Journl of IOLOGICL RESERCHES ISSN: 08526834 E-ISSN:2337-389X Volume 22 No. 2 June 2017 Originl rticle The utiliztion of ink cp mushroom (Coprinus cinereus) on plm oil mill effluent degrdtion Yohnes ernrd Suowo Deprtment of Microiology, Reserch Centre of iology, The Indonesin Institute of Sciences strct reserch on the utiliztion of ink cp mushroom (Coprinus cinereus) on Plm Oil Mill Effluent (POME) degrdtion ws conducted. Plm oil industry is n griculturl industry which keeps on developing in Indonesi. side from producing plm oil, this industry lso genertes POME. POME hs high level of OD nd COD vlue thus it is dngerous for the environment. Coprinus cinereus ws exmined to degrde POME ecuse of its ility to produce ligninses enzymes. The im of this reserch ws to evlute ligninses enzymes produced y C. cinereus fter the ddition of inducers nd its ility in degrding POME. Coprinus cinereus ws cultivted on Defined Medium (DM) then the myceli ws hrvested Inducers used in this study were CuSO 4, KNO 3 nd sucrose. The suspension of Coprinus cinereus myceli ws used to degrde POME. The result showed tht Coprinus cinereus could produce Lccse, Mngn Peroxidse nd Lignin Peroxidse on DM. The ddition of 200 µm CuSO 4 nd 10 mm KNO 3 on DM could increse lccse ctivity y 62.92 %. Coprinus cinereus hs ility to degrde Poly R-478 nd POME. The tretment of 200 µm CuSO 4 on medi could increse the degrdtion of Poly R-478 y 19.32% fter 9 dys of incution. The ddition of 15 g/l sucrose on POME could increse decoloriztion y 75.26 % nd COD reduction y 91.26 % fter 27 dys of incution. Ink Cp Mushroom (Coprinus cinereus) cn e used for POME degrdtion. The ddition of sucrose nd heting tretment would ccelerte COD reduction. Keywords: Coprinus cinereus, inducer, POME, Poly R-478, degrdtion Received: 08 Ferury 2017 Revised: 16 pril 2017 ccepted: 25 My 2017 Introduction s n griculturl country, the sector of griculture plys n importnt role in Indonesin economy nd plm oil plnttion hs een promising industry. The plnttion re hs een incresing through the yers nd present in lmost ll of Indonesin min islnds. ccording to Ministry of griculture sttistics, totl re used for this crop in 2015 reched pproximtely 11,444,808 hectre with the production cpcity ws 30,948,931 tons (nonymous, 2014). The plm oil products will e used to cover the country s need nd exported. esides producing plm oil, this industry lso genertes solid wste nd effluent. Plm oil mill effluent (POME) is wste wter chrcterized y rown in color, suspension-like with high colloid nd unplesnt smell (hmd nd Chn 2009). POME originted from the remnnt of plm fresh fruits milling. POME is hzrdous to the environment ecuse it hs high content of orgnic compound (COD= 40,000-50,000 mg/l, OD= 20,000-25,000 mg/l, oil, ft nd complex polymer (crohydrte, protein, lipid, minerl nd nitrogen compound)) (Iegulm-Njoku nd chi, 2014). The milling of ton of fresh fruits plm unch will generte wstewter y 50 % of totl wste while empty fruit unch milling will produce 23 % of totl wste. The production of POME in Indonesi Corresponding uthor: Y Suowo Deprtment of Microiology, Reserch Centre of iology, The Indonesin Institute of Sciences. Jl. Ry ogor KM 46, Ciinong, ogor. Phone : 085883336936 Fx : 021-8765062 e-mil : yosuowo@yhoo.com reched 28.5 million tons/yer (Irvn et l., 2012). The relese of POME to surrounding wterody without proper wste tretment will impct lrgely to the environment (Wu et l., 2007). There re some species of white-rot fungi tht produce ligninses enzymes including Phnerochet chrysosporium, Coriolus hirsutus, Phlei rdit, Coriolus versicolor, gricus isporus, Pleurotus ostretus, Trmetes versicolor, Trmetes villos, Pycnoporus cinnrinus. White-rot fungi secreted one or more extrcellulr enzymes to degrde lignin: Lignin Peroxidse (LiP) (EC 1.11.1.14), Mn-dependent Peroxidse (MnP) (EC 1.11.1.13) nd Cu-contining