RESULT AND DISCUSSION 1 Research Results

Bogor, 21-22 October 2015 263 Where : P = Rainfall Q = Streamflow I = Interception T = Transpiration E = Evaporation It = Infiltrationdeep leakage ΔS = Change in soil water storage Because of in the annual water balance, the values of It and ΔS are zero, while the value of ET is the sum of T and E values, wich are parts of the Interception I. Therefore, the equation will be: P = Q + ET 5 3. RESULT AND DISCUSSION 3.1 Research Results 3.1.1 Annual Rainfall Based on the rainfall data over the last 15 years period 1997-2011, in the area of study in PT. Arara Abadi, Perawang has an average rainfall of 2,361 mm.year -1 , with the number of rain days an average of 148 days.year -1 . The annual rainfall is quite volatile, with the wettest year occurred in 2008 3,127 mm and the driest occurred in 1997 1,748 mm. The distribution of average of monthly rainfall Figure 2. shows the rainfall occurs throughout the year, and not clearly differentiated between the dry and rainy seasons. That is because the climate in area of study including Riau Province is the humid tropics. The average of monthly rainfall is 196.0 mm. The driest month occurred in February and July, and the wettest occurred in March and October. The precipitation has large temporal variations both annual and monthly, characterized by the standard deviation is quite large 375 mm. Although in the study area has relatively flat topography 95.61 of the area is on a slope of 0-8, but it has a spatial variation of rainfall due to the nature of convective rain tropical convective rainfall. Figure 2: Monthly rainfall and standard deviation at research location for period of 1997-2011 100 200 300 400 500 600 700 Jan Feb Mar Apr Mei Jun Jul Agt Sep Okt Nop Des R a in fa ll m m Month Anual rainfall: 2,361 mm Standard deviation: 375 mm Bogor, 21-22 October 2015 264 3.1.2 Interception Losses The rainfall interception losses, throughfall and stemflow at each of the plantation ages are presented in Table 2. In age of 2 and 3 years are observed during 5 months, by 45 and 40 rain events respectively. Whereas in age of 4, 5 and 6 years old, it was observed for 4 months, by 43, 46 and 44 rain events respectively. Table 2: Rainfall interseption, throughfall and stemflow at each of plantation age Plantation Age year Ranifall mm n Throughfall mm Stemflow mm Interseption mm 2 1,054 45 847.4 80.4 39.3 3.7 167.4 15.9 3 1,008 40 783.6 77.7 36.1 3.6 188.3 18.7 4 797 43 633.9 79.5 32.5 4.1 130.6 16.4 5 824 46 684.6 83.1 32.5 3.9 106.9 13.0 6 765 44 634.6 83.0 28.3 3.7 102.1 13.3 Remark: n = number of rainfall event 3.1.3 Water and Sediment Yield The recapitulation of calculation results on the water and sediment yields for 5 years of observation are presented in Table 3. Table 3: Water and sediment yields at E. Pellita forest plantations Parameters Years 2008 2009 2010 2011 2012 Plantation age year 2 3 4 5 0-1 Rainfall mm 2,813.0 2,679.0 2,783.0 1,663.0 2,219.0 Water yield mm 1,467.6 1,245.9 948.7 474.6 1,134.1 Sediment yield ton. ha -1 5.06 3.49 2.59 1.42 14.66 Remark: The E. pellita plantations were clear-cutted on March 2012 Based on the table above, the magnitude of the water and sediment yields declined from 2008 to 2011. These fact was because of both the decreasing anual rainfall, and increasing age of E. Pellita plantations. The older age of the plantations form a forests community are increasingly tightly, especially start from the age of 2 or 3 years after the activities of weeding were done. The declining of water yield is also allegedly because the older the plantations age the higher value of evapotranspiration water consumption. 3.1.4 Evapotranspiration anda water balances The annual water balance in E. Pellita plantation forest for period of 2008 to 2012 is presented in Table 4. Table 4: Water balances of micro catchment at E. Pellita forest plantation Parameters Years 2008 2009 2010 2011 2012 Plantation age year 2 3 4 5 0-1 Rainfall mm 2,813.0 2,679.0 2,783.0 1,663.0 2,219.0 Total runoff mm 1,467.6 1,245.9 948.7 474.6 1,134.1 Evapotranspiration, ET mm 1,345.4 1,433.1 1,834.3 1,188.4 1,084.9 ET to rainfall 47.8 53.5 65.9 71.5 48.9 Bogor, 21-22 October 2015 265 The table above shows that the water use by plantation forest that reflected by the value of ET increased from 2008 to 2010, along with the increasing age of the plant plant age 2 to 4 years. Then, the value of ET decreased in 2011 due to decreasing annual rainfall, and then again decreased in 2012 because of clear-cutting activities. 