Degradation of polychlorinated biphenyl mixtures by the lignin-degrading fungusPhanerochœte chrysosporium

Degradation of lower-chlorinated and higher-chlorinated PCB congeners (Delor 103 and Delor 105 as equivalents of Aroclor 1242 and Aroclor 1254, respectively) by the white-rot fungusPhanerochœte chrysosporium was investigated in N-limited and non-limited media. No degradation of either Delor 103 or Delor 105 was found in a N-limited medium 9 d after their addition whereas in the non-limited medium during the same period their levels dropped by 55 and 58%, respectively. The degradation was non-specific and no significant differences in the degradation of di-, tri-, tetra-, penta-, hexa-, and hepta-congeners were found. No activity of Mn-dependent peroxidase (MnP) or lignin peroxidase (LiP) was detectable in the non-limited medium. We assume that the degradation of PCBs byP. chrysosporium is relatively non-specific, takes place under non-limited conditions and is independent of the activities of MnP or LiP.     

黄孢原毛平革菌木素过氧化物酶类在天然木素降解中作用的研究

通过诱变得到十一株木素过氧化物酶酶活降低的黄孢原毛平革菌（Ｐｈａｎｅｒｏｃｈａｅｔｅｃｈｒｙｓｏｓｐｏｒｉｕｍ）突变株,用灰色理论分析了其木素过氧化物酶类的产生与木素降解能力间的相关性,并从中筛选到一株木素过氧化物酶缺陷、锰过氧化物酶酶活明显降低的突变株,其木素降解能力为原始菌株的８０％左右。该菌粗酶液作用于纤维素酶酶解杉木木素和天然褐腐木素,可产生小分子的木素降解产物,此反应不需Ｈ２Ｏ２参与。红外光谱分析表明粗酶液对木素的作用主要为氧化作用,因此推测此突变株粗酶液中含有不同于木素过氧化物酶和锰过氧化物酶的与木素氧化降解有关的酶类     

Solid state fermentation of Achras zapota lignocellulose by Phanerochaete chrysosporium

The biological transformation of lignocellulose of Achras zapota by white rot fungi, Phanerochaete chrysosporium, in solid state fermentation (SSF) was studied for 28 days. The kinetic transformation of lignocellulose was monitored through the determination of acid soluble and acid insoluble lignin content, total organic carbon (TOC) and chemical oxygen demand (COD). The lignolytic enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP) were quantified on weekly intervals. The degradation of lignin and other structural moieties of A. zapota lignocellulose were confirmed by high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The degradation of lignin was increased after 7 days of fermentation with the release of water soluble and fermentable products. The LiP and MnP activities were increased in the first week of SSF and lignin degradation was also set to increase. This was accompanied with increase in COD by 94.6% and TOC by 80% and lignin content was decreased by 76%. The maximum activities of the enzymes LiP and MnP in extracellular fluid of SSF under nitrogen limitation, at pH 5.0, at temperature 37 degrees C and at 60% humidity were 2100 U/L and 1200 U/L.     

Amelioration of ligninolytic enzyme production by Phanerochaete chrysosporium in airlift bioreactors

Maximum activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) in free cultures of Phanerochaete chrysosporium (ATCC 24725) were 258 U l^–1 and 103 U l^–1, respectively, in an airlift bioreactor. Immobilisation of the fungus on an inert carrier as well as several design modifications of the bioreactor employed gave MnP activities around 500–600 U l^–1 during 9 days' operation. The continuous operation of the latter led to MnP and LiP activities about 140 U l^–1 and 100 U l^–1, respectively, for two months, without operational problems. Furthermore, the extracellular liquid secreted decolourised the polymeric dye Poly R-478 about 56%.     

Biodegradation of polychlorinated biphenyls by plant cells

The PCB biodegradative ability of plant cells cultivated in vitro in media containing a mixture of PCB congeners, Delor 103, is demonstrated. For experiments we used submerged cultures of Armoracia rusticana, Solanum aviculare, Atropa bella-donna, transformed hairy root or embryogenic cultures of Solanum nigrum. Transformation of PCB was followed by gas chromatography after cultivations of the above-mentioned cultures with Delor 103 (10 mg 100 ml⁻¹). The overall PCB metabolizing capability and also degradation of individual congeners greatly differed from strain to strain. The highest capability to metabolize PCB was assayed with differentiated cultures of Solanum nigrum. Beside the capability of PCB degradation, total peroxidase activity in the medium and the cell extract was also followed. Differentiated or hairy root cultures exhibiting higher degradation abilities of PCB also showed increase of peroxidase activities.     

Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw

The white rot fungus Phlebia radiata 79 (ATCC 64658) produces lignin peroxidase (LiP), manganese peroxidase (MnP), glyoxal oxidase (GLOX), and laccase in the commonly used glucose low-nitrogen liquid medium. However, the enzymes which this fungus utilizes for selective removal of lignin during degradation of different lignocellulosic substrates have not been studied before. Multiple forms of LiP, MnP, GLOX, and laccase were purified from P. radiata culture extracts obtained after solid-state fermentation of wheat straw. However, the patterns of extracellular lignin-modifying enzymes studied were different from those of the enzymes usually found in liquid cultures of P. radiata. Three LiP isoforms were purified. The major LiP isoform from solid-state cultivation was LiP2. LiP3, which has usually been described as the major isoenzyme in liquid cultures, was not expressed during straw fermentation. New MnP isoforms have been detected in addition to the previously reported MnPs. GLOX was secreted in rather high amounts simultaneously with LiP during the first 2 weeks of growth. GLOX purified from P. radiata showed multiple forms, with pIs ranging from 4.0 to 4.6 and with a molecular mass of ca. 68 kDa.     

Rapid decolorization of azo dyes by crude manganese peroxidase from Schizophyllum sp. F17 in solid-state fermentation

The production of ligninolytic enzymes by the fungus Schizophyllum sp. F17 using a cost-effective medium comprised of agro-industrial residues in solid-state fermentation (SSF) was optimized. The maximum activities of the enzymes manganese peroxidase (MnP), laccase (Lac), and lignin peroxidases (LiP) were 1,200, 586, and 109 U/L, respectively, on day 5 of SSF. In vitro decolorization of three structurally different azo dyes by the extracellular enzymes was monitored to determine its decolorization capability. The results indicated that crude MnP, but not LiP and Lac, played a crucial role in the decolorization of azo dyes. After optimization of the dye decolorization system with crude MnP, the decolorization rates of Orange IV and Orange G, at an initial dye concentration of 50 mg/L, were enhanced to 76 and 57%, respectively, after 20 min of reaction at pH 4 and 35°C. However, only 8% decolorization of Congo red was observed. This enzymatic reaction system revealed a rapid decolorization of azo dyes with a low MnP activity of 24 U/L. Thus, this study could be the basis for the production and application of MnP on a larger scale using a low-cost substrate.     

Effect of carbon and nitrogen limitation on lignin peroxidase and manganese peroxidaseproduction by Phanerochaete flavido-alba

The ligninolytic enzymes lignin peroxidase (LiP) and manganese dependent peroxidase(MnP), were detected in extracellular fluids of Phanerochaete flavido-alba FPL 106507cultures under carbon or nitrogen limitation. MnP activities were found to be higher than LiPactivities under all growth conditions tested. Higher titres of both peroxidases were obtainedunder carbon limitation in excess nitrogen. Isoelectric points (pIs) observed after FPLC and IEFof concentrated extracellular fluids revealed more acidic pIs for LiP enzymes obtained innitrogen-limited cultures than those in carbon-limited cultures. However, the change in thelimiting growth factor does not significantly affect MnP pIs.     

Optimum stability conditions of pH and temperature for ligninase and manganese-dependent peroxidase from Phanerochaete chrysosporium. Application to in vitro decolorization of Poly R-478 by MnP

Summary In the present work, the two main factors affecting enzymatic stability, i.e. pH and temperature, were analysed in order to determine the optimum ones to maintain ligninase (LiP) and manganese-dependent peroxidase (MnP) activities for prolonged periods of time. The optimum pH and temperature range obtained was around 4.2 and 34 °C for the former and 4.5 and 32 °C for the latter. Under these conditions LiP and MnP showed a half-life time of about 100 and 500 h, respectively. In addition, extracellular liquid containing mainly MnP (200 U/l) was able to decolorize about 20% of the polymeric dye Poly R-478 in 15 min. The decolorization was carried out at a pH of 4.5 (6 mM sodium malonate) and a temperature of 30 °C.     