Phenoloxidse, Lccse (EC 1.10.3.2) (Songulshvili et l., 2007). Lccse will oxidize orgnic nd inorgnic compounds such s phenol, ctechol, hydroquinone, 2,6-dimethoxyphenol, nd syringldzine (Thruston, 1994) nd lso min romtic nd scorte (Mdhvi nd Lele, 2009). Shneider et l. (1999) reported tht C. cinereus produces lccse with the highest titer vlue from ll five detected enzymes. The ctivity ws higher during mycelium growth thn during fruiting ody formtion (Rymond et l., 2015). Coprinus cinereus lso produces peroxidse such s lignin peroxidse nd mngn peroxidse (Heinzkill et l., 1998). Some orgnisms secrete lccse in smll mount, ut the yield cn e incresed y the tretment of chemicl compounds such s romtic nd phenolic compounds, metl, lcohol nd detergent (Leonowicz et l., 2001). The effect of copper on lccse synthesis ws studied on Trmetes versicolor (Collins nd Doson, 1997) nd Pleurotus ostretus (Plmieri et l., 2003). The tretment of 200 µm CuSO 4 on sumerged culture of Volvriel volvce resulted high lccse ctivity (Chen http://dx.doi.org/10.23869/phjr.22.2.20175 Pulished y PI Est Jv. Open ccess www.erklhyti.org 61
Suowo et l., 2003). The tretment of 10 mm KNO 3 on sumerged culture of Gnoderm lucidum incresed the production of lccse nd MnP (Songulshvili et l., 2007). The tretment of sucrose on sumerged culture of Pleurotus ostretus incresed the ctivity of lccse (Suowo, 2015). Recently, the utiliztion of ink cp mushroom (Coprinus cinereus) to degrde POME nd inducer tretment to increse ligninses ctivity on the fungi hs not een conducted yet. The ojective of this reserch ws to evlute ligninses enzymes produced y Coprinus cinereus fter the ddition of inducers on medi nd to oserve its ility to degrde POME. Method Microorgnism The isolte of Coprinus cinereus derived from culture collection of Microiology Deprtment, Reserch centre of iology, The Indonesin Intitute of Sciences. The isolte ws preserved on slnt gr (PD) nd incuted t 4 ºC. Medi Medi used were: Defined Medium (DM) (Songulshvili et l., 2007), Potto Dextrose roth (PD), en sprout extrct (TE), Poly R-478 medi (Glenn nd Gold 1983). DM composition: 10 g glucose; 1 g NH 4NO 3; 0.8 g KH 2PO 4; 0.2 g N 2HPO 4; 0.5 g MgSO 4.7H 2O; 2 g yest extrct. PD: 400 g potto, 20 g Dextrose. en Sprout Extrct (TE): 100 g en sprout; 60 g sucrose. Medi Poly R-478: 0.60 g KH 2PO 4; 0.50 g MgSO 4.7H 2O; 0.40 g K 2HPO 4; 0.22 g (NH 4) 2 trtrte; 40.0 g sorose; 0.20 g Poly R-478 (Sigm); 15.0 g gr (Oxoid No 3); 10.0 ml minerl solution stock; dded with distilled wter until 1 L. Minerl solution stock: 7.4 g CCl 2.2H 2O; 1.2 g Ferri citrte; 0.7 g ZnSO 4.7H 2O; 0.5 g MnSO 4.4H 2O; 0.1 g CoCl 2.6H 2O; 10.0 mg Thimin HCl; dded with distilled wter until 1 L. Inoculum Preprtion C. cinereus ws cultivted on DM, 20 ml of myceli suspension ws inoculted into 180 ml DM medium, then incuted in shker t room temperture, 150 rpm for 7 dys. Suspension of myceli ws hrvested y centrifugtion t 9,000 rpm, 4 for 15 min then ws wshed with doule distilled wter. The suspension of myceli (5 ml) ws inoculted in DM in finl volume of 50 ml. The culture ws incuted in shker t 150 rpm, room temperture for 4 dys. The superntnt ws hrvested y centrifugtion t 9,000 rpm, room temperture for 15 min. Then, the superntnts were used for further nlyses. Ligninses Enzymes ctivity of C. cinereus Lccse ctivity (Lc) ws determined vi the oxidtion of 2,2-zino-is(3 ethylenzthizoline)-6-sulfonte (TS) (Ppinutti et l., 2003). The rection mixture consists of 0.5 ml citrte uffer of ph 6, 0.1 ml TS 1 mm, 0.4 ml superntnts. TS oxidtion ws monitored y the increse in sornce t 420 nm. Mngnese Peroxidse ctivity (MnP) ws mesured y monitoring the oxidtion of guicol spectrophotometriclly (Yoshid et l., 1996). The rection mixture contined 0.1 ml guicol 4 mm, 0.1 ml lctte uffer 50 M of ph 4.5, 0.2 ml MnSO 4 1 mm, 0.3 ml distilled wter, 0.1 ml H 2O 2, 0.2 ml superntnts. Guicol