3.2 Discussion The Table 2 shows that the amount of water lost through interception interception losses at E. pellita plantation forest of 2 to 6 years old ranged from 13.0 to 18.7 of the rainfall, or an average of 15.8. Compared with other species of Eucalyptus, the value is slightly varied. Pudjiharta 1999 mentions that the interception of E. urophylla is in a similar range 8.8 to 17.3, whereas in E. signata indicate larger numbers, namely 22 Lima, 1976 in Pudjiharta , 2001. Likewise, the amount of stemflow and throughfall of the E. pellita are in the similar range of numbers to other species of Eucalyptus. During the period of E. pellita plantation age of 2 to 5 years old, the average of annual water yield is 1034.2 mm, or approximately 40.3 of the rainfall numbers. The average of sediment yield is small with an average of 3.14 tonnes.ha -1 .yr -1 . This sediment yield is allegedly mostly from the open sourced at the right-left stream called stream bank erosion. Erosion rate on forest land is relatively small because of the rapid growth of shrubsweeds that cover the surface of the ground sooner. Although the sediment yield is small, but if compared with the sediment yields in natural forests is still far greater. Fritsch and Sarrailh 1986 in Bruijnzeel 1997 reported the sediment yield from the rain forest in France was very small 0.05 to 0.75 tonnes.ha -1 .yr -1 . Likewise, the research by Malmer 1990 in Bruijnzeel 1997 in the rainforest of Malaysia also indicates the small rates are 0.05 to 0.2 tonnes.ha -1 .yr -1 , while the study by Douglas 1967 in Bruijnzeel 1997 shows the almost equal average rate of 0.25 tonnes.ha -1 .yr -1 . The E. pellita trees were harvested in March 2012, then land preparation in April, and new plantation for next rotation in May. Thus, starting April to December 2012 can be regarded as a phase of plant age 0 to less than one year. The sediment yield in 2012 showed a considerable increasing significantly. It shows that logging operation can increase the sediment yield in water bodies due to the opening of vegetation cover on soil K Brown, 2010; Khanal and Parajuli, 2013. Based on Table 4, it shows that the water use by E. pellita plantation forest the value of ET increased from 2008 to 2010 along with the increasing age of the trees age of 2 to 4 years. The water use by E. pellita plantation increased follow sigmoid growth curve on the first step when plantation has increased, especially in age of 3 years Samraj et al., 1998, and then tend to be stable after the plantation reaches the age of 8 years Bosch and Hewlett, 1982; Bren et al., 2010. The study by Robert et al. 2001 concluded the same results, by comparing the tree transpiration of E. sieberi in age of 14, 45 and 160 years old. The results showed that tree transpiration decreased of 2.2 mm.day -1 , 1.4 mm.day -1 and 0.8 mm.day -1 , respectively. Buckley et al. 2012 also said that the water use of E. delegatensis R. Baker Alphine ash in regrowth phase are more than twice as much more than 460 ± 100 mm.day -1 than mature plants more than 7 years. In 2011, the rate of ET decreased affected by a drastic reduction in rainfall compared to previous years. The influence of rainfall on the ET is through the provision of water in the root zone as soil moisture as one of the factors affected the value of ET. The precipitation rate in 2011 decreased far below compared the annual average value in the last 15 years 2,300 Bogor, 21-22 October 2015 266 mm. The rate of ET in 2011 was 1,188.4 mm, or 71.5 of the annual rainfall. The percentage value is greater than the value of evapotranspiration that occurred in 2010 65.9. In 2012, during the 0-1 years old of plantation showed the small value of ET 48.9 of rainfall, but the total runoff increased in large number 1,134.1 mm. This indicates that logging activities by reduction of forest cover in the watershed area has led to an increase in the river flow Zhao et al., 2009, Sørensen et al., 2009; Nóbrega et al ,. 