Search

Biological pretreatment of lignocellulose by white-rot fungi is an environmentally friendly alternative to chemical and physical approaches to enhance enzymatic saccharification. However, inefficient lignin degradation and substantial cellulose consumption during fungal pretreatment can cause low sugar yields. In this study, the combined action of CuSO4 and MnSO4 effectively improved the degree and selectivity of delignification during solid-state fungal pretreatment of poplar wood by Phanerochaete chrysosporium. Compared to pretreatment without this supplementation, a 1.9 fold higher lignin degradation and 2.4 times higher delignification selectivity value were obtained due to the increased and prolonged manganese peroxidase activity. Enzymatic saccharification of supplemented pretreated wood resulted in a 2.9 times higher glucose yield compared to the non-supplemented system. This study has demonstrated that the combined application of Mn2+ and Cu2+ can significantly improve the fungal pretreatment process and that the beneficial effect of Cu2+ on delignification is not restricted to laccase-producing fungi.

The effect of different organic, inorganic nitrogen sources and trace elements on growth and ligninolytic enzymes production by Grammothele fuligo has been investigated. Amongst all the nitrogen compounds used, the most favourable for growth was ammonium oxalate. It showed maximum Lignin Peroxidase activity (80.6 IU/mL) with ammonium chloride. The optimum Manganese Peroxidase activity (4.13 IU/mL) was observed with ammonium acetate. DL-alanine served as the best organic nitrogen source for the growth. The highest MnP (36.7 IU/mL) and laccase (3921.5 IU/mL) activities were revealed in medium supplemented by DL-tryptophan. However, their positive effects on enzyme accumulation were due to a higher biomass production. The higher concentrations of trace elements were found to be fungistatic for its growth viz. B, Co, Cu, Fe (400 ppm) and Co (100 ppm). It exhibited maximum LiP activity (456.9 IU/mL) with 10−3 ppm Fe and MnP activity (3.30 IU/mL) with 10−6 ppm B and 10−3 ppm Ca. The maximum laccase activity (653.5 IU/mL) was observed with 10−6 ppm Cu. This is the first report on nitrogen sources and trace elements effect on ligninolytic enzymes production of Grammothele. The results will facilitate research to understand the nature of the fungus and to increase its enzymes production under controlled conditions.

Harvesting whole-tree biomass for biofuel combustion intensifies removal of nutrients from the ecosystem. This can be partly abated by applying ash from the combustion back to the system, as the ash is rich in nutrients. Ash is very alkaline and ash application raises soil pH, which in turn can stimulate microbial activity and thus decomposition and mineralization. Our aim was to test if ash induced decomposition activity was associated with enhanced turnover of recalcitrant, i.e. relatively old, organic pools. Two experiments were conducted in the same coniferous plantation after the application of 0, 3, 4.5 and 6 t ash ha −1, and 0, 3, 9, 15 and 30 t ash ha −1, respectively. We used natural abundance of 15N in mosses, mites and ectomycorrhizal fungi 26 months after ash application, as well as temporal variation in δ 15N values of ectomycorrhizal fungi, as an indicator of decomposition of recalcitrant organic matter in the first experiment. Furthermore, in the second experiment we used measurements of extracellular manganese peroxidase activity almost 4 years after ash application as an indication of potential decomposition of lignin, an important component of recalcitrant organic matter. The δ 15N signature increased significantly for ectomycorrhizal fungi, dead moss, Nothroid and Gamasida mites, and manganese peroxidase activity tended to increase, with increasing ash doses. This suggests that ash application stimulates turnover of recalcitrant organic matter, which can increase the available pool of nitrogen in the system. This will potentially enhance the fertilizer value of ash. However, the δ 15N in ectomycorrhizal fungi tended to peak at 18 months after ash application, before decreasing, suggesting that the turnover of recalcitrant organic matter is reduced again with time.

