8.MI701-Raden Haryo Bimo Setiarto Available online at http://jurnal.permi.or.id/index.php/mionline DOI: 10.5454/mi.7.4.8ISSN 1978-3477, eISSN 2087-8587 Vol 7, No 4, November 2013, p 192-197 *Corresponding author; Phone/Fax: +62-81-327025330/+62- 21-87907612 , Email: haryobimo42@yahoo.com Rice straw is a by-product of rice production in Indonesia. It becomes a problem because it contains lignocellulose compound that is very difficult to decom- pose (Arantes et al. 2012). On the other hand, it can potentially be converted into organic fertilizer. During harvesting months, rice straw is produced in huge quantity and cannot be optimally utilized. In the fact, rice straw can be used to increase soil organic nutrient by composting process. Long term utilization of rice straw compost can recover soil fertility because it contains lignocelullolytic microbes as bio-remedy agents (Chang et al. 2012). Rice straw compost contains phosphate (6.86 ppm), carbon (35.83%), water (35.83%), nitrogen (1,57%), phosphor (0.02%), iron (4.04 ppm), and zinc (0.09 ppm). Based on this composition, each ton rice straw compost contains soil nutrition equivalent to 41.2 kg Urea and 4.5 kg SP36 (Liu et al. 2012). It can substitute more than half of chemical fertilizer stock. Lignocelullolytic enzymes such as laccase, cellulase, and xylanase can be produced by lignocelullolytic fungi isolated from spent mushroom substrate that had been used to grow champignon (Agaricus) (Dixon and Webb 1979). The lignocellulolytic fungi have a main role of converting bio-mass of rice straw into compost. So, they can degrade and use lignin, cellulose, and hemicelluloses respectively as carbon source and hydrogen source for The main problem in soil conservation is the lack of carbon source from organic material. Rice straw from spent mushroom substrate (SMS) can be used as organic fertilizer to supply organic carbon for soil. It can also improve soil structure and increase macro-elements and micro-elements required by plants. This research focused on analyzing lignocellulolytic activity and Indole Acetic Acid (IAA) concentration produced by 14 fungal strains isolated from rice straw that had previously been used as substrate for champignon (Agaricus sp.). Four strains were isolated (JPF 2, JPF 5, JPF 13, and JPF 14) and then characterized. JPF 14 isolate had the highest -1 laccase activity (1.767 U mL ) and produced the highest concentration of indole acetic acid (IAA) hormone (6.78 -1 -1 mg mL ). JPF 13 isolate had the highest amylase activity (0.746 U mL ). JPF 2 isolate had the highest cellulase -1 -1 activity (0.502 U mL ). JPF 5 isolate had the highest xylanase activity (0.560 U mL ). Based on their ability to grow on certain pH and temperature, isolate JPF 2 was classified as mesophylic-acidophylic microbe, isolates JPF 5 and JPF 14 were psycrophylic-acidophylic microbes, and isolate JPF 13 was psycrophylic-alkalophylic microbe. Key words: Champignon, indole acetic acid hormone, lignocellulolytic fungi, rice straw, spent mushroom substrate Jerami padi merupakan salah satu substrat yang kaya lignoselulosa dan memiliki potensi untuk dapat diolah menjadi pupuk organik. Penambahan kompos jerami akan menambah kandungan bahan organik tanah, sehingga dapat mengembalikan kesuburan tanah. Tujuan penelitian ini adalah seleksi dan karakterisasi jamur pendegradasi lignoselulosa dan penghasil hormon asam indol asetat (IAA) yang diisolasi dari jerami padi limbah budidaya jamur Champignon (Spent Mushroom Substrate). Hasil yang diperoleh menunjukkan bahwa dari 4 isolat jamur lignoselulolitik