2010; Zegre 2011; Khanal and Parajuli, 2013; Shamsuddin et al., 2014. Based on these results, in general, within a cycles of plantation 6 years, showed that the water consumption water use of E. pellita plantation in Perawang-Riau was in an average of 59.7 47.8 - 71.5. The magnitude indicates that the amount of water use by plantations is still below of the rainfall numbers. Ecologically, the annual water balance shows that the condition of the research area in Perawang-Riau is in a safe condition. However, from the aspect of the water and sediment yields, it is need to be aware especially at the phases of post-logging, land preparation and new plantation, due to the increased both river flow and sediment yield significantly. 4. CONCLUSION From the results and discussion above, it can be conclude that the water use of E. pellita plantation is high enough i.e: 1,188 – 1,834 mm.year -1 , but it is still below the average of the annual rainfall, so that the potential of water deficit drought can still be avoided for Riau Regions. The loss of water from the ecosystem both through the rainfall interception or runoff are less enough. The interception rate is around 13.3 – 18.7 of rainfall, whereas runoff rate is around 1,663 – 2,813 mm.year -1 . The average of annual sediment yield is as low as 3.14 ton.ha -1 .year -1 . The critical growth phase prone to hydrological hazards was identified at post-logging until 1 year old plants phase. So that, in these phases, there are required the soil and water conservation practices. REFERENCES Al-Kaisi, Broner, I. 1998. Crop water use and growth stages. Crops Series No. 4.715. Colorado State University-Press. USA. www.colostate.eduDeptsCoopExt. Accessed on 21 Mei 2001. Bosch, J.M., Hewlett, J.D. 1982. A review of Catchment Experiments to Determine the Effect of Vegetation Changes on Water Yields and Evapotranspiration. J. Hydrol., 55, 3- 23. Bren, L., Lane, P., Hepworth, G. 2010. Longer-term water use of native eucalyptus forest after logging and regeneration: The Coranderrk experiment. Journal of Hydrology, 384, 52- 64. Brown, K. 2010. Effectiveness of Forestry Best Management Practices in Minimizing Harvesting Impacts on Streamflow and Sediment Loading in Low-Gradient Headwaters of The Gulf Coastal Plain. Master Thesis. The School of Renewable Natural Resources. Louisiana State University. Bruijnzeel, L.A. 1997. Hydrology of forest plantations in the tropics. In Nambiar, E.K.S. and A.G. Brown Eds., Management of Soil, Nutrient and Water in Tropical Plantation Forest. pp.125-167. ACIAR Monograph No. 43. Canberra, Australia. Bruijnzeel, L.A. 2004. Hydrologycal fuctions of tropical forests: Not seeing the soil for the trees?. Agriculture, Ecosystems and Environment, 104, 185-228. Bogor, 21-22 October 2015 267 Buckley, T.N., Turnbull, T.L., Pfautsch, S., Gharun, M., Adams, M.A. 2012. Differences in water use between mature and post-fire regrowth stands of subalpine Eucalyptus delegatensis R. Baker. Forest Ecology and Management, 270, 1 –10. Calder, I.R. 1992. Water use of Eucalyptus: a review with special reference to South India. Agricultural water management, 11, 333-402. Colin, N., Reynolds, B., Neal, M., Wickham, H., Hill, L., Williams, B. 2004. The water quality of stream draining a plantayion forest on Gley Soils: The Nant Tanllwyth, Plynlimon Mid-Wales. Hydrology and Earth System Science, 83, 485-502. Feller, M.C. 1981. Water balances in Eucalyptus regnans, E. obliqua and P. radiata forests in Victoria. Australian Forestry, 44, 153-161. Khanal, S., Parajuli, P.B. 2013. Evaluating the impacts of forest clear cutting on water and sediment yields using SWAT in Mississippi. Journal of Water Resource and Protection, 5, 474- 483. http:dx.doi.org10.4236jwarp.2013.54047 Lima, W.D.P., Zakia, M.J.B., Libardi, P.L., Filho, A.P.D. 1990. Comparative evapotranspiration of Eucalyptus, Pine and natural τCerrado” vegetation measure by the soil water balance method. IPEF International 1, Piracicaba, 5-11. Ministry of Forestry MoF. 2004. Keputusan Menteri Kehutanan No. 101Menhut-II2004 Tentang Percepatan Pembangunan Hutan Tanaman Untuk Pemenuhan Bahan Baku Industri Pulp dan