Degradation of 4-chlorophenol by the white rot fungus Phanerochaete chrysosporium in free and immobilized cultures

4-Chlorophenol (4-CP) degradation was investigated by suspended and immobilized Phanerochaete chrysosporium conducted in static and agitated cultures. The best results were achieved when experiment was carried out in a rotating biological contactor instead of an Erlenmeyer flask, for both batch degradation and repeated batch degradation. The relative contribution of lignin peroxidase (LiP) versus manganese peroxidase (MnP) to the 4-CP degradation by P. chrysosporium was investigated. 4-CP degradation slightly increased and a high level of MnP (38 nKat ml(-1)) was produced when P. chrysosporium was grown at high Mnll concentration. High LiP production in the medium had no significant effect on 4-CP degradation. 4-CP degradation occurred when P. chrysosporium was grown in a medium that repressed LiP and MnP production. This result indicates that LiP and MnP are not directly involved in 4-CP degradation by P. chrysosporium.     

>Decolourisation and depolymerisation of solubilised low-rank coal by the white-rot basidiomycete Phanerochaete chrysosporium

Peroxidases secreted by the white-rot basidiomycete Phanerochaete chrysosporium can oxidise a wide range of recalcitrant compounds including lignin and aromatic xenobiotics. Since low-rank coals such as brown coal and lignite retain structural features of the parent lignin, we investigated the possibility that P. chrysosporium is capable of acting on a brown coal, with the production of useful low-molecular-mass compounds. In nitrogen-limiting liquid medium containing 0.03% solubilised Morwell brown coal, P. chrysosporium was found to convert about 85% of the coal after 16 days incubation to a form not recoverable by alkali-washing and acid-precipitation. The modal molecular mass of the residual coal macromolecules was reduced from the initial 65kDa to 32 kDa. Extensive bleaching of the coal coincided with the presence of extracellular lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP), although both LiP and MnP activity were lower in cultures containing coal. These reductions are accounted for by interference with the enzyme assays by solubilised coal and by binding of MnP to precipitated coal. LiP was about eight times more sensitive than MnP to inhibition by solubilised coal. In nitrogen-sufficient medium containing solubilised coal, neither coal modification nor LiP activity were observed, suggesting that LiP is an essential component of the bleaching process.     

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.     

Removal of Aroclor 1254 by the white rot fungus Coriolus versicolor in the presence of different concentrations of Mn(IV)oxide

Lignin peroxidase (LiP) plays an active role in the biodegradation of lignin and phenolic structures resembling lignin. The role of other enzymes in the biodegradation of recalcitrant compounds, e.g. manganese(II)-peroxidase, is uncertain. Solid manganese(IV)oxide addition improved the production of manganese(II)-dependant peroxidase (MnP) and H₂O₂ and increased the rate of biodegradation of Aroclor 1254 in a nitrogen-limited medium by the white rot fungus Coriolus versicolor. MnP activity was detected 48 h after the addition of MnO₂ to the cultures and was absent in cultures that did not receive MnO₂. The rate of Aroclor 1254 removal by C. versicolor was influenced by the concentration of MnO₂. 34.5 mM concentrations only increased the H₂O₂ production. Removal of Aroclor 1254 in the absence of MnO₂ still took place which implied the presence of (LiP) or nonspecific absorption. The cultures containing 57.5 mM MnO₂ removed ca. 84% of the initial 750 mg l⁻¹ Aroclor in 6 days of incubation. Cultures with no MnO₂ and 34.5 mM removed 79 and 76%, respectively. Cultures with MnP or LiP as the dominant enzyme species removed penta- and hexachlorobiphenyls at a slower rate than tri- and tetrachlorobiphenyl.     

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium.

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.     

Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

The relationship between lignin peroxidase and manganese peroxidase production capacities and cultivation periods of mushrooms

Mushrooms are able to secrete lignin peroxidase (LiP) and manganese peroxidase (MnP), and able to use the cellulose as sources of carbon. This article focuses on the relation between peroxidase-secreting capacity and cultivation period of mushrooms with non-laccase activity. Methylene blue and methyl catechol qualitative assay and spectrophotometry quantitative assay show LiP secreting unvaryingly accompanies the MnP secreting in mushroom strains. The growth rates of hyphae are detected by detecting the dry hyphal mass. We link the peroxidase activities to growth rate of mushrooms and then probe into the relationship between them. The results show that there are close relationships between LiP- and/or MnP-secretory capacities and the cultivation periods of mushrooms. The strains with high LiP and MnP activities have short cultivation periods. However, those strains have long cultivation periods because of the low levels of secreted LiP and/or MnP, even no detectable LiP and/or MnP activity. This study provides the first evidence on the imitate relation between the level of secreted LiP and MnP activities and cultivation periods of mushrooms with non-laccase activity. Our study has significantly increased the understanding of the role of LiP and MnP in the growth and development of mushrooms with non-laccase activity.     

高效木质素降解菌的筛选及其对玉米秸秆的降解效果

本研究利用愈创木酚和苯胺蓝固体培养基对菌株进行初筛,利用形态学和分子生物学对筛选出的菌株进行鉴定,以黄孢原毛平革菌Phanerochaete chrysosporium CGMCC 5.0776为对照,利用其对玉米秸秆进行预处理并测定木质素和纤维素的降解率,测定筛选菌株在预处理玉米秸秆过程中漆酶、锰过氧化物酶（manganese peroxidase,MnP）和木质素过氧化物酶（lignin peroxidase,LiP）活性。结果表明:利用愈创木酚和苯胺蓝固体培养基,从16株白腐真菌菌株中筛选出2株具有较高漆酶或MnP活性的菌株,鉴定其为桦栓孔菌Trametes betulina (L.) Pilát（ZT-153）和亚黑管孔菌Bjerkandera fumosa (Pers.) P. Karst.（ZT-307）,测定T. betulina ZT-153和B. fumosa ZT-307对玉米秸秆酸不溶木质素降解效率分别为13.60%和21.87%,较对照P. chrysosporium CGMCC 5.0776高1.58%和9.85%,对纤维素的降解率较低,分别为4.10%和4.50%。2株菌株在预处理玉米秸秆过程中,T. betulina ZT-153表现出漆酶和MnP活性,B. fumosa ZT-307只表现出LiP活性。其中B. fumosa ZT-307对玉米秸秆酸不溶木质素的降解效率最高,在秸秆资源的综合利用方面具有较好的潜力和应用前景。     

Production of manganese-dependent peroxidase in a new solid-state bioreactor by Phanerochaete chrysosporium grown on wood shavings. Application to the decolorization of synthetic dyes

The production of manganese-dependent peroxidase (MnP) byPhanerochœte chrysosporium in a new solid-state bioreactor, the immersion bioreactor, operating with lignocellulosic waste, such as wood shavings, was investigated. Maximum MnP and lignin peroxidase (LiP) activity of 13.4 and 8.48 μkat/L were obtained, respectively. Thein vitro decolorization of several synthetic dyes by the extracellular liquid produced in the above-mentioned bioreactor (containing mainly MnP) was carried out and its degrading ability was assessed. The highest decolorization was reached with Indigo Carmine (98%) followed by Bromophenol Blue (56%) and Methyl Orange (36%), whereas Gentian Violet was hardly decolorized (6%).     

Characterization of polychlorinated biphenyl-degrading bacteria isolated from contaminated sites in Czechia

Biphenyl-utilizing polychlorinated biphenyls (PCB)-degrading bacteria were isolated from sites highly contamined by PCBs, and their degradation abilities were determined using GC for typical commercial PCB mixtures (Delor 103 and Delor 106). Out of twelve strains which utilized biphenyl as a sole source of carbon and energy, strainsPseudomonas alcaligenes KP2 andP. fluorescens KP12, characterized by the BIOLOG identification system and the NEFERM test, were shown to significantly co-metabolize the PCB mixture Delor 103. DNA-DNA hybridization was used to compare both strains with well-known PCB-degradersBurkholderia cepacia strain LB400 andRalstonia eutropha strain H850. The strain KP12 employs the samemeta-fission route for degradation of chlorobenzoates as a chlorobiphenyl degraderPseudomonas cepacia P166. Both isolates KP2 and KP12 belong to different phylogenetic groups, which indicates that the same geographical location does not ensure the same ancestor of degradative enzymes. We confirmed that also highly chlorinated and the most toxic congeners, which are contained in commercial PCB mixtures, can be biotransformed by members of indigenous bacterial-soil community under aerobic conditions.