terpilih (JPF 2, JPF 5, JPF 13, dan JPF 14), isolat JPF 14 memiliki aktivitas enzim -1 lakase tertinggi yaitu sebesar 1.767 U mL dan mampu menghasilkan hormon IAA (asam indol asetat) dengan -1 kadar tertinggi yaitu 6.78 mg mL . Isolat jamur JPF 13 memiliki aktivitas enzim amilase tertinggi dengan nilai -1 -1 0.746 U mL . Isolat jamur JPF 2 memiliki aktivitas enzim selulase tertinggi yaitu sebesar 0.502 U mL . Isolat -1 jamur JPF 5 memiliki aktivitas enzim xilanase tertinggi dengan nilai 0.560 U mL . Selain itu berdasarkan pertumbuhan pada suhu dan pH tertentu, isolat jamur JPF 2 dapat dikelompokkan sebagai mikroba mesofilik- asidofilik, isolat jamur JPF 5 dan JPF 14 dapat dikelompokkan sebagai mikroba psikrofilik-asidofilik, isolat jamur JPF 13 dapat dikelompokkan sebagai mikroba psikrofilik-alkalofilik. Kata kunci: hormon asam indol asetat, jamur Champignon, jamur lignoselulolitik, limbah jerami padi fungal SHORT COMMUNICATION The Lignocellulolytic Activity and Ability to Produce Indole Acetic Acid Hormone of Fungal Inoculant Isolated From Spent Mushroom (Agaricus sp.) Substrate RADEN HARYO BIMO SETIARTO* AND IWAN SASKIAWAN Microbiology Division, Research Center for Biology LIPI Jalan Raya Jakarta-Bogor Km 46, Cibinong Science Center, Bogor 16911, Indonesia growth (Dewi 2002). Cellulase is an enzyme that hydrolyzes β-1,4 glucoside chain on cellulose and derivatives into glucose. It has a multi-enzyme system consisting of endoglucanase (EC.3.2.1.4), selobiohydrolase (EC.3.2 .1.91), and β-glucosidase (EC.3.2.1.21) (Ahamed 2008; Bhat 1997). Xylanase is an enzyme group which can hydrolyze polymer xylan into xylose. It can be classified into β-xilosidase, exoxylanase, and endoxylanase. Exoxylanase can cut the chain of xylan polymer at the end of reduction, so it can produce xylose as the main product as well as other short chain oligosaccharides (Da Silva et al. 2005, Goddess 2002, Linko et al. 1984). Laccase (benzendiol: oxygen oxidoreductase, EC 1.10.3.2) is an extracellular enzyme that uses oxygen to carry out oxidation reaction in aromatic and non aromatic compound. It belongs to oxidase enzyme class which requires metal ion. The Laccase enzyme requires oxygen and produces water as one of the side products. The main substrate of laccase is lignin and oxidation reaction will not produce hydrogen peroxide compound (Couto et al. 2006; Couto et al. 2007; Kirk and Farrel 1997; Kruus 2000). Lignin peroxidase, and Manganese peroxidase are two ligninase enzymes which can degrade lignin in addition to the laccase enzyme (Jeffries 1994, Kruus 2000). Fungi isolated from spent champignon substrate not only can produce lignocellulolytic enzyme, but also Indole Acetic Acid (IAA) hormone to induce and increase plant growth (Aryantha et al. 2002). IAA is an endogenic auxin hormone that is available in plant body. It is a regulatory growth hormone that was first found as the main signal for plant growth (Ahmad et al. 2005). It is formed in the root and crops meristem tissue. Moreover, it can push elongation cell in coleoptil and plant internode. Elongation cells especially exist in vertical turn near the amplification cell. The IAA hormone also has a role in activating cell component, producing cell wall, and reorganizing into matrix whole cell (Bric et al. 1991, Levean et al. 2004). The IAA hormone produced by fungi will be absorbed by plant, that the plant grow faster and bigger (Arshad and Frakenburg 1991). Based on previous research, so this study will concern to selecting fungi isolates from spent Champignon substrate which can produce high