Kertas. Murtiono, U.H., Supangat, A.B., Susanti, P.D., Sulasmiko, E., Sugianto, A. 2012. Kajian Erosi dan Neraca Air pada Berbagai Jenis Vegetasi sebagai Dasar Pemodelan Tata Air. Laporan Hasil Penelitian. Balai Penelitian Teknologi Kehutanan Pengelolaan DAS. Badan Litbang Kehutanan. Surakarta. unpublished. Nóbrega, R.S., Souza, E.P., Sousa, F.A.S. 2010. The impacts of changes in land cover on water resources in The Western Amazon. Journal Of Environmental Hydrology, 18, http:www.hydroweb.com Pudjiharta, Ag. 2001. Pengaruh hutan tanaman industri Eucalyptus terhadap tata air di Jawa Barat. Jurnal Puslitbang Konservasi Alam, Tahun 2001. Bogor. Roberts, S., Vertessy, R., Grayson, R. 2001. Transpiration from Eucalyptus sieberi L. Johnson forests of different age. Forest Ecology and Management, 143, 153 –161. Seyhan, E. 1977. Fundamentals of hydrology. in Indonesian by. S. Subagyo. 1993. Dasar- dasar hidrologi Edisi cetakan kedua, 380 pp., Gajah Mada Univ. Press. Yogyakarta. Shamsuddin,S.A., Yusop, Z., Noguchi, S. 2014. Influence of plantation establishment on discharge characteristics in a small catchment of tropical forest. International Journal of Forestry Research, Article ID 408409, 1-10. http:dx.doi.org10.11552014408409 Smith, M.K. 1974. Throughfall, stemflow and interception in Pine and Eucalypt Forest. Australian Forestry, 36, 190-197. Sørensen, R., Ring, E., Meili, M., Högbom, L., Seibert, J., Grabs, T., Laudon, H., Bishop, K. 2009. Forest harvest increases runoff most during low flows in two boreal streams. Ambio, 387, 357-363. Supangat, AB., Junaedi, A., Kosasih Nasrun. 2008. Kajian tata air hutan Acacia mangium dan Eucalyptus pellita. Laporan Hasil Penelitian. Balai Penelitian Hutan Penghasil Serat. Badan Litbang Kehutanan. Kuok. unpublished. Supangat, AB., Junaedi, A., Kosasih Irwan. 2011. Dampak penanaman jenis penghasil kayu pulp terhadap tata dan kualitas air. Laporan Hasil Penelitian. Balai Penelitian Hutan Penghasil Serat. Badan Litbang Kehutanan. Kuok. unpublished. Waterloo, M.J. 1994. Water and nutrient dynamics of Pinus caribaea plantation forests on former grassland soils in Viti levu, Fiji. PhD dissertation. Vrije Universiteit van Amsterdam, Amsterdam, the Netherlands. Bogor, 21-22 October 2015 268 Zhao, F.F., Zhang, L., Xu, Z.X. 2009. Effects of vegetation cover change on streamflow at a range of spatial scales. Proceedings 18 th World IMACS MODSIM Congress, Cairns, Australia 13-17 July 2009. http:mssanz.org.aumodsim09 Zegre, N. 2011. Evaluating the hydrologic effects of forest harvesting and regrowth using a simple rainfall-runoff model. Journal of Environmental Hydrology, 19, 1-10. Bogor, 21-22 October 2015 269 PAPER C5 - Double Layer Immobilized Manganese Peroxidase from Pleurotus ostreatus Effectively Enhanced the Biodecolorization of Remazol Brilliant Blue R Dye Dede Heri Yuli Yanto 1 , Sanro Tachibana 2 1 Research Center for Biomaterials, Indonesian Institute of Sciences LIPI Jl. Raya Bogor Km. 46 Cibinong 16911, Bogor, Indonesia. 2 Department of Applied Bioscience, Faculty of Agriculture, Ehime University 3-5-7 Tarumi, Matsuyama 7908566, Ehime, Japan Corresponding Email: dedebiomaterial.lipi.go.id ABSTRACT The ability of white rot fungi to degrade lignin using relatively non-specific extracellular enzyme has encouraged the use of these fungi in decolorization processes of highly colored textile dye wastewaters. In this study, a manganese peroxidase MnP enzyme isolated from Pleurotus ostreatus was applied to decolorize Remazol Brilliant Blue R RBBR, as a model of textile dye, using an immobilization form in Ca-alginate beads. The result showed that single layer immobilization of MnP decolorized only 32 of RBBR 100 ppm. However, double layer immobilized enzymes that placed the MnP in the inner layer of the bead and Mn 2+ , Tween80, and H 2 O 2 as mediators in outer layer effectively enhanced the biodecolorization 2- fold compare than the single layer immobilization. MnP activity affected the decolorization yield of the RBBR with 4.5 U of MnP could decolorized the RBBR 70 in 10 h. In addition, application of the immobilized enzyme in bioreactor system improved the decolorization rate. This study is important for the further application of enzyme immobilization for