DECOLORIZATION OF TEXTILE WASTEWATER USING Marasmius sp. IN MODIFIED PACKED BED BIOREACTOR

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1 DECOLORIZATION OF TEXTILE WASTEWATER USING Marasmius sp. IN MODIFIED PACKED BED BIOREACTOR Guswandhi, a Panjaitan S.P.J, a Setiadi T., a Suhardi S.H b a Department of Chemical Engineering, b Centre for Life Sciences Faculty of Industrial Technology, Institut Teknologi Bandung Jl. Ganesa 10 Bandung, Indonesia tjandra@che.itb.ac.id ABSTRACT Textile industry produces wastewater that requires specific treatment because of its color composition. The objective of this research is to evaluate the performance of the culture used in degrading indigo dyes. The variable of this research is the immersion time between the fungus culture and dyes (15 minute and 30 minute) with 6 hours between each immersion; and the variety of dyes (indigo carmine and indigo bromine). The research is done using an Indonesian white rot fungi, Marasmius sp., immobilized in luffa medium. Through this research, it is shown that significant decolorization occurred after four days of treatment. Color analysis was done at indigo s maximum wavelength (λ = 661 nm). It showed an average absorbance decrease from 1,556 A to 0,701 A. Laccase assay showed a maximum activity of 44,17 U/L for indigo carmine treatment and 14,71 U/L for indigo bromine treatment. Overall, this research shows a promising potency for the treatment of textile waste water in a modified packed bed bioreactor using Marasmius sp. Key words : textile waste water treatment, white rot fungi, mycotreatment I. INTRODUCTION In Indonesia, textile industries grow with the rate of 0.85% per annum. This growth brings benefits as well as problems. Due to the increase of production, the wastewater produced also increases which in the end become potential pollutant to the environment. One of the problem in textile wastewater is its dye composition. In textile industry, dye is one of the main raw material and about 10-15% of the dye used can not be reused and has to be discharged. Dye in the wastes can cause problem to the society due to its characteristics. It can cause skin and eye irritation and also cancer. Another problem that can be caused by dye is mutagenic [1]. Chemical coagulation is frequently practiced in Indonesian textile industries and sometime it is the only mean to treat the wastewater. The main disadvantage of this method is that it produces a lot of sludge which need to be disposed of. Moreover, according to the current Indonesian Government Regulation, textile sludge may be classified as a hazardous waste, therefore the sludge should be treated in a proper way. This means that the sludge disposal causes a substantial increase in the cost of wastewater treatment Another alternative in treating the wastewater is to use microorganisms. Recently, there are a number of researchers done on the possibility of using white rot fungus to treat textile wastewater which has been proved to be superior to bacteria s [2, 3, 4, 5]. Böhmer et al. [5] reported that more than 90% dyes decolorization can be achieved by a white rot fungi culture in a modified packed bed bioreactor. In this bioreactor, textile wastewater was fed into the reactor contained a white rot fungi culture for some period called immersion time. The wastewater then discharged from the reactor, so that the culture had time to grow and produced more enzymes. After some period of time, wastewater was fed again into the reactor to have further treatment. In this study, an Indonesian white rot fungi, namely Marasmius sp. would be used to decolorize indigo dyes in the similar bioreactors The objective of this research is to study the effect of different immersion time (15 minute and 30 minute) to the performance of treatment system. Another objective of this research is to study the performance of Marasmius sp. in treating indigo carmine and indigo bromine and its stability to treat the dyes in four cycles Yogyakarta-Indonesia, 4-5 th December 20071

II. MATERIALS AND METHODS A. Operation conditions and bioreactor configuration An Indonesian white rot fungi namely Marasmius sp. was grown in potato dextrose agar.

Then, luffa was sterilized using an autoclave at 121 o C for 15 minute After the culture was ready, luffa was put into the bioreactor having a working volume of 5 L.

Between immersion, the culture and wastewater were separated for six hours. Each experiment was carried out in four cycles with every cycle lasted for eight days.