concentration IAA hormone and high activity of lignocellulolytic enzyme. Moreover, this research focused on producing organic fertilizer starter inoculant made of lignocellulo- lytic fungi isolated from spent champignon substrate which can produce IAA hormone. The starter inoculant was then used to produce compost from rice straw as spent mushroom substrate. Concentration of IAA hormone and activity of laccase, cellulase, and xylanase enzymes from fungal isolates were used as the parameters for selection and characterization process. Fungi isolated from spent champignon substrate were grown on potato dextrose agar (PDA) medium. PDA medium contained 400 g potato extract boiled in 1 L of aquadest, 7 g bacto agarose, and 20 g dextrose. Next, the fungal isolates were grown in 50 ml Potato Dextrosa Broth (PDB) medium (37 °C, 72 h, 120 rpm -1 min ) to analyze the lignocellulolytic activity. Extraction process was done using buffer phosphate 0.2 M (pH 7) with ratio of culture : buffer phosphate (1:2) (v/v). Then, it was centrifuged using Kubota High Speed Refrigerated Centrifuge model 6500, capacity (6 x 500 mL), and dimension (500 x 740 x 940 mm) with parameter (20 000 x g, 10 min, 0-4 °C). Crude lignocellulolytic enzyme and IAA hormone were isolated on supernatant fraction. Next, it was filtered using Whatman filter paper grade 41 (diameter porous 20-25 µm) to get crude extracts of laccase, cellulase, xylanase enzymes, and IAA hormone. Assay of laccase enzyme activity was conducted based on previous report (Bourbonais and Paice, 1990). Three hundred seventy five µL sample of crude extract containing laccase enzyme was mixed with 375 µL (1.8 mM) ABTS (2,2-azinobis-3-ethylbenzthiazoline-6- sulfonate) in sodium acetic buffer (pH 4.5). The mixture was then incubated in water bath at 37 °C for 10 min. Enzymatic reaction was stopped using 250 µL 1 % (b/v) SDS (sodium dodecyl sulphate). Then, it was measured by using Spectrophotometer UV-Vis (λ=420 nm). One International Unit of laccase enzyme activity was defined as the total laccase enzyme that can oxidize 1 µmol ABTS each minute. Assay of cellulase and xylanase enzyme activity was conducted based on previous report (Miller 1959). One hundred twenty five (125) μL sample of crude extract containing cellulase enzyme was mixed with 0.5 % (b/v) CMC (carboxy methyl celullose) liquid substrate in 0.05 M acetic acid buffer pH 5. Then, It was incubated in water bath at 37 °C for 10 min. Later, 500 μL DNS (3,5-dinitro salisilic acid) was added. Then, the mixture was heated in water bath (100 °C, 5 min) to stop enzymatic reaction. Before measurement using Spectrophotometer UV-Vis (λ= 540 nm), 5 mL aquadest was added to dilute th sample. One International Unit of cellulase enzyme activity was defined as the total μmol product of glucose as the Volume 7, 2013 Microbiol Indones 193 results of cellulase enzyme hydrolysis each minute. For xylanase, 150 μL crude xylanase extract was mixed with 0.5% (b/v) suspension of beechwood xylan in 0.05 M acetic acid buffer pH 5. Then, the mixture was incubated in water bath at 37 °C for 10 min. Later, 200 μL DNS (3,5- dinitro salisilic acid) was added. Then, the mixture was heated in water bath (100 °C, 5 min) to stop enzymatic reaction. Two mL aquadest was added to dilute the sample before being measured with Spectrophotometer UV-Vis (λ=540 nm). One Internati- onal Unit of xylanase activity was defined as the total μmol xylose produced from hydrolysis by xylanase enzyme each minute. Assay of indole acetic acid (IAA) hormone was conducted based on previous report (Gordon