decolorization of textile dye effluents. Keywords: Decolorization, immobilized enzymes, Manganese peroxidase Pleurotus ostreatus, textile dyes. 1. INTRODUCTION The ability of white rot fungi to degrade lignin using relatively non-specific extracellular enzyme makes them one of the most important microorganism that have been used for the bioremediation of recalcitrant compounds and decolorization of highly colored wastewaters. Khan et al. 2013. In addition, ligninolytic fungus easily degraded most xenobiotic compounds contained in effluents Dhanjal et al. 2013; Hadibarata et al. 2012. Decolorization of dyes strongly correlates with their chemical structure, which is due to their resistance to decolorization Hsueh et al. 2009. RBBR, an anthracene derivative, represants an important class of often toxic and recalcitrant organopollutants, it structurally resembles certain polycyclic aromatic compounds which are substrates of ligninolytic peroxidases. RBBR was decolorized more easily than azo dyes because of their chemical structure. Enzyme immobilization technology is an effective means to make enzyme reusable and to improve its stability Fágáin, 2003. According to the known reports, several different types of supporters were applied to immobilize enzyme, including activated carbon, controlled porosity glass, and chitosan microspheres Jiang et al. 2005, Wang et al 2008 reported the beads with immobilized laccase containing Na-Alginate, gelatin, and PEG were at the concentration of 2.0, 2.0, and 0.5 mv, respectively, had preferable decoloring capability after cross- Bogor, 21-22 October 2015 270 linking with 0.6 glutaraldehyde, and the removal percentage of Reactive Red B-3BF RRB exhibited about 50 even during the tenth decolorization cycles. Ligninolytic cultures of certain white rot fungi have been shown to produce ligninolytic enzymes such as manganese peroxidase MnP, lignin peroxidase LiP and laccase. These all enzyme has been reported to decolorized dyes. However, many studies showed that MnP should have contributed more to decolorization than other enzymes such as LiP and laccase Moldes et al. 2003; Champagne and Ramsay, 2005. Pleourotus ostreatus PL1 produces extracellular MnP under solid state fermentation in wood meals. Therefore, in this study, decolorization of RBBR dye by double layer immobilized enzyme from P. ostreatus PL1 using Ca-alginate as a supporter, Mn 2+ , tween 80 and H 2 O 2 implicated to the beads was conducted due to be the enzyme protection from excessive H 2 O 2 involved during decolorization. 2. EXPERIMENTAL METHOD 2.1. Chemicals and Microorganism RBBR dye was used as a substrate and was provided by Sigma St. Louis, USA. Chemical structure of RBBR is shown in Figure 1. Tween 80, manganese II sulfate, hydrogen peroxide, calcium chloride, ammonium sulfate, and sodium alginate were purchased from Wako Pure Chemical Industries, Ltd. Japan. P. ostreatus PL1 used in this study was supplied from culture collection of Department of Applied Bioscience, Faculty of Agriculture, Ehime University, Japan and has been reported as petroleum hydrocarbons degrader fungi Yanto and Tachibana, 2014. Figure 1: Chemical structure of Remazol Brilliant Blue R dye. 2.2. Crude enzyme extraction Crude enzyme from isolate P. ostreatus PL1 was obtained based on methods described in our previous study Yanto et al. 2014. The fungus was pre-grown in wood meals 1 kg containing glucose 100 g and shitake nutrient 150 g with final water content 60 for 1 month. Crude enzyme was extracted from the pre-grown fungus using homogenizer 10,000 rpm for 10 minutes with malonate buffer pH 4.5. After filtration, filtrate was subjected to centrifuge at 8,000 rpm, 4 o C for 20 minutes. Supernatant was collected and placed in container. Ammonium sulfate 75 was added into the container, stirred for 1 hour then centrifuged at 8,000 rpm, 4 o C for 20 minutes. Pellet was collected and diluted in malonate buffer while stirred. Crude enzyme powder was obtained after freeze-drying for 3 days. Enzymatic analysis showed that the crude enzyme mainly consisting manganese peroxidase MnP activity 0.5 ug crude enzyme and neither laccase nor lignin peroxidase activities were detected. 