2 II. MATERIALS AND METHODS A. Operation conditions and bioreactor configuration An Indonesian white rot fungi namely Marasmius sp. was grown in potato dextrose agar. Then, it was inoculated into luffa as a growth support which had been added Kirk medium [6]. Luffa was cleaned and washed before being used. Then, luffa was sterilized using an autoclave at 121 o C for 15 minute After the culture was ready, luffa was put into the bioreactor having a working volume of 5 L. The wastewater contained 100 ppm of dyes either indigo carmine or indigo bromine. The immersion time was varied i.e. 15 minute and 30 minute. Between immersion, the culture and wastewater were separated for six hours. Each experiment was carried out in four cycles with every cycle lasted for eight days. In the end of every cycle, dye with the same amount was added. The addition of dyes was done three times. The diagram of the bioreactor system is given in Figure 1. Packed Bed bioreactor waste flows Waste container : flow : pump Fig 1. The diagram of the modified packed bed bioreactor system. B. Sample analysis The first sample was taken in the beginning of new cycle. The following samples were taken everyday after four times of immersion. Then, samples were analyzed for its color intensity, protein concentration, and laccase activity. Color intensity was analyzed using Ultrospec 3000 pro UV/Visible spectrofotometer. Protein concentration analysis was done according to Bradford [7]. In the laccase enzyme analysis, ABTS (2,2'-AZINO-bis [3-ethylbenziazoline-6-sulfonic acid]) was used as substrate to determine its activity [8]. III. RESULTS AND DISCUSSION A. Decolorization In each run, decolorization processes can be observed visually. Figure 2 shows dyes decolorization along with time (days). It was found that a significant decolorization occurred after four days of the process. Figures 3-6 show absorption spectra in visible wavelength ( nm). In the beginning of every cycle, a peak of absorbance in the wavelength of 661 nm was found. This peak decreased during the treatment and was not detected in the end of 8 days. This shows that the treatment of dyes using Marasmius sp. is able to cleave the benzene ring of the dye. (a) (b) (c) (d) Yogyakarta-Indonesia, 4-5 th December 20072

3 Fig 2. Samples of: (a) indigo carmine, 15 minutes immersion; (b) indigo carmine, 30 minutes immersion; (c) indigo bromine, 15 minutes immersion; and (d) indigo bromine, 30 minutes immersion. The sample on the first left was the original dyes, then followed by the following-day samples. Fig 3. Absorption spectra of dyes in visible wavelength for indigo carmine in the 15 minute immersion process: (a) initial dye; (b) eighth-day sample Fig 4. Absorption spectra of dyes in visible wavelength for indigo carmine in the 30 minute immersion process: (a) initial dye; (b) eighth-day sample Fig 5 Absorption spectra of dyes in visible wavelength for indigo bromine in the 15 minute immersion process: (a) initial dye; (b) eighth-day sample Fig 6 Absorption spectra of dyes in visible wavelength for indigo bromine in the 30 minute immersion process: (a) initial dye; (b) eighth-day sample B. Color Intensity Decrease Color intensity decrease was analyzed at the maximum wavelength of 661 nm. Figures 7 and 8 show the absorbance difference ( A) of indigo carmine and indigo bromine in every cycle, respectively. The absorbance difference was the difference between the absorbance value of initial dye and eighth-day sample. In Figure 7, it can be seen that the least degree of decolorization occurred in the first cycle, it might due to the fungi had not been acclimatized to the dyes. In the later cycles, the fungi had been adapted to the condition, therefore the better decolorization took place. A decrease in decolorization activity in the third and fourth cycles was observed (Figure 7). This shows a decrease in the activity of fungi in the system, it probably was caused by the nutrient depletion. It made the growth of the fungus hindered and decreased the performance of the system. It can also be seen in Figure 7 that the 15 minute immersion process gave better decolorization activities than the 30 minute one. It indicated that the longer immersion time was not suitable for the decolorization process. Fig 7. Absorbance difference of indigo carmine in every cycle ( : 15 minute immersion process; : 30 minute immersion process) Fig 8. Absorbance difference of indigo bromine in every cycle ( : 15 minute immersion process; : 30 minute immersion process) Yogyakarta-Indonesia, 4-5 th December 20073