and Webber 1950). Zero point five mL crude extract of IAA hormone was mixed with 1 mL Salkowski reagent (0.5 M FeCl .6H O in H SO ). The mixture was then 3 2 2 4 incubated in dark room for 30 min until color is formed. Then, the absorbance was measured using Spectropho- tometer UV-Vis (λ=530 nm). ConcentrationIAA hormone was calculated by linear regression analysis using IAA standard curve. The standard curve was made using varying concentration of IAA from 0.005 -1 mg mL to 0.050 mg mL . In general, all 14 fungal isolates showed low cellulase and xylanase activities. Nevertheless, their -1 laccase activities and concentrations of IAA hormone differ from one another. Fungal isolate JPF 2 had the -1 highest cellulase activity (0.502 U mL ) (Table 1). This results was higher than cellulase activity expressed by -1 Aspergillus sp. (0.04 U mL ), Fusarium sp. (0.05 U -1 -1 mL ), Aspergillus flavus (0.34 U mL ), and -1 Botryotrichum sp. (0.0098 U mL ) (Table 1) as reported by Kerem et al. (1992). However, it was still lower than the celllulase activity showed by -1 Trichoderma reesei (1.66 U mL ) and Aspergillus -1 niger (1.69 U mL ) (Table 1) grown on rice straw medium (Ahamed and Vermette 2008). Fungal isolate JPF 5 showed the highest xylanase -1 activity (0.560 U mL ). It was still lower than that of Aspergillus niger grown on rice straw medium, of -1 which the activity was 15.33 U mL (Da Silva et al. 2005). The highest laccase activity was shown by -1 fungal isolate JPF 14 (1.767 U mL ), followed by -1 fungal isolate JPF 4 (1.691 U mL ), of which the activity was still higher than that of Omphalina (1.162 -1 U mL ) isolated from empty bunch of palms (Kruus 2000). Nevertheless, the activities shown by the fungal isolates were lower than that of Marasmius sp. (4.560 -1 -1 U.mL ) and Pleurotus ostreatus (4.394 U mL ), which were isolated from industrial pulp paper (Couto and Toca Herera 2007; Kirk and Farrel 1997). Fungal isolate JPF 14 not only showed the highest Fungal isolate Cellulase activity -1 (U mL ) Xylanase activity -1 (U mL ) Laccase activity -1 (U mL ) IAA hormone concentration -1 (mg mL ) JPF 1 0.340 0.412 0.988 5.34 JPF 2 0.502 0.476 0.654 4.23 JPF 3 0.344 0.464 1.467 5.17 JPF 4 0.380 0.493 1.691 5.48 JPF 5 0.423 0.560 0.756 5.20 JPF 6 0.353 0.458 0.935 4.00 JPF 7 0.315 0.431 0.965 3.91 JPF 8 0.438 0.527 0.969 5.64 JPF 9 0.387 0.533 1.113 5.53 JPF10 0.302 0.453 1.603 3.98 JPF 11 0.385 0.508 0.403 4.09 JPF 12 0.473 0.541 0.889 6.11 JPF 13 0.243 0.479 1.281 4.84 JPF 14 0.371 0.482 1.767 6.78 Table 1 Cellulase, xylanase, laccase activities and IAA hormone concentration expressed by 14 fungal isolated from spent champignon (Agaricus sp.) substrate Microbiol Indones194 SETIARTO AND SASKIAWAN PDA (potato dextrose agar) medium and incubated at room temperature for 5 d. To make starter inoculant F0, fungal isolates were transferred from PDA medium -1 into bean sprout extract-sugar (60 g L ) medium and incubated at room temperature for 7 d. Then, the starter inoculant F0 was transferred again into sterile cassava dregs (onggok) to make starter inoculant F1 and incubated for 14 d. After incubation, it was dried and mixed again with sterile cassava dregs with ratio (1:20) (w/w). Then, it was incubated for another 14 d to make starter inoculant F2, which will be used to inoculate rice straw (spent champignon substrate) to produce organic fertilizer. Population density of the starter inoculant F2 was estimated by using TPC (total plate count) method (Table 2). The population density of inoculant F2 made from isolates JPF 2 and JPF 13 