2.3. Experimental design In the first set of experiment, decolorization of RBBR was conducted by using two types of immobilized MnP in a bioreactor system. The first type is a single layer immobilized MnP Bogor, 21-22 October 2015 271 containing a mixture of Mn 2+ 1 mM, H 2 O 2 1 mM, and Tween 80 1 in the beads. The second is a double layer immobilized MnP containing a mixture of Mn 2+ 1 mM, H 2 O 2 1 mM, and Tween 80 1 placed in the second layer of the beads. The difference of single and double layer immobilized MnP was described in Figure 2. Second, experiment was conducted to understand the effect of reaction medium using water and malonate buffer solution. Third, efficiency decolorization of RBBR was observed by applying different activities of MnP in the beads 1.5, 3.0, and 4.5 U. Immobilization of MnP in the beads was preapared by mixing the crude enzyme from P. ostreatus PL1 and sodium alginate solution 1.5 wv. Sodium alginate 1.5 wv was added to the crude enzyme solution and stirred until homogeny. The mixture was dropped into the calcium chloride solution 0.1 M and stirred until the formation of calcium alginate bead was complete. The beads were washed with distilled water until they were free of chloride ions and were then stored in a refrigerator at 4 o C for one day before being used for the decolorization study. Decolorization study was conducted using the bioreactor system MasterFlex ® LS ® Cole-Parmer Instrument Company with two easy- loaders model 7518-10 and tubing size no. 16. MnP beads were placed into the bioreactor column Ø = 2.5 cm, h = 10 cm, v = 49 cm 3 and the dye-simulating textile effluent 100 mg L -1 was flowed from an Erlenmeyer flask 100 mL into the beads at a flow rate of 1.5 mLminute. The experimental design of the vertical bioreactor system is shown in Figure 3. Figure 2: Immobilized MnP beads used in this study. Single 1 and double 2 layer immobilized MnP RBBR decolorization was determined by monitoring the decrease in absorbance at the wavelength of the maximum absorbance max = 595 nm. The absorbance of the dye solution was monitored for 3, 6, 9, 12, 24, 48, and 72-hour reactions in bioreactor with a UV-Vis spectrophotometer Shimadzu UV-1600. The decolorization efficiency R, was calculated as Equation 1. R = 1-A final A initial x 100 1 where A initial is the initial absorbance and A final is the observed absorbance. 3. RESULTS AND DISCUSSION Production of crude enzyme from P. ostreatus PL1 was first conducted by preparing pre-grown PL1 in wood meal. PL1 grew fast and completely cover all the surface of wood meal until the inner part in one month incubation. After crude enzyme extraction and enzymatic activities determination, it is found that only manganese peroxidase MnP activity was detected in the crude enzyme but no laccase and lignin peroxidase were detected. Our previous study showed MnP MnP H 2 O 2 Tween 80 Mn 2+ Mn 2+ H 2 O 2 Tween 80 1 2 Bogor, 21-22 October 2015 272 that fungus P. ostreatus PL1 has the capability to decolorize RBBR in malt extract agar MEA and malt extract liquid medium. However, the use of crude enzyme in particular by using immobilization technique is needed to conduct in order to understand enzyme play role in decolorization of RBBR or other dyes. Immobilization by using Ca-alginate as supporter has been conducted in our previous study and showed high efficiency biodecolorization for some azo dyes using bioreactor system Yanto et al. 2014. In this study, different type of immobilized MnP was conducted by using single and double layer beads. Figure 3: Bioreactor system used in this study Figure 4: Effect of immobilization type of MnP in the beads on decolorization of RBBR using bioreactor system As shown in Figure 4, single layer immobilization of MnP decolorized only 32 of RBBR 100 ppm. However, double layer immobilized enzymes that placed the MnP in the inner layer of the bead and Mn 2+ , Tween 80, and H 2 O 2 as mediators in outer layer effectively 10 20 30 40 50 60 70 80 20 40 60 80 D ec ol or iz at ion Time hour Double Single Control of single layer Control of double layer Description: 1. RBBR sample 2. Precision pump tubing no.16 3. Immobilize packed column 4. Pump with two easy-loaders 1 2 4 3 Bogor, 21-22 October 2015 273 enhanced the