 and minimum activity (83%) by M7. M1, M3, and M5 decolorized RB 5 dye solution by 90%, 93%, and 90%, respectively. Table 5 shows the effect of medium concentration on RB 5 (350 mg/l) decolorization using the Latin square method. RB 5 was decolorized by over 90% in the most mediums, expect M8. Of the various media examined,

Fig. 3. Morphological changes of F. trogii cultivated in basal (a) and optimized medium (b).

C. Park et al. / Enzyme and Microbial Technology 40 (2007) 1758–1764

M1 (5% fructose, 0.088% peptone, and 0.03% Na2 HPO4 ) was found to be the most effective at decolorizing of RB 5. Results from batch cultures using basal and optimum media are shown in Fig. 2. Experiments were performed at 28 ◦ C and 150 rpm for 8 days using a 400 mg/l dye concentration. As seen in Fig. 2(a), the decolorization activity rapidly increased and reached over almost 80% and 95% for basal and optimum media, respectively. From the results shown in Fig. 2(b), maximal laccase activity of the optimum medium was determined to be higher than that of the basal medium. Enzyme activities during cultivation with basal and optimum media were maximal after 7 days of cultivation. In optimal medium, enzyme activity increased dramatically between 4 days and 5 days. Maximal laccase activities of 202.8 U/ml and 48.1 U/ml were obtained at day 7 of cultivation with optimum and basal medium, respectively. Optimal medium produced a higher enzyme activity, which was about 4.2-fold greater than that of the control. 3.4. Relationship between the morphology of F. trogii and decolorization Pellet morphology has been reported to be one of the key factors to determine fermentation productivity [10,16,17]. To investigate the relationship between morphological changes and decolorization by F. trogii, the pellet morphologies of F. trogii was characterized under basal and optimized conditions (Fig. 3). In terms of the morphology of F. trogii cultivated in basal medium (Fig. 3(a)), pellet and clump predominated during fermentation. However, in the case of optimized medium, the pellets broke up during the early period of fermentation, and then differentiated into clump and free mycelia over the 7-day incubation (Fig. 3(b)). Fig. 4 shows changes in mean pellet area under basal and optimized conditions. In the case of basal medium, as pellet growth increased, mean area increased from 0.127 mm2 at day 1 to 0.802 mm2 at day 4, and thereafter, it decreased to 0.452 mm2 at day 8 when most pellets differentiated into clumps and free mycelia. However, in optimized medium, the mean area increased slightly from 0.215 mm2 at day 1 to 0.3 mm2 at day 3 then it decreased to 0.097 mm2 at day 8. It was

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Fig. 5. Variation in the fractal dimension of F. trogii cultured in basal and optimized medium.

difficult to characterize the morphological changes of F. trogii using morphological factors such as hairiness, circularity and perimeter, because the morphological changes of F. trogii were complex, i.e., they included pellets, clumps, and free mycelia. So, in this study, fractal analysis was used to more quantitatively define these morphological changes. As shown in Fig. 5, fractal dimensions increased as fermentation proceeded in basal and optimized medium. In basal medium, the fractal dimension gradually increased from 1.357 at day 1 to 1.741 at day 8. However, in optimized medium, a significant increase in fractal dimension was observed during days 4 and 5. From the above results, changes in fractal dimension appear to be related to decolorization efficiency. As comparing to result shown in Fig. 2, it was found that changes in fractal dimension resembled those enzyme activity. This result suggests that simple fractal analysis can predict the relationship between physiological function and morphological changes with a complex morphology, as has been described by several researchers [19–21]. Overall, based on our morphology study on F. trogii, we concluded that morphological changes and increase of decolorization efficiency were induced by medium optimization. Moreover, this phenomenon was successfully explained by morphological study and fractal analysis. 4. Conclusions

Fig. 4. Variation in mean pellet area in F. trogii cultured in basal and optimized medium.

F. trogii is an efficient fungal strain to produce an enzyme for decolorization, and laccase can be obtained in very high yield at the optimal condition. For more efficient decolorization, various factors have been considered, such as optimization of major medium ingredients, observation of fungal growth, increase of enzyme activity, investigation of decolorization rate, and so on. In addition, this study showed that morphological changes were induced by medium optimization and this phenomenon was successfully explained by morphology and fractal analysis. Further investigations should be focused on the immobilization of selected enzymes for industrial application of the enzymatic decolorization.

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Acknowledgments The authors would like to acknowledge the support by the National Research Laboratory Program of Korea Ministry of Science and Technology (MOST) and Korea Institute of Industrial Technology (KITECH).

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