Soil microorganisms play an important role in the degradation of PAHs and use various metabolic pathways for this process. The effect of soil pH, different soil amendments and the co-cultivation of fungi on the degradation of PAHs in soil and on the activity of ligninolytic enzymes was evaluated. For that purpose, three fungi were studied: Trichoderma viride, Penicillium chrysogenum and Agrocybe aegerita. Biodegradation assays with a mixture of 200 ppm PAHs (fluorene, pyrene, chrysene, and benzo[a]pyrene---50 ppm each) were set up at room temperature for 8 weeks. The maximum laccase activity by solid state fermentation---SSF (7.43 U/g) was obtained by A. aegerita on kiwi peels with 2 weeks and the highest manganese peroxidase activity (7.21 U/g) was reached in 4 weeks, both at pH 7. Fluorene, pyrene, and benzo[a]pyrene achieved higher degradation rates in soil at pH 5, while chrysene was more degradable at pH 7. About 85--90\% of the PAHs were degraded by fungal remediation. The highest degradation of fluorene was achieved by co-cultivation of A. aegerita and P. chrysogenum, remaining 14\% undegradable. Around 13\% of pyrene stay undegradable by A. aegerita and T. viride and by A. aegerita and P. chrysogenum, both systems supported in kiwi peels, while 11\% of chrysene remained in soil by the co-cultivation of these fungi, supported by peanut shells. Regarding benzo[a]pyrene, 13\\% remained in soil after treatment with A. aegerita. Despite the increase in degradation of some PAHs with co-cultivation, higher enzyme production during degradation was observed when fungi were cultivated alone., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the research project PTDC/AAG-TEC/5269/2014, the strategic funding of UID/BIO/04469/2013 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Ziva Vipotnik is a recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) - Doctoral Program in Applied and Environmental Microbiology (DP_AEM); operation NORTE-08-5369-FSE000060; co-financed by North 2020 through the European Social Fund (ESF)., info:eu-repo/semantics/publishedVersion

To investigate the effects of the mixing of litters on their remediation efficiency in petroleum-contaminated soil, litters from two common plants in the petroleum-contaminated region of Northern Shaanxi, China, Bothriochloa ischaemum (L.) Keng and Sophora davidii Kom. ex Pavol., and their mixture were mixed with 45 g/kg petroleum-contaminated soil. Based on these, a 150-day simulated remediation experiment was conducted at 25 °C and consistent moisture conditions. The effects on the degradation of petroleum components and the restoration of nutrient contents, pH, and enzymatic activity in the disturbed soil were detected. The effects of the litter treatments on the community structure and carbon source utilization characteristics of soil microorganisms were also studied. The results indicated that all litter treatments significantly accelerated the degradation of petroleum components, while the mixing of litter exhibited significant synergistic effects, leading to significantly higher degradation rates of saturated hydrocarbons, aromatic hydrocarbons, and nonhydrocarbon substances than the observed rates in the single-litter treatments and the predicted rates based on the single-litter treatments. Litter treatment significantly increased the N and P contents and enzymatic activity of contaminated soil. The effects of mixed litter on soil chemical and biological properties fell between the effects of the 2 types of single-litter treatments. However, the mixing of litters exhibited significant synergistic effects in supplementing available P and increasing sucrase, dehydrogenase, lignin peroxidase, and laccase activity, while it exhibited significant antagonistic effects in supplementing nitrate nitrogen and increasing urease, phosphatase, polyphenol oxidase, and manganese peroxidase activity. Litter treatment significantly altered the community structure of soil microorganisms. The relative abundances of some petroleum-degrading microbial phyla or genera in mixed litter-treated soil were significantly different from those in single litter-treated soils, which might contribute to the strengthened remediation effects of mixed litter treatment. The results might provide a theoretical basis for the more effect utilization of biomass resources in the remediation of petroleum-contaminated soil.