oxidtion ws monitored y the increse in sornce t 465 nm. Lignin Peroxidse ctivity (LiP) ws estimted y the method of Tien nd Kirk (1983). The ssy is sed on the oxidtion of vertryl lcohol to vertryl ldehyde in the presence of H 2O 2. Rection mixture contined 0.1 ml of 8 mm vertryl lcohol, 0.2 ml cette uffer 50 mm of ph 3, 0.45 ml distilled wter, 0.05 H 2O 2 5 mm, 0.2 ml superntnts. The incresing of sornce ws monitored t 310 nm, with three replictions. Ligninses Enzymes ctivity on Three Medi Three medi is often used for growing sidiomycetes fungi in the lortory, nmely: PD, TE nd DM. These medi will e used to evlute the ctivity of ligninses enzymes of C. cinereus. The suspension of myceli (20 ml) ws inoculted into PD, TE nd DM in finl volume of 200 ml. The cultures were incuted in shker 150 rpm, room temperture for 16 dys. Ligninses enzymes (Lc, MnP, LiP) ctivity ws mesured on dy: 0, 2, 5, 7, 9, 12, 14, nd 16. 2 ml of liquot ws tken for centrifugtion t 9,000 rpm t room temperture. The superntnts were used for further nlyses like the methods mentioned ove, with three replictions. The ility of C. cinereus on Poly R-478 Decoloriztion Poly R-478 is model compound tht is often used to determine the ility of microil decoloriztion of dye nd mesuring the ligninses ctivity. The ddition of inducers such s CuSO 4, KNO 3 nd sucrose in the Poly R- 478 medi to enhnce the ligninses ctivity of C. cinereus. Medi were tested include: T0 with composition: Poly R-478 + suspension of myceli; T1: Poly R-478 medi + CuSO 4 + suspension of myceli; T2: Poly R- 478 medi + KNO 3 + suspension of myceli; T3: Poly R-478 medi + sucrose + suspension of myceli; T4: Poly R-478 medi + CuSO 4 + KNO 3 + sucrose + suspension of myceli. The suspension of C. cinereus myceli (20 ml) ws inoculted into the medi in finl volume of 200 ml. The cultures were incuted in shker 150 rpm t room temperture for 9 dys. 2.0 ml of smples ws centrifuged t 9,000 rpm, room temperture for 15 mins. The decoloriztion of Poly R-478 dye ws mesured y the percentge reduction of sornce t 520 nm, with three replictions. The ility of C. cinereus on POME Degrdtion Plm Oil Mill Effluent (POME) contins lignin nd cellulose. Lignin cn e degrded y lccse secreted y C. cinereus, tht resulted in POME decoloriztion nd the decrese of COD level. POME used in this reserch ws otined from PT ngk Putr Mndiri in ngk Islnd. Experimentl design: 62
The utiliztion of ink cp mushromm (Coprinus cinereus) Induction effect: Two medi tretments were used nd. Medi ws composed of 100 ml POME + 15 g/l sucrose, while medi ws composed of 100 ml POME + 15 g/l sucrose + 200 µm CuSO 4 + 10 mm KNO 3. oth medi were sterilized then inoculted with 10 ml of myceli suspension. oth cultures were incuted in shker (150 rpm) t room temperture for 27 dys. POME decoloriztion nd the decrese of COD level were oserved on dy 0 nd 27 with spectrophotometer t 600 nm (Li et l., 2009) Steriliztion effect: Medi ws composed of 100 ml POME + 15 g/l sucrose + 200 µm CuSO 4 + 10 mm KNO 3. Steriliztion tretments were done. Medi D ws sterilized in n utoclve (121, 1 tm, 15 min), while medi E ws not sterilized. oth medi were inoculted with 10 ml of myceli suspension then incuted in shker (150 rpm t room temperture for 27 dys). POME decoloriztion nd the decrese of COD level were oserved on dy 0 nd 27 with spectrophotometer t 600 nm (Li et l., 2009). Shking effect: Medi ws composed of 100 ml POME + 15 g/l sucrose + 200 µm CuSO 4 + 10 mm KNO 3. oth medi were sterilized then inoculted with 10 ml of myceli suspension. Shking tretments were done. Culture F ws incuted in shker (150 rpm t room temperture for 27 dys), while culture G ws not shked during incution. POME decoloriztion nd the decrese of COD level were oserved on dy 0 nd 27 with spectrophotometer t 600 nm (Li et l., 2009). The significnce etween tretments ws determined using T-Test nlysis