biodecolorization 2-fold 66 compare than the single layer immobilization. Comparing with the control both single and double layer, immobilized MnP showed high efficiency to decolorize RBBR significantly different indicating decolorization occurred via enzymatic reaction. In addition by observing the colour of the beads after reaction which is no RBBR dye remain in the bead corroborated this finding. This study found that interaction of MnP directly with manganese II sulfate, hydrogen peroxide and Tween 80 affected the activity of enzyme. However, double layer immobilized enzyme protected the MnP damage from interaction with peroxide and keep the enzyme in the inner layer without any leak out from the bead. Therefore double layer beads were more preferable to apply for decolorization of dyes using bioreactor system. Biodecolorization efficiency by immobilized enzymes can be influenced by the medium used in bioreactor. In this study, the use of water and malonate buffer were applied to know the effect of medium in biodecolorization of RBBR. Figure 5 shows that malonate buffer more preferable to use as medium in decolorization than distilled water and double layer immobilized bead shows high efficiency for biodecolorization than single layer. As already reported by several study that MnP activity was optimum in pH 4.5, it can be understand that distilled water which is pH around 7 can affect the biodecolorization efficiency by enzyme. Figure 5: Effect of reaction medium on decolorization of RBBR by immobilized MnP in bioreactor system Figure 6: Effect of MnP activity on decolorization of RBBR using bioreactor system 10 20 30 40 50 60 70 80 Control single Single Control double Double Control Single Single Control double Double W at er B u ff er Decolorization 20 40 60 80 100 20 40 60 80 D ec ol or iz at io n Time hour 4.5 U 3.0 U 1.5 U Control Bogor, 21-22 October 2015 274 As shown in Figure 6, MnP activity affected the biodecolorization of RBBR using bioreactor system. The higher activity of the MnP used for decolorization the higher biodecolorization of the RBBR. MnP activity 4.5 U could decolorized 70 RBBR in 10 h. The role of MnP in decolorization of some dyes has been reported by Svobodova et al. 2006 that significant MnP was detected in high efficiency decolorization of some dyes by Irpex lacteus. 4. CONCLUSION The study summarized that double layer immobilization of MnP which that placed the MnP in the inner layer and Mn 2+ , Tween 80, and H 2 O 2 as mediators in outer layer effectively enhanced the biodecolorization 2-fold compare than the single layer immobilization in biodecolorization of RBBR. MnP activity affected the decolorization yield of the RBBR with 4.5 U of MnP could decolorized the RBBR 70 in 10 h. In addition, application of the immobilized enzyme in bioreactor system improved the decolorization rate. This study is important for the further application of enzyme immobilization for decolorization of textile dye effluents. REFERENCES Champagne, P.P., Ramsay, J.A. 2005. Contribution of manganese peroxidase and laccase to dye decolorization by Trametes versicolor. Applied Microbiology and Biotechnology, 693, 276 –285. Dhanjal, N.I.K., Mittu, B., Chauhan, A., Gupta, S. 2013. Biodegradation of textile dyes using fungal isolates. Journal of Environmental Science and Technology, 62, 99 –105. Fágáin, C.O. 2003. Enzyme stabilization – recent experimental progress. Enzyme Microbiology and Technology, 33, 137 –149. Hadibarata, T., Yusoff, A.R.M., Kristanti, R.A. 2012. Decolorization and metabolism of anthraquionone-type dye by laccase of white-rot fungi Polyporus sp. S133. Water Air Soil Pollution, 223, 933 –941. Hsueh, C.C., Chen, B.Y., Yen, C.Y. 2009. Understanding effects of chemical structure on azo dye decolorization characteristics by Aeromonas hydrophila. Journal of Hazardous Materials, 167, 995 –1001. Jiang, D.S., Long, S.Y., Huang, J., Xiao, H.Y., Zhou, J.Y. 2005. Immobilization of Pycnoporus sanguineus laccase on magnetic chitosan microspheres. Biochemical Engineering Journal, 25, 15 –23. Khan, R., Bhawana, P., Fulekar, M.H. 2013. 