The aim of this research is to isolate and identify fungi with high lignin-degrading abilities that are autochthonous to southern Serbian region. Two novel fungal isolates identified as Trametes hirsuta F13 and Stereum gausapatum F28 were selected to assess their ligninolytic enzyme activities and the efficiency of lignin removal from beech wood sawdust. Obtained results show that both isolates are good sources of industrially valuable enzymes with a potential for application in various biotechnological and industrial processes. Both isolates showed laccase, manganese-dependent peroxidase, and versatile peroxidase activities, while only S. gausapatum F28 had lignin peroxidase activity. This is the first record of the ability of S. gausapatum species to produce lignin peroxidase. T. hirsuta F13 showed higher laccase activity than S. gausapatum F28, while S. gausapatum F28 had higher manganese peroxidase activity. Also, T. hirsuta F13 exhibited much higher laccase activity under submerged cultivation conditions than solid-state cultivation conditions, which is rare for fungi. This is important for industrial processes since the submerged fermentation is a dominant technique in industry. The test of the efficiency of lignin removal showed that both isolates are efficient lignin decomposers. After five weeks of incubation on beech wood sawdust, the total lignin losses were 33.84 % with T. hirsuta F13 and 28.8 % with S. gausapatum F28.

The aim of this work was to study the potential of the non-lignolytic filamentous fungus Penicillium sp. CHY-2, isolated from Antarctic soil, for the biodegradation of eight different aliphatic and aromatic hydrocarbons such as octane, decane, dodecane, ethylbenzene, butylbenzene, naphthalene, acenaphthene, and benzo[a]pyrene. Among all the compounds, CHY-2 showed the highest level of degradation for decane (49.0%), followed by butylbenzene (42.0%) and dodecane (33.0%), and lower levels of degradation for naphthalene (15.0%), acenaphthene (10.0%), octane (8.0%), ethylbenzene (4.0%), and benzo[a]pyrene (2.0%) at 20 °C. The addition of carbon sources such as glucose (5 g L−1) and Tween-80 (5 g L−1) enhanced decane degradation by about 1.8-fold and 1.61-fold respectively at 20 °C. The metabolites produced during the degradation of decane were identified by gas chromatography-mass spectrometry (GC-MS). Furthermore, the enzyme manganese peroxidase (MnP) from CHY-2 was purified. MnP was found to consist of monomers with a molecular mass of 36 kDa. The purified MnP had an optimum pH of 5.0 and temperature of 30 °C. The Km and Vmax values of MnP towards Mn2+ were 1.31 μM and 185.19 μM min−1 respectively. These results indicated that the strain CHY-2 can be used for the degradation of hydrocarbons and could have promising applications in treatment of hydrocarbon contaminated sites.

Nanodiamonds (NDs) are produced with large scale and applied in many areas, thus the environmental impacts and hazards of NDs should be systematically investigated. In this study, we evaluated the interaction between detonation NDs and white rot fungus Phanerochaete chrysosporium and the impact on the fungus decompositions activities. NDs did not influence the biomass gain of P. chrysosporium and the culture medium pH values. The mycelia of P. chrysosporium were destroyed upon the direct contact with NDs, while the rest retained the fibrous structure. Ultrastructural observations suggested that small aggregates of NDs seldom entered the fungus cells, but the break of cell wall and the loss of cytoplasm were induced by NDs. Under both optical and electron microscopes, the aggregation of colloidal ND particles was observed, which was the possible reason of low toxicity. High concentrations of NDs inhibited the laccase activity and manganese peroxidase activity of P. chrysosporium, which led to the decrease of decomposition activity for pollutants. Colloidal ND particles were not well dispersed in sawdust degradation evaluations, so no inhibitive effect was observed for wood degradation. The toxicological mechanism of NDs was assigned to oxidative stress. The results collectively suggested that NDs had low toxicity to white rot fungi and could be applied safely. The colloid dispersion/aggregation of nanoparticles in biological systems should be carefully considered during the design of safe nanomaterials.