t the level of P<0.05, with three replictions Results C. cinereus (ink cp) cultivted in DM medium could produce three ligninses enzymes: Lccse, Mngnese Peroxidse nd Lignin Peroxidse. Induction using CuSO 4 nd KNO 3 incresed lccse enzyme ctivity y 62.92% (Tle 1). Tle 1. Ligninses ctivity of C. cinereus Enzymes Without induction (UmL -1 ) CuSO 4 nd KNO 3 induction (UmL -1 ) Rise of enzymes ctivity (%) Lccse 237.84 ±18.10 387.01±187.70 62.92 MnP 692.83±448.92 8201.78±432.08 1083.80 LiP 2867.38±193.59 6667.55±637.62 132.53 The ctivity of Ligninses Enzymes Produced C. cinereus secreted three types of ligninses enzymes on PD, DM nd TE medi. Ligninses ctivity levels on those three medi were vries. Lccse secreted on DM nd PD ws twice s much of tht on TE. MnP secreted on PD ws three times higher thn tht on TE nd four times higher thn tht on DM. LiP production did not show significnt difference mong three medi (P < 0.05), lthough the highest production on PD ws oserved (Tle 2) Tle 2. Ligninses ctivity of C. cinereus on three medi Enzymes Enzyme concentrtion (UmL -1 ) DM PD TE Lccse 377.75 431.19 168.11 MnP 263.88 1286.21 383.58 LiP 3709.92 7042.56 4737.29 DM= Defined Medium, PD= Potto Dextrose roth; TE= ensprout extrct. Numers followed y corresponding letters on the sme row t difference column showed no significnt difference ( P < 0.05) The ility of C. cinereus on Poly R-478 Degrdtion Poly R-478 is chemicl mteril model tht often used to understnd out ligninses ctivity. Degrdtion of Poly R-478 is indicted y color chnge to lighter shde. In this study, the lccse ility of C. cinereus to degrde Poly R-478 ws oserved. The decreses of Poly R-478 concentrtion on medi were vry, the highest ws oserved on medi with the ddition of CuSO 4 (T1), followed sucrose (T3). While medi with the ddition of KNO 3 (T2) showed the lowest reduction (Tle 3) Tle 3. Poly R-478 degrdtion y C. cinereus No Poly R-478concentrtion(mg/L) Poly R-478 Reduction (%) Dy 0 Dy 9 1 T0 74.13 66.50 10.50 c 2 T1 73.32 59.15 19.33 e 3 T2 71.72 69.10 3.59 4 T3 73.82 62.19 15.83 d 5 T4 68.54 63.76 6.84 T0 : medi + fungi; T1 : medi + CuSO 4 + fungi; T2 : medi + KNO 3 + fungi; T3 : medi + sucrose + fungi;t4 : medi + CuSO 4 + KNO 3 + sucrose + fungi. Numers followed y corresponding letters on the sme column showed no significnt difference ( P < 0.05). 63
Suowo The ility of C. cinereus on Plm Oil Mill Effluent Degrdtion. POME contins lignin nd cellulose which resulted in drk rown color nd high COD level. The ppliction of ligninses enzymes produced y C. cinereus ws expected to reduce the rown color nd COD level. The ddition of sucrose into POME () showed higher color reduction (75.26 %) thn ddition of sucrose, CuSO 4, nd KNO 3 (). The COD level reduction on tretment nd ws 85.31% nd 72.82%, respectively (Figure 1). Color reduction (% ) 80 70 60 50 40 30 20 10 0 COD reduction (% ) 90 85 80 75 70 65 Figure 1. Color reduction percentge () nd COD reduction percentge () on POME inoculted with C. cinereus. : POME + sucrose, : POME + sucrose + CuSO 4 + KNO 3 rs mrked with sme letter showed no significnt difference (P<0.05) Steriliztion tretments on POME did not improve the process of decoloriztion. Color decoloriztion oserved in sterilized POME (D) inclined to e higher thn unsterilized POME (E), ut the difference ws not significnt (P < 0.05). The decrese of COD level ws significntly different on sterilized POME (D) nd unsterilized POME (E) (P < 0.05), with the decrese on D ws higher thn tht on E (Figure 2). 100 80 COD reduction (% ) 60 40 20 0 D E Figure 2. Color reduction percentge () nd COD reduction percentge () on sterilized POME (D) nd unsterilized POME (E) inoculted with C. cinereus. rs mrked with sme letter showed no significnt difference (P<0.05) Shking tretment lso influenced POME degrdtion y C. cinereus. The color reduction of POME with shking tretment (F) showed higher (46.13 %) thn without shking (G). The COD level reduction showed contrdictory result. POME without shking (G) (69.31 %) showed higher COD level reduction thn POME with shking (F) (Figure 3). 64
