the i. lacteus strain kr 35w was maintained on mgpy ( 1% malt extract , 1% glucose , 0.5% peptone , and 0.5% yeast extract ) agar slants at 4. the fungal inocula were prepared in 250 ml erlenmeyer flasks , containing 100 ml of mgpy medium , for 7 days . four mycelial agar discs ( 0.9 cm ) were obtained from a fresh mgpy agar culture for use as the inocula . the liquid inocula were gently homogenized , and used at a 10% ( v / v ) dilution . the stationary cultures were performed using 50 ml medium in 250 ml erlenmeyer flasks , containing 100 ppm of tnt ( supelco , usa ) , at 28 , as described in the literature ( shin , 2004 ) . to estimate the degradation rate of tnt , the contents of each flask were extracted three times with 30 ml of methylene chloride . the resulting extracts were resuspended with 10 ml of methanol and analysed by reverse - phase hplc ( shimadzu , japan ) with a shim - pack clc - ods ( m ) column ( 4.6  250 mm ) . elution was performed by a linear gradient of 20% acetonitrile containing 1% acetic acid , increased to 90% after 60 min at a flow rate of 1 ml / min . gc - ms to identify and verify tnt metabolites was performed using gc - ms ( hp5980 series gc - msd ; hewlett packard ) . the hp5-ms column ( 60 m , 0.25 mm i.d . the temperature program started at 50 and was held for 1 min in splitless mode . then the splitter was opened and the oven was heated to 120 at a rate of 12/min . the second temperature ramp was up to 200 at a rate of 15/min and the final temperature ramp was up to 300 at a rate of 20/min , this temperature being maintained for 15 min . the i. lacteus strain kr 35w was maintained on mgpy ( 1% malt extract , 1% glucose , 0.5% peptone , and 0.5% yeast extract ) agar slants at 4. the fungal inocula were prepared in 250 ml erlenmeyer flasks , containing 100 ml of mgpy medium , for 7 days . four mycelial agar discs ( 0.9 cm ) were obtained from a fresh mgpy agar culture for use as the inocula . the liquid inocula were gently homogenized , and used at a 10% ( v / v ) dilution . the stationary cultures were performed using 50 ml medium in 250 ml erlenmeyer flasks , containing 100 ppm of tnt ( supelco , usa ) , at 28 , as described in the literature ( shin , 2004 ) . to estimate the degradation rate of tnt , the contents of each flask were extracted three times with 30 ml of methylene chloride . the resulting extracts were resuspended with 10 ml of methanol and analysed by reverse - phase hplc ( shimadzu , japan ) with a shim - pack clc - ods ( m ) column ( 4.6  250 mm ) . elution was performed by a linear gradient of 20% acetonitrile containing 1% acetic acid , increased to 90% after 60 min at a flow rate of 1 ml / min . gc - ms to identify and verify tnt metabolites was performed using gc - ms ( hp5980 series gc - msd ; hewlett packard ) . the hp5-ms column ( 60 m , 0.25 mm i.d . the temperature program started at 50 and was held for 1 min in splitless mode . then the splitter was opened and the oven was heated to 120 at a rate of 12/min . the second temperature ramp was up to 200 at a rate of 15/min and the final temperature ramp was up to 300 at a rate of 20/min , this temperature being maintained for 15 min . a hplc - chromatogram of the solution containing tnt at the beginning of the experiment showed a single peak , which eluted after 39.7 min ( fig . after incubation with i. lacteus , the intensity of tnt was decreased according to the incubation time and another two peaks were appeared . tnt concentration decreased with a significant rate and about 95% of tnt disappeared after 48 h. during the degradation of tnt by i. lacteus , the residual tnt and degradation products were monitored through gc - ms analysis . as shown in fig . 2 , tnt was converted to about six degradation products over 24 h that eluted after 16.9 , 17.6 , 17.7 , 17.8 , 18.1 , and 18.6 min respectively . the abundances of tnt and other degradations products were decreased to timedependent manner , suggesting that i. lacteus may have an ability to degrade tnt and intermediates completely . two intermediates in fig . 2 were identified as 4-amino-2,6-dinitrotoluene ( 4-adnt , peak 2 ) and 2-amino-4,6-dinitrotoluene ( 2-adnt , peak 3 ) by comparison with their corresponding standards using retention times and molecular mass ions ( fig . numerous authors have demonstrated the biodegradation of tnt by the white - rot fungi ( fernando et al . , 1990 ; michels and gottschalk , 1995 ; van aken et al . , 1999 ) . the initial steps in the degradation of tnt involved the reduction of nitro groups ( parrish , 1977 ; rieble et al . , 1994 ) . further degradation of these compounds and mineralization occurred under ligninolytic conditions , suggesting the involvement of ligninolytic enzymes ( parrish , 1977 ) . among these enzyme systems the preparations of mnp from white - rot fungi , nematoloma frowardii and phlebia radiata , were able to mineralize tnt and a mixture of reduction products from tnt ( scheibner et al . , 1997 ; scheibner and hofrichter , 1998 ; van aken et al . , 1999 ) . however , lip catalyzed oxidation of early tnt metabolites leading to the corresponding nitroso - dinitrotoluenes , but not capable of oxidizing the 4-adnt and 2-adnt ( michels and gottschalk , 1995 ) . although we could identify only two metabolites , 4-adnt and 2-adnt in this experiment , the degradation process of tnt by i. lacteus was seemed to be similar to that of other white - rot fungi . previously , we have reported that mnp of i. lacteus played a major role in the decolorization of textile industry wastewaters ( shin , 2004 ) and purified mnp ( 53 kda ) catalyzed oxidation of various dyes ( shin et al . , white - rot fungus i. lacteus is a good candidate for the biodegradation of tnt as well as its reduction metabolites . furthermore the mnp of this fungus together with lip may involve in the mineralization of tnt .
white - rot fungus irpex lacteus degraded tnt significantly in proportion to the culture time . 
 after 48 h incubation , about 95% of tnt was degraded . 
 two reduced metabolites were identified as 4-amino-2,6-dinitrotoluene ( 4-adnt ) and 2-amino-4,6-dinitrotoluene ( 2-adnt ) which was further degraded .