Deadwood is an important structural component in forest ecosystems and plays a significant role in global carbon and nutrient cycling. Relatively little is known about the formation and decomposition of CWD by microbial communities in situ and about the factors controlling the associated processes. In this study, we intensively analyzed the molecular fungal community composition and species richness in relation to extracellular enzyme activity and differences in decomposing sapwood and heartwood of 13 temperate tree species (four coniferous and nine deciduous species, log diameter 30–40 cm and 4 m long) in an artificial experiment involving placing the logs on the forest soil for six years. We observed strong differences in the molecular fungal community composition and richness among the 13 tree species, and specifically between deciduous and coniferous wood, but unexpectedly no difference was found between sapwood and heartwood. Fungal species richness correlated positively with wood extractives and negatively with fungal biomass. A distinct fungal community secreting lignocellulolytic key enzymes seemed to dominate the decomposition of the logs in this specific phase. In particular, the relative sequence abundance of basidiomycetous species of the Meruliaceae (e.g. Bjerkandera adusta) correlated with ligninolytic manganese peroxidase activity. Moreover, this study reveals abundant white-rot causing Basidiomycota and soft-rot causing Ascomycota during this phase of wood decomposition.

White-rot fungi are distinguished by their ability to efficiently degrade lignin via lignin-modifying type II peroxidases, including manganese peroxidase (MnP) and lignin peroxidase (LiP). In the present study, time-of flight secondary ion mass spectrometry (ToF-SIMS) was used to evaluate lignin modification in three coniferous and three deciduous wood preparations following treatment with commercial preparations of LiP and MnP from two different white-rot fungi. Percent modification of lignin was calculated as a loss of intact methoxylated lignin over nonfunctionalized aromatic rings, which is consistent with oxidative cleavage of methoxy moieties within the lignin structure. Exposure to MnP resulted in greater modification of lignin in coniferous compared to deciduous wood (28 vs. 18 % modification of lignin); and greater modification of G-lignin compared to S-lignin within the deciduous wood samples (21 vs. 12 %). In contrast, exposure to LiP resulted in similar percent modification of lignin in all wood samples (21 vs 22 %), and of G- and S-lignin within the deciduous wood (22 vs. 23 %). These findings suggest that the selected MnP and LiP may particularly benefit delignification of coniferous and deciduous wood, respectively. Moreover, the current analysis further demonstrates the utility of ToF-SIMS for characterizing enzymatic modification of lignin in wood fibre along with potential advantages over UV and HPCL-MS detection of solubilized delignification products.

Noncovalent second-shell interactions are important in controlling metal-binding affinity and activity in metalloenzymes, but fine-tuning these interactions in designed metalloenzymes has not been fully explored. As a result, most designed metalloenzymes have low metal-binding affinity and activity. Here we identified three mutations in the second coordination shell of an engineered Mn(II)-binding site in cytochrome c peroxidase (called MnCcP.1, containing Glu45, Glu37, and Glu181 ligands) that mimics the native manganese peroxidase (MnP), and explored their effects on both Mn(II)-binding affinity and MnP activity. First, removing a hydrogen bond to Glu45 through Tyr36Phe mutation enhanced Mn(II)-binding affinity, as evidenced by a 2.8-fold decrease in the KM of Mn(II) oxidation. Second, introducing a salt bridge through Lys179Arg mutation improved Glu35 and Glu181 coordination to Mn(II), decreasing KM 2.6-fold. Third, eliminating a steric clash that prevented Glu37 from orienting toward Mn(II) resulted in an 8.6-fold increase in kcat/KM, arising primarily from a 3.6-fold decrease in KM, with a KM value comparable to that of the native enzyme (0.28 mM vs 0.19 mM for Pleurotus eryngii MnP PS3). We further demonstrated that while the effects of Tyr36Phe and Lys179Arg mutations are additive, because involved in secondary-shell interactions to different ligands, other combinations of mutations were antagonistic because they act on different aspects of the Mn(II) coordination at the same residues. Finally, we showed that these MnCcP variants are functional models of MnP that mimic its activity in both Mn(II) oxidation and degradation of a phenolic lignin model compound and kraft lignin. In addition to achieving KM in a designed protein that is similar to the that of native enzyme, our results offer molecular insight into the role of noncovalent interactions around metal-binding sites for improving metal binding and overall activity; such insight can be applied to rationally enhance these properties in other metalloenzymes and their models.