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Biodecolorization of textile dyes by immobilized enzymes in a vertical bioreactor system. Procedia Environmental Sciences, 20, 235 –244. Bogor, 21-22 October 2015 276 PAPER C6 - Biodecolorization of Textile Dye by Isolated Tropical Fungi Maulida Oktaviani 1 , Dede Heri Yuli Yanto 1 1 Research Center for Biomaterials, LIPI, Jl. Raya Bogor Km 46, Cibinong, Bogor 16911. Corresponding Email: maulida.oktaviani26gmail.com ABSTRACT Extensive research on fungi with enormous secretion of ligninolytic enzyme has been continously growing. In this first report, the ability of isolated tropical fungi to decolorize the dye remazol brilliant blue R RBBR and also the ligninolytic enzyme activity were investigated. The screening result showed that 2 isolates of fungi collected from Cibinong area, Biom3 and Eco3, formed clear areas on solid media containing RBBR pH 4.5 when grow for 4 days. Decolorization zone produced by Biom3 and Eco3 were 80 mm and 68.5 mm, respectively. Therefore, the fungi isolates were tested for their ability to degrade RBBR 100 ppm in liquid media and compared with D7 strain collection of RC Biomaterials, LIPI. The results showed that the decolorization by Biom3 and D7 after 3 days of incubation were 23.4 and 71.7, respectively. The decolorization increased up to 48.0 and 97.4¸ respectively, when culture media was added by glucose. The addition of glucose as carbon source was important for growth of the fungi and influenced the decolorization process of RBBR. Enzyme assays showed no LiP was detected in both culture medium of Biom3 and D7. Laccase was not produced by Biom3 isolate, comparing to D7 that produced 158±41 Ul of laccase during biodecolorization. MnP as high as 50.2 Ul and 351.4 Ul was detected in culture medium of Biom3 and D7. The addition of 0.1 mM Cu 2+ increased efficiency of decolorization and enzyme activity. Keywords: biodecolorization; ligninolytic enzyme; remazol brilliant blue R; tropical fungi 1. INTRODUCTION In the recent years, the main issue developed with the growth of textile industries is increasing in the use of synthetic dyes and that consequences of environmental pollution due to release of the wastewater into the river during manufacturing and usage Hu et. al., 2009. The discharge of these effluent into the environment in large quantities results reduced dissolve oxygen DO concentration and killing aerobic organism Chander Arora, 2007. Therefore, removing dyes from industrial wastewater is very important before discharge of wastewater into the environment Ayed et. al., 2011. There are various methods for textile wastewater treatment for the removal of dye by physical, chemical, and biological approach. However, physical and chemical treatment are usually inefficient in removing dye, costly, and little adaptable to a wide range of dye wastewater. Biological treatment involve degradation of the dye by microorganism, such as bacteria, fungi and algae, that they reduce the effect of contaminants. Decolorization by microorganism have been focussed by many researchers because the capability of microorganism in decolorizing a wide range of dyes Fu Viraraghavan, 2001; Kaushik Malik, 2009. Fungi, especially white rot fungi basidiomycetes, have been reported to degrade various xenobiotic compounds and dyes due to production of extracellular oxidases including laccase, Mn peroxidase MnP, and lignin peroxidase LiP. Therefore, the utilization and extensive Bogor, 21-22 October 2015 277 research of fungi and their enzymes can be a prospective solution for the treatment of industrial dye containing effluents Wesenberg et. al., 2003. Indonesia as a tropical country has high biodiversity of microorganism, including fungi. However, until now, only limited information of the Indonesian fungi that have capability on dye decolorization. In addition, the development of textile industries in Indonesia grow rapidly and dye has not included as one textile industry effluent that must be regulated Setiadi et. al., 1999. Thus, gaining knowledge and extensive research about the potential ability of tropical Indonesian fungi to degrade various dye effluent should be conducted. This paper presents a study in screening of the degrading textile dye ability of fungi isolated from Cibinong, Bogor, Indonesia. 2. EXPERIMENTAL METHOD 2.1 Materials