EXPERIMENTAL METHOD 1 Materials Prosiding INAFOR III 2015 FullIssue low rest

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 The remazol brilliant blue R RBBR was obtained from Sigma-Aldrich USA. Yeast extract and peptone were obtained from Hi Media Chemicals Mumbai, India, while other chemicals were procured from Merck Germany. 2.2 Microorganisms Microorganisms used in this study were fungi D7 collection of Lab of Biomass Process Technology and Bioremediation RC Biomaterials and fungal isolate selected among screened samples collected from Cibinong Science Centre area, Cibinong, Bogor. The screening was conducted based on the growth and ability of isolate to decolorize remazol brilliant blue R RBBR and lignin incorporated into agar medium. A piece of mushroom 1 cm was placed onto solid medium and incubated for 7 days at 25 ˚C for 7 days. The compositions per litre of distilled water of solid medium used were the following: Bottom layer: Dzapek-Dox modified broth 35 g, KH 2 PO 4 1 g, yeast extract 2 g, peptone 2 g, agar 20 g, lignin 2 g, glucose 10 g. Top Layer: Malt extract 5 g, benomyl 10 mg, RBBR 1 g, agar 10 g. pH medium was adjusted to 4.5 and autoclaved for 15 minutes at 121˚C prior to use. The formation of clear zone was observed during fungi growth within 4 days.The potential isolates showing clear zone were selected for further studies. Stock culture of potential isolates were maintained on malt extract agar pH 4.5 at 4˚C and sub-cultured after one months. 2.3 Dye decolorization in liquid medium The isolated fungi were grown individually on Dzapek-Dox broth medium. Twenty ml of sterilized dzapek-dox modified broth medium in 100 ml Erlenmeyer flask were inoculated with three plugs from actively growing colony of fungi culture and incubated at static condition at 25˚C for 7 days. After 7 days, one ml of RBBR was added to the fungi culture to final concentration of 100 ppm. Uninoculated flasks served as a control. The decolorization efficiency was observed for 24, 48, and 72 h. All the experiments were carried out in duplicate. 2.3.1 Effect of carbon source on dye decolorization Effect of carbon source was evaluated by growing the isolated fungi in Dzapek-Dox broth medium with glucose and peptone pH 4.5 prior to decolorization experiment. The composition of the medium per litre of distilled water, pH 4.5 were Dzapek-Dox modified broth 20 g, glucose 20 g, and peptone 1 g. All the experiment were carried out in duplicate. 2.3.2 Effect of copper ion on fungi growth, dye decolorization and enzymatic activity Addition of CuSO 4 .5H 2 O to the growth medium was studied in order to determine effect of copper ion Cu 2+ on dye decolorization and enzyme activity. Fungal culture was exposed to the various concentration of Cu 2+ prior to decolorization experiment. The experiment was Bogor, 21-22 October 2015 278 carried on in 100 ml Erlenmeyer flask, composed of 20 ml of malt extract medium 20 gL, glucose 20 gL, peptone 1 gL, and supplemented with CuSO 4 .5H 2 O to give nominal metal ion concentration of 0.1, 1, and 2 mM. The pH of the medium was adjusted to 4.5. 2.4 Dry mycelium weight dmw After incubation, fungal mycelia obtained were removed from a medium by filtered using pre- weighed filter paper, while the filtrate was used for decolorization and enzyme assay. The mycelia then was left to dry to constant weight at 105˚C. Dry mycelium weight dmw was expressed in terms of g of biomass per liter of culture Erkurt et. al., 2007. 2.5 Decolorization assay The supernatant of filtered fungal mycelium were used for decolorization using spectrophotometric examination. Dye decolorization were determined by measuring the absorbance change of RBBR at 592 nm with a UV-vis spectrophotometer Shimadzu 2010. Decolorization efficiency R, was determined according to the following formula: R = 1 – A observed A initial x 100 1 Where, A initial is the initial absorbance and A observed is observed absorbance Yanto et. al., 2014. 2.6 Enzyme assays The enzymatic activities laccase, MnP, and LiP secreted in the culture were determined using method according to Minussi et. al. 2001 with a little modification. Laccase activity was measured by monitoring the oxidation of syringaldazine at 525 nm. One unit of laccase activity was defined as the amount of enzyme necessary to oxidase one µmol of substrate in 1 minute. The reaction mixture for laccase assay contained 0.9 ml culture filtrate, 0.75 ml 0.1 M sodium acid buffer, and 0.1 ml 0.5 mM syringaldazine. LiP activity was determined by the oxidation of veratrylic alcohol at 310 nm. One unit of LiP was defined as the amount of enzyme that oxidised one µmol of verattrylic alcohol per minute per litre of culture filtrate. The reaction mixture for LiP assay was composed of 1 ml culture filtrate, 0.3 ml 2 mM H 2 O 2 , and 2 ml LiP buffer. Manganese peroxidase activity were determined by the formation of Mn 3+ in sodium malonate buffer pH 4.5 in the presence of H 2 O 2 at 470 nm. One unit of MnP was defined as the amount of enzyme required to form 1 µmol of Mn 3+ in 1 minute at 25˚C Kariminiaae- Hamedaani et. al., 2007. The reaction mixture for MnP assay was consisted of 1 ml culture filttrate, 1.75 ml 50 mM malonic buffer pH 4.5, 0.125 ml 20 Mm 2.6 dimethoxy phenol, 0.125 ml 20 mM MnSO 4 , and 0.3 ml 2 mM H 2 O 2 . Enzyme activity UL was calculated according to the following formula: Enzyme activity = Abs ɛ x V mixture µl : 10 6 x 10 6 x 60 t : V enzyme µl : 10 3 2 Where, ɛ for laccase, LiP, and MnP were 6500, 9300, and 49600, respectively. 3. RESULT AND DISCUSSION 3.1 Isolation and Screening of Fungal Strain