The present study was carried out to isolate rubber degrading bacteria and to check the enzymes responsible for degradation, so that the isolates can be effectively used in rubber degradation. Different bacteria were isolated from soil samples and natural rubber samples collected from a rubber processing unit, and were screened for their ability to degrade natural rubber by plate assay. A biodegradation experiment was carried out in the laboratory to confirm degradation. Enzymes responsible for natural rubber degradation were characterized, crude enzyme was extracted by submerged fermentation process and its activity was determined. After extraction of crude enzyme, it was partially purified by dialysis process, and then, the enzyme activity and specific activity were determined. Then, the ability of these enzymes to degrade natural rubber discs were checked. The ability of natural rubber degrading microorganisms to degrade rubber products such as rubber gloves discs and rubber tire discs, was tested. Among the isolated bacterial species, Bacillus subtilis was predominant and showed growth on the natural rubber discs. The biodegradation experiment showed 48.6 % weight loss. It was screened that laccase and manganese peroxidase enzymes are responsible for natural rubber degradation. When enzyme activity was determined, Bacillus subtilis, showed more manganese peroxidase activity compared to laccase activity. Laccase enzyme activity was 0.0138 IU and manganese peroxidase activity was 0.0155 IU. The molecular weights of laccase enzyme (64 kDa) and of manganese peroxidase enzyme (68 kDa) were determined. Biodegradation of rubber by microorganisms was confirmed by Schiff’s staining, SEM, FTIR and NMR studies.

The release of carbonaceous nanomaterials (CNMs) into the environment inevitably affects the surrounding microenvironment, biochemical processes of microbes, and even entire ecosystems. However, the knowledge of the impact of CNMs on the environmental microbial activities and further natural organic matter biodegradation is still less known. Here we investigated the impact of three CNMs (single-walled carbon nanotubes, graphene, and oxidized graphene) on extracellular enzymes activities of a fungal strain (Cladosporium sp.) and lignin biodegradation. All three CNMs increased the activities of three non-specific enzymes (laccase, manganese peroxidase, and lignin peroxidase) to different degrees, among which manganese peroxidase activity increased significantly, with an average promotion of 20% within 18 days. Meanwhile, structural characterization revealed the transformations of CNMs caused by fungal biodegradation. CNMs stimulation fungal enzyme activities further enhanced lignin degradation. Adsorption experiments and electrochemistry analysis demonstrated the mechanism that CNMs acted as adsorbent of extracellular enzymes as well as electron conductors, which enhanced extracellular direct electron transfer between enzymes and lignin and facilitated lignin conversion and degradation. Our results highlight the potential effect of CNMs on the lignin degradation by fungi, and require consideration when evaluating the environmental effects of CNMs on microbial communities or even carbon cycling across whole ecosystems.

Two dominant cyanobacterial species, Phormidium lucidum and Oscillatoria subbrevis, isolated from submerged polyethylene carry bags in domestic sewage water were found to be capable of degrading low-density polyethylene (LDPE) sheets efficiently. The FT-IR, SEM, NMR, CHN content, thermal, and tensile strength of PE were monitored for structural, morphological, and chemical changes of PE. The CHN analysis corroborated about 4% carbon utilization by the cyanobacterial species from the PE. The rapid growth of cyanobacterial species on the PE surface suggested that the microorganisms continued to gain energy from the PE. The reduction in lamellar thickness, weight, and crystallinity of the cyanobacterial-treated PE pointed to an efficient biodegradation process without any pro-oxidant additives or pretreatment. Alteration in bond indices computed from FT-IR spectroscopy revealed changes in functional group and side chain features indicating biodegradation. The enhanced laccase and manganese peroxidase activity corroborated the biodegradation. The 13C-NMR spectroscopy of the PE is consistent with short branching providing further evidence of biodegradation. Scanning electron microscopy and optical microscopy exhibited large grooves on the surface suggesting significant disruption of polyethylene structure.