was 5 -1 5 5.6 x 10 CFU mL , fungi isolate JPF 5 was 4.6 x 10 -1 5 -1 CFU mL , and JPF 14 was 2 x 10 CFU mL . TPC 3 method was performed by serial dilution factors 10 , 4 5 6 10 , 10 , and 10 . TPC (total plate count) method indicated that the total microbial population on organic fertilizer starter 5 -1 inoculant F2 was between 2 x 10 CFU mL and 5.6 x 5 -1 10 CFU mL . Microbes growing on starter inoculant F2 of organic fertilizer was dominated by fungi isolate. So, it can be assumed that there is no contamination on the production process of organic fertilizer starter inoculant. Lignocellulolytic fungi was applied as starter to produce compost organic fertilizer from rice straw (Chang et al. 2012). Laccase, cellulase, and xylanase enzymes are very important in composting to produce organic fertilizer from rice straw. Laccase enzyme helps to degrade lignin from rice straw. Cellulase and xylanase enzyme have an important role main role in hydrolyzing cellulose and hemicelluloses, respectively, to become glucose and xylose (Liu et al. 2012). Glucose and xylose are sources of carbon and hydrogen for fungi, which are essential nutrition source to induce plant growth. In addition, there is also another important reason why fungal is used as starter for organic fertilizer, that is the fungal ability to produce endogenous auxin hormone such as indol acetic acid (IAA) hormone (Ahmad et al. 2005). Combination of lignocellulolytic enzyme and IAA hormone will produce organic fertilizer that is nutrition rich and containing high concentration of growth hormone regulator, which is necessary to increase the soil fertility and can accelerate the growth of roots, branches, bars, and leaves (Isikhuemhen and Mikiashvilli 2012). laccase activity, but also produced the highest -1 concentration of IAA hormone (6.78 mg mL ) in comparison to the other fungal isolates. The second best IAA producer is JPF 12 with IAA hormone -1 concentration 6.11 mg mL . All 14 fungal isolates isolated from spent champignon substrate produced with high concentrations of IAA hormone between -1 -1 3.91 mg mL and 6.78 mg mL . This was much higher than the amount produced by Rhyzobium sp. (14.40 μg -1 mL ), which formed symbiosis with plant root, and -1 Pseudomonas putida (1.1225 μg mL ) (Levean and Lindow 2004; Ahmad et al. 2005). Fungal isolates having high activity of lignocellulo- lytic enzyme and IAA hormone were characterized by measuring the biomass dry weight at varying temperature and pH.m Cultivation was performed on -1 shaker incubator (72 h, 100 rpm min ) with pH variation from 4.0 to 8.0 (with interval pH 1.0) and temperature variation between 20 °C and 60 °C (with interval temperature 10 °C). After incubation, the fungal biomass was separated from medium by centrifugation (20 000 x g, 10 min, 0-4 °C) using Kubota high speed refrigerated centrifuge model 6500, capacity (6 x 500 mL), dimension (500 x 740 x 940 mm) followed by filtration using Whatman filter paper grade 41 before being weighed. The optimum growth temperature and pH of the lignocellulolytic fungi is defined as the condition giving the best dry weight. Characterization of Optimum Growth pH and Temperature of the Selected Lignocelullolytic Fungi (isolates JPF 2, JPF 5, JPF 13, and JPF 14) was conducted by measuring the cellular dry weight (Fig 1,2). Cultivation 4 strains of lignocellulolytic fungi was -1 performed on shaker incubator (72 h, 100 rpm min ) with pH variation from 4.0 to 8.0 (with interval pH 1.0) and temperature variation between 20 °C and 60 °C (with interval temperature 10 °C). Fungi isolate