4 Figure 8 shows absorbance difference of indigo bromine after being treated in the process. The result shows that at 15 minute immersion, the maximum decolorization occurred in the third cycle. This can be an indication that in the first cycle, the culture had not been adapted to the presence of wastewater. In the later cycles, fungi had been adapted and gave a greater decolorization. In the fourth cycle, there was a decrease in the decolorization performance. The reason for the decrease was probably due to the nutrient was depleting, the similar phenomenon was also observed in treating the indigo carmine. In the 30 minute immersion, the maximum decolorization occurred in the first cycle. The decolorization decreased in the second cycle, increased in the third cycle, and decreased again in the final cycle. Figure 8 also shows that the 30 minute immersion gave relatively more stable results. However, it always observed that there was a trend of decreasing occurred at the fourth cycle. C. Laccase Assay The decolorization of dyes by white rot fungi has been reported due to the extra cellular enzymes produced [4] and it has been studied that the laccase was the enzyme having the ability to degrade dyes [3]. The enzyme activity and absorbance value during indigo carmine and indigo bromine treatment along with time (days) is presented in Figure 9 and 10, respectively. It can be seen that the highest laccase activity in treating indigo carmine was U/L for 15 minute immersion process and U/L for 30 minute one (Figure 9). The highest laccase activity for treating indigo bromine was U/L for 15 minute immersion process and U/L for 30 minute one (Figure 10). In treating both of dyes, it shows that the 30 minute immersion could increase the laccase production. Moreover, it was also observed that the laccase activity in certain times was very low, and even it was not detected quantitatively. If the laccase activity was compared with the decrease of color intensity, there was no correlation observed. There were times when the decolorization occurred although the laccase activity was low. This was probably due to Marasmius sp. produced another extracellular enzymes beside laccase that also might degrade the dyes. Although the highest laccase activity was found in the 30 minute immersion process, the laccase production in the 15 minute one was more stable. In the 15 minute immersion process, the laccase was still present in a relatively high concentration until the end of the treatment. Meanwhile in the 30 minute one, the laccase sometimes could not be quantified, because of its low concentration. (a) (b) Fig 9. The absorbance value and enzyme activity for indigo carmine along with time (days): (a) 15 minute immersion process; (b) 30 minute immersion process ( : absorbance value; : enzyme activity) Yogyakarta-Indonesia, 4-5 th December 20074

5 (a) (b) Fig 10. The absorbance value and enzyme activity for indigo bromine along with time (days): (a) 15 minute immersion process; (b) 30 minute immersion process ( : absorbance value; : enzyme activity) D. Protein Analysis Protein analysis using Bradford assay resulted in the data of total protein concentration. Bradford assay detects all enzymes in the wastewater, both the active one and also deactivated one and this assay does not detect degraded proteins. Protein concentration data are shown in Figure 11. It shows that protein concentration increased in every cycles. The highest protein increase was observed in the second cycle. In the third and fourth cycle, there was also protein increase but not as much as the increase in the second cycle. This suggests that the highest protein production by Marasmius sp. occurred in the beginning of treatment. The rate of protein production decreased with time until it became relatively stable in the end of the fourth cycle. The decrease of protein concentration was probably caused by the degradation of protein at the end of cycle. Figures 12 and 13 show that there was no correlation between the protein concentration and laccase enzyme activity. It was observed that protein concentration tends to increase while enzyme activities did not follow that trend. This suggested that the laccase was not the only enzyme responsible for degradation processes of dyes. It has been reported that the process of decolorization using white rot fungi Marasmius sp. followed a consecutive reaction. Laccase was a biocatalyst that took part in decolorization processes. White rot fungi such as Marasmius sp. had been well known for the varied enzymes produced. One of the enzymes produced was the peroxidase enzyme group, such as lignin peroxidase, manganese peroxidase, H 2 O 2, and laccase. Beside this group, Marasmius sp. showed positive response to non spesific qualitative analysis of peroxidase using guaiacol as substrate [9]. (a) (b) Fig 11. Protein concentration in each cycle: (a) indigo carmine; (b) indigo bromine ( : 15 minute immersion process; : 30 minute immersion process) Fig 12. Protein concentration and laccase activity along with time: (a) indigo carmine, 15 minute immersion; (b) indigo carmine, 30 minute immersion ( : protein concentration; : laccase activity) Yogyakarta-Indonesia, 4-5 th December 20075

Fig 13. Protein concentration and laccase activity along with time: (a) indigo bromine, 15 minute immersion; (b) indigo bromine, 30 minute immersion ( : protein concentration; : laccase activity) E.

Besides studying the performance of decolorization by the living cultures, this study also studied the adsorption of dyes by luffa.

The color intensity was analyzed sphectrophotometrically at the maximum wavelength. Decrease in color intensity was calculated as a difference between initial and final absorbance of the samples ( A).