To identify white rot fungi with high potential in biological pretreatment of lignocellulosic biomass, preliminary screening was carried out on plates by testing different strains for their ability to oxidize guaiacol and decolorize the dyes azure B and Poly R-478. Of the 86 strains screened, 16 were selected for secondary screening for their ligninolytic ability; however, low manganese peroxidase activity and no lignin peroxidase activity were detected. Strain BBEL0970 proved to be the most efficient in laccase production and was subsequently identified as Trametes versicolor by analysis of the ribosomal DNA internal transcribed spacer gene sequence. In combining laccase production with biological pretreatment, the replacement of glucose with barley straw significantly improved the laccase activity by up to 10.3 U/mL, which provided evidence toward potential utilization of barley straw in laccase production by BBEL0970. Simultaneously, comparison by thermogravimetric analysis of the untreated and pretreated barley straw in liquid fermentation of laccase also demonstrated the high potential of BBEL0970 in biological pretreatment of lignocellulosic biomass. This work sheds light on further exploration on the integrated process of low-cost laccase production and efficient biological pretreatment of barley straw by T. versicolor BBEL0970.

The effect of different basal media, temperature, pH, days of incubation, carbon and nitrogen sources was studied on growth, reproduction and ligninolytic enzymes production of Dictyoarthrinium synnematicum Somrith. The fungus reproduced by chlamydospores and produced maximum growth in Glucose-peptone medium. It produced chlamydospores only at 24℃ and pH range of 5-9. The optimum mycelial growth rate was observed at 24℃ and pH 5 after 16 days of incubation. It showed gradual increase in the production of Lignin peroxidase up to 30 days, Manganese peroxidase up to 18 days of incubation after which it decreased and Laccase up to 22 days. The optimum growth and laccase activity were attained in lactose supplemented medium whereas the highest Lignin peroxidase and Manganese peroxidase activity was observed with glucose. It produced chlamydospores with fructose, glucose, maltose, sucrose and xylose. Sodium nitrate is the best inorganic nitrogen source for growth. Resting spores were formed in inorganic and organic nitrogen sources used in basal medium. Ammonium phosphate and potassium nitrate supported highest activity of Lignin peroxidase whereas best Manganese peroxidase activity was exhibited with ammonium oxalate and laccase with sodium nitrate. The fungus did not express ligninolytic enzymes activity with any organic nitrogen source.

In this study it has firstly reported the using of immobilized fungal mycelia in fungal co-culture studies to enhance activity of lignin-modifying enzymes. For this purpose, the mycelia of Pleurotus ostreatus OBCC 6043 and Lentinus tigrinus OBCC 3007 were immobilized on nylon scouring pad as carrier material. . The immobilized mycelia of the fungi were compared in terms of their laccase and manganese peroxidase activities in mono- and co-culture conditions. The maximum laccase activities of pure Pleurotus ostreatus OBCC 6043 and Lentinus tigrinus OBCC 3007 cultures were determined as 53.73 and 27.58 U/L, respectively, while the maximum manganese peroxidase activities were 12.54 and 52.02 U/L. In co-culture conditions, distinct enhancement was observed in laccase and manganese peroxidase activities as 319.28 and 554.33 U/L, respectively. In the case of laccase, enzyme activity was 5.94 and 11.58 times higher than that of Pleurotus ostreatus OBCC 6043 and Lentinus tigrinus OBCC 3007 mono-cultures, respectively. On the other hand, manganese peroxidase activity could be improved distinctly, 44.21 and 10.66 fold higher values than the corresponding ones. The results of the present co-culture study are significantly higher than most of the reported results in the literature, not only for laccase but also for manganese peroxidase activity., Bu çalışmada lignin modifiye edici enzimlerin fazla miktarda üretilmesi için immobilize fungus misellerinin birlikte kültür edilmesi ilk kez olarak