JPF 2 grew optimum at temperature of 30 °C with weight of biomass cell 1.3985 g and pH 5 with weight of biomass cell 1.0887 g. Then, fungi isolate JPF 5 grew optimum at temperature of 20 °C with weight of biomass cell 1.3584 g and pH 5 with weight of biomass cell 1.5375 g. Meanwhile, fungi isolate JPF 13 grew optimum at temperature of 30 °C with weight of biomass cell 1.1986 g and pH 8 with weight of biomass cell 1.9778 g. Later, fungi isolate JPF 14 grew optimum at temperature of 20 °C with weight of biomass cell 1.2978 g and pH 6 with weight of biomass cell 0.9113 g. For Production of Lignocelullolytic Fungi Starter Inoculant for Organic Fertilizer, the experiments was conducted as follow. Fungal isolates were cultivated on Volume 7, 2013 Microbiol Indones 195 REFERENCES Ahamed AP, Vermette. 2008. Culture-based strategies to enhance cellulase enzyme production from Trichoderma reesei RUT-C30 in bioreactor culture conditions. Biochem Eng J. 40(3):399-407. doi:10.1016 /j.bej.2007.11.030. Ahmad F, Ahmad L, Khan MS. 2005. Indole acetic acid production by the indigenous isolates of Azotobacter and Pseudomonas in the presence and absence of ACKNOWLEDGMENTS Author extends full gratitude for all researchers and technicians at Microbial Biochemistry Laboratory Research Center for Biology LIPI, who had supported the ongoing of this project. This research activity was funded by Project DIPA PN V 2012 Microbiology Division, Research Center for Biology, Indonesian Institutes of Sciences. 2.5 2 1.5 1 0.5 0 W ei g h t b io m as s fu n g i ce ll ( g r) 0 2 4 6 8 10 pH variation W ei g h t b io m as s fu n g i ce ll ( g r) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 10 20 30 40 50 60 70 Temperature variation (°C) Fig 2 Characterization for optimum temperature for growth of fungi isolates JPF 2 ( ), JPF5 ( ), JPF 13 ( ), and JPF 14 ( ). Fig 1 Characterization for optimum pH for growth of fungi isolates JPF 2 ( ), JPF5 ( ), JPF 13 ( ), and JPF 14 ( ). Fungi isolates -1 Results of TPC (CFU mL ) Average TPC results -1 (CFU mL ) 1 2 3 JPF 2 5 7 x 10 4 x 10 5 6 x 10 5 5.6 x 10 5 JPF 5 3 x 10 5 6 x 10 5 5 x 10 5 4.6 x 10 5 JPF 13 5 x 10 5 8 x 10 5 4 x 10 5 5.6 x 10 5 JPF 14 2 x 10 5 3 x 10 5 1 x 10 5 2 x 10 5 Table 2 Population density of organic fertilizer starter inoculant F2 calculated based on TPC method Microbiol Indones196 SETIARTO AND SASKIAWAN Dixon M, Webb EC. 1979. Enzyme. Third Edition. New York: Academic Press. Gordon SA and Weber RP. 1997. Colorimetric estimation of indole acetic acid. Plant Physiol. 26:192-195. doi:10.1104/pp.26.1.192. Hanafiah KA. 2005. Biologi Tanah: Ekologi dan Mikrobiologi Tanah. [Soil Biology: Ecology and Soil Microbiology]. Jakarta : PT. Raja Grafindo Persada. Isikhuemhen OS, Mikiashvilli NA. 2012. Lignocellulolytic enzyme activity, substrate utilization, and mushroom yield by Pleurotus ostreatus cultivated on substrate containing anaerobic digester solids. J Ind Microbiol Biotechnol. 36(11):1353-1362. doi:10.1007/s10295- 009-0620-1. Jeffries TW. 1994. Biodegradation of lignin and hemicelluloses. in: C. Ratledge (ed.). Biochemistry of Microbial Degradation. Netherlands:Kluwer Academic Publishers. p 233-277. doi:0.1007/978-94-011-1687- 9_8. Kerem Z, Friesem D, Hadar Y. 1992. Lignocellulose degradation during solid-state fermentation: Pleurotus ostreatus versus Phanerochaete chrysosporium. Appl Environ Microbiol. 58(4):1121-1127. Kirk TK, Farrell RL. 1997. Enzymatic combustion: the microbial degradation of lignin. Annu Rev Microbiol. 