The utiliztion of ink cp mushromm (Coprinus cinereus) Color reduction (% ) 47 46 45 44 43 42 41 40 39 COD reduction (% ) 69,5 69 68,5 68 67,5 67 66,5 38 F G 66 F G Figure 3. Color reduction percentge () nd COD reduction percentge () on POME with shking (F) nd POME without shking (G) inoculted with C. cinereus. rs mrked with sme letter showed no significnt difference (P<0.05) Discussions Defined Medium ws used y Songulshvili et l. (2007) in the inoculum preprtion of some sidiomycetes fungi such s Cerren mxim, Fomes fomentrius, Funli trogii, Gnoderm dspersum, Gnoderm lucidum, Hypsizygus mrmoreus, et ceter. It is lso used in the lccse production y Gnoderm lucidum. In this reserch, DM medi ws used to exmine the ility of C. cinereus to produce ligninses. The result showed tht C. cinereus secreted three types of ligninses, which were lccse, mngnese peroxidise, nd lignin peroxidise. The ddition of CuSO 4 nd KNO 3 s inducers into DM medi ws done to increse lccse ctivity. With inducer ddition, ctivity of lccse incresed y 62,92 % (387.01 U/mL). The presence of Cu ffected lccse production while KNO 3 influenced the production of iomss. ccording to ldrin nd Griel (2002), Cu not only induced lccse gene ut lso ffected the ctivity nd stility of enzyme. The ddition of KNO 3 s source of N incresed iomss production y 41-69 % nd incresed the production of lccse nd MnP (Songulshvili et l., 2007). ccording to Chen et l. (2003) Volvriell volvce in high N concentrtion showed high lccse ctivity nd vice vers. In this reserch, lccse ctivity produced y C. cinereus ws 237.84 ± 18.10 U/mL. It ws higher thn tht on Rymond et l. (2015) which cultivted C.cinereus in textile wste. Lccse ctivity during mycelium growth ws higher thn during fruiting ody formtion. The highest lccse ctivity ws 39,45 ±12,05 U/g. This difference is cused y the type of sustrtes used nd the type of strins. The cultivtion of C. cinereus on DM, PD nd TE ws conducted to determine the est medi to produce ligninse. C. cinereus ws le to grow nd produced three ligninses (lccse, LiP, MnP) on ll three medi. The highest production of lccse ws oserved on PD nd DM, while the lccse ctivity in oth medi showed no significnt difference. The highest production of MnP ws oserved on PD while the production of LiP on ll three medi showed no significnt difference. Generlly, PD ws the most effective medi for ligninses production y C. cinereus. Poly R-478 contining medi ws used to determine the ility of ligninses produced y C. cinereus to degrde Poly R-478. It ws oserved tht there ws difference in Poly R-478 degrding ility fter the ddition of inducers. The presence of inducers incresed ligninses ctivity especilly lccse. These re medi with the highest decline of Poly R-478 concentrtion to the lowest: medi + CuSO 4 (T1), medi + sucrose (T3), medi without inducer (T0). In the medi dded with KNO 3, either individully or mixed, degrdtion occurred in much slower rte (T2 nd T4). The ddition of CuSO 4 incresed lccse ctivity tht resulted in higher Poly R- 478 degrdtion rte. The presence of CuSO 4 lso incresed the ctivity of MnP (Tle 1) which lso ccelerted the degrdtion of Poly R-478. The study of Moreir et l. (2001) used pure enzymes showed tht MnP ws the mjor fctor responsile for the reduction of color in Poly R-478. The ddition of sucrose cn lso increse the degrdtion rte of Poly R-478. Sucrose is one of the source of C on growth nd production of ntifungl gent of Gymnopilus spectilis (Vhidi et l., 2006). The ddition of KNO 3 ostructed the degrdtion of Poly-R. It ws shown in T0 (without inducer) whose degrdtion level ws higher thn tht in medi + KNO 3. High concentrtion of N in the medi inhiits Poly R decoloriztion process y C. cinereus corresponding to Moreir et l. (2004) tht used Trmetes versicolor to decolorize Poly-R478. The est decoloriztion efficiency ws otined in culture with limited nitrogen in n eroic environment. nother fctor is the derivte (NO 2 - ) of 65