6 Fig 13. Protein concentration and laccase activity along with time: (a) indigo bromine, 15 minute immersion; (b) indigo bromine, 30 minute immersion ( : protein concentration; : laccase activity) E. Decolorization by Sterilized Cultures Decolorization of wastewater occurred not only because of enzyme activities but also because of an adsorption process by luffa. Besides studying the performance of decolorization by the living cultures, this study also studied the adsorption of dyes by luffa. The study was conducted with the same operating condition as the fungi culture condition. The only difference was that the luffa grown by Marasmius sp. had been sterilized. The color intensity was analyzed sphectrophotometrically at the maximum wavelength. Decrease in color intensity was calculated as a difference between initial and final absorbance of the samples ( A). The study showed the highest A of 0,612 A for indigo bromine and 0,758 A for indigo carmine. These values are sometimes higher that the absorbance difference in the research with living cultures. The reason of the high value was probably due to the difference in the permeability of cell wall. In the main research, the culture was alive, thus the fungi developed some impermeable cover on luffa resulting the adsorption of dyes is relatively low. In the dye adsorption study, luffa has been sterilized first therefore the cell wall was broken and the dyes could be easily adsorbed on the luffa. Nevertheless, a more detailed study is needed to find out the reason of the high value of adsorption by the sterilized culture. F. Scanning Electron Microscopy (SEM) The scanning electron microscopy of the luffa before, after inoculated with Marasmius sp. and at the end of fourth cycle show in Figure 14. From the pictures, it can be seen that Marasmius sp. was well immobilized in the luffa. In the end of fourth cycle, the quantity of Marasmius sp. still grew on the luffa had decreased a lot. This suggested that a number of Marasmius sp. was not strongly immobilized to luffa, therefore some of Marasmius sp. was washed out by the wastewater. (a) (b) (c) Fig 14. Scanning electron microscopy: (a) luffa before inoculated; (b) luffa after inoculated with Marasmius sp.; (c) luffa and Marasmius sp. at the end of fourth cycle. IV. CONCLUSION From this study, it can be summarized as follows: a. It was found that a significant decolorization occurred after four days of the process. b. The dye decolorization in 15 minute immersion processes gave a more stable result than those of 30 minute ones, moreover the 15 minute immersion process gave more stable enzyme production. c. The laccase assay showed a maximum activity of U/L for treating indigo carmine and U/L for indigo bromine, however no correlation was observed between the laccase activity and the decrease of color intensity. d. Marasmius sp. probably produced other extracellular enzymes, beside laccase, that could degrade the dyes. Yogyakarta-Indonesia, 4-5 th December 20076

7 e. Marasmius sp. degraded indigo carmine dyes better than that of indigo bromine. ACKNOWLEDGMENT This research was funded by collaborative research scheme between Centre for Life Science ITB and Research Group on Design and Development Chemical Engineering Product ITB on ligninolytic enzymes utilization for industrial processes. REFERENCES [1] Mathur, N., Bhatnagar P. and P. Bakre., Assessing Mutagenicity of Textile Dyes From Pali (Rajasthan) Using Ames Bioassay, Applied Ecology and Environmental Research 4(1), 2005, pp [2] Couto, R. S., Sanroman, M. A., Hofer, D. and Gübitz, G.M., Investigation of Several Bioreactor Configuration for Laccase Production by Trametes Versicolor Operating in Solid-State Conditions, Biochemical Engineering Journal, 15, 2003, pp [3] Couto, R. S., Sanroman, M. A., Hofer, D. and Gübitz, G.M., Production of Laccase by Trametes hirsute Grown in an Immersion Bioreactor and its Application in the Decolorization of Dyes from a Leather Factory, Eng Life Sci 4, No. 3, 2004, pp [4] Champagne, P. P. and Juliana A. R., Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor, Appl Microbiol Biotechnol 69, 2005, pp [5] Böhmer, U. Suhadi S. H.and Thomas B, Temporary Lift up Immersion of white rot fungi on palm oil fibre and pine wood chips to decolourize reactive textile dyes, Unpublished Report, T.U. Dresden, [6] Tien, M. and T.K.Kirk, Lignin peroxidase of Phanerochaete chrysosporium. In: Methods in Enzymology. W.A. Wood and S.T. Kellogg, eds. Academic Press. Inc. San Diego, Ca. Vol. 161B, 1988, pp [7] Bradford, M. M., (1976). A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Analytical Biochemistry, 72, 1976, pp [8] Bar, M., Kinetics and Physico-Chemical Properties Of White-Rot Fungal Laccases, Department of Microbiology and Biochemistry, University of the Free State, Bloemfontein, [9] Martinez et al., Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack lignin, International Microbiology, 8, 2005, pp Yogyakarta-Indonesia, 4-5 th December 20077

8 Yogyakarta-Indonesia, 4-5 th December 20078

Optimisation of Laccase Production using White Rot Fungi and Agriculture Wastes in Solid State Fermentation

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