41:465-501. doi:10.1146/annurev.micro.41.1.465. Kruus K. 2000. Laccase: a useful enzyme for industrial applications. Kemia Kemi. 27:184-187. Kubata BK, Takamizawa K, Kawai K, Suzuki T, Horitsu H. 1995. Xylanase IV, an exoxylanase of Aeromonas caviae ME-1 which produces xylotetraose as the only low-molecular-weight oligosaccharide from xylan. Appl Environ Microbiol. 61:1666-1668. Lehninger A. 1982. Dasar-Dasar Biokimia Jilid 1. [Principle of Biochemistry Part 1]. Alih Bahasa Maggy T. Jakarta: Erlangga. Levean JH, Lindow SE. 2004. Utilization of plant hormone indole acetic acid for growth by Pseudomonas putida strain 1290. American Society for Microbiology. 1(5):2365-2370. Linko M, Vilkari L, Suiko MI. 1984. Hydrolysis of xylan and fermentation of xylose to ethanol. Biotechnol Adv. 2(2):233-252. doi:10.1016/0734-9750(84)90007-7. Liu D, Zhang R, Yang X, Wu H, Xu D, Tang Z, Shen Q. 2012. Thermostable cellulase production of Aspergillus fumigatus Z5 under solid-state fermentation and its application in degradation of agricultural wastes. International Biodet Biodegrad. 65(5):717-725. doi:10.1016/j.ibiod.2011.04.005. Miller GL. 1959. Use of dinitrosalicylic acid reagent of determination of reducing sugar. Anal Chem. 31(3):246- 248. doi:10.1021/ac60147a030. tryptofan. Turk J Biot.29:29-34. Alexopolous CJ, Mims SW, and Blackwell M. 1996. Introductory Mycology, 4th Ed. New York: John Wiley and Sons, Inc. Arantes V, Milagres AMF, Filley TR, Goodell B. 2012. Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions. J Ind Microbiol Biotechnol. 38(4):541-555. doi:10.1007/s10295-010-0798-2. Aryantha IN, Lestari DP, Pangesti NPD. 2002. Mikrobia penghasil fitohormon. [Microbes producer phytohor- mone]. Bandung: Institut Teknologi Bandung. Arshad M and Frankerburger WT. 1991. Microbial production of plant hormones. Plant Soil. 133(2):1-8. doi:10.1007/BF00011893. Bhat MK, Bhat S. 1997. Cellulose degrading enzymes and their potential industrial applications. Biotechnol Adv. 15(3-4):583-620. doi:10.1016/S0734-9750(97)00006-2. Bourbonnais R, Paice MG, Reid DI, Lanthier P, Yaguchi M. 1995. Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2’- azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization. Appl Environ Microbiol. 61:1876-1880. Bourbonnais R dan Paice MG. 1990. Demethylation and delignification of kraft pulp by Trametes versicolor laccase in the presence of ABTS. Applied Microbial Technol. 36(6):823-827. doi:10.1007/BF00172202. Bric JM, Bostock RM, Silverstone SE. 1991. Rapid in situ assay for indole acetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol. 57(2):535-538. Chang AJ, Fan J, Wen X. 2012. Screening of fungi capable of highly selective degradation of lignin in rice straw. Int Biodeter Biodegr. 72: 26-30. doi:10.1016/j.ibiod.2012. 04.013. Couto SR, Toca Herrera JL. 2006. Industrial and biotechnological applications of laccases: a review. Biotechnol Adv. 24(5):500 -513. doi:10.1016/j.biotech- adv.2006.04.003. Couto SR, Toca Hererra JL. 2007. Laccase production at reactor scale by filamentous fungi. Biotechnol Adv. 25(6):558-569. doi:10.1016/j.biotechadv.2007.07.002. Da Silva R, Lago ES, Merheb CW, Machione MM, Park YK, Gomes E. 2005. Production of xylanase and CMCase on solid state fermentation in different residues by Thermoascus auranticus Miehe. Braz J Microbiol. 36(3):235-241. doi:10.1590/S1517-838220050003000 06. Deacon JW. 1997. Modern Micology. New York:Blackwell Science. Dewi. 2002. Hidrolisis limbah hasil pertanian secara enzimatik. [Hydrolysis agricultural product waste using enzyme]. Akta Agrosia. 5(2):67 -71. Volume 7, 2013 Microbiol Indones 197 1: 192 2: 193 3: 194 4: 195 5: 196 6: 197