Suowo cknowledgement The uthor cknowledge with grtitude for the support nd chnce given y the Hed of Reserch Centre for iology, Indonesin Institute of Sciences nd Prof. Dr. I Mde Sudin M.Sc to conduct this reserch nd to ll tht hve helped nd involved in this reserch. References KNO 3 which not only toxic ut lso cn ffect ligninses ctivity (Moreir et l., 2004) The ddition of sucrose into POME () incresed the ctivity of lccse nd MnP, resulted in ccelerted POME degrdtion. This cn e seen from reduction of COD level nd color of POME. Sucrose is source of C nd energy for C. cinereus which resulted in higher ctivity. This result is in ccondnce to Suowo (2015) which stted tht the ddition of CuSO 4 nd sucrose incresed lccse ctivity which led to ccelerted decoloriztion nd degrdtion of POME y Pleurotus ostretus. While the ddition of CuSO 4, KNO 3 nd sucrose simultneously did not effect on POME degrdtion. POME degrdtion is complex process involving mny enzymes such s ligninses, cellulses, lipses, etc. NO 2 - which derivted from KNO 3 ws toxic nd ntimicroil effect on soil microil ctivity (ncroft et l., 1979). Heting in utoclve reduced initil microes which resulted in condition tht only enzymes from C. cinereus present to degrde POME, ut decoloriztion level from tretment D nd E showed no significnt difference. While significnt difference of COD level cn e oserved on medi with nd without steriliztion. Thus, it cn e concluded tht POME steriliztion do not hve effect on decoloriztion ut ffect COD level. The increse of lccse ctivity due to microe ctivity ws oserved. The result is in ccordnce with ldrin (2004) which reported tht lccse ctivity of Trmetes versicolor nd Pleurotus ostretus incresed fter intercting with soil fungi, yests, cteri or unsterilized soil. The reduction of COD level in tretment D ws the highest (91.26 %) fter 27 dys. The COD reduction in this study took longer time compre to POME degrdtion using Cndid rugos nd Geotrichum cndidum with the result of Cndid rugos: COD (48.6 %), OD (74.5 %) O&G (41.8 %) nd G.cndidum: COD (59.1 %), OD (75.7 %), O&G (59.1 %) fter 144 h (Iegulm-Njoku nd chi, 2014). Shker tretment during incution ffected decoloriztion of POME. POME with shker tretment (F) showed fster decoloriztion. Shking influences the vilility of oxygen in the medi, the distriution of nutrition nd the homogenity of sustrtes. On the contrry, POME without shker tretment (G) showed higher decline of COD level compre to shking condition (F). Degrdtion of lignicellulose occured fster in sttic condition which might e cused y the unifiction of enzymes in sttic stte. The result is contrdictory with the study of Rohill et l (2012) using vrious microorgnisms (spergillus flvus,. niger,. oryze,. tereus) which reported tht dye decoloriztion ws more effective on shker. sed on this reserch, it cn e concluded tht C. cineres produced three ligninses enzymes (Lccse, LiP nd MnP) on DM. The ddition of 200 µm CuSO 4, 10 mm KNO 3 nd 15 g/l sucrose incresed lccse. PD nd DM were suitle medi for C. cinereus to produce ligninses enzymes compred to TE. The ddition of CuSO 4, KNO 3 nd sucrose simultneously into Poly-R medi did not effect on Poly R-478 degrdtion. POME supplemented with sucrose incresed decoloriztion nd reduction of COD level. Steriliztion nd shking tretments incresed color reduction. For future reserch, POME degrdtion using other sidyomycetes tht hve higher lccse ctivity nd other inducers might show etter result. hmd L., nd Chn CY. 2009. Sustinility of plm oil industries: n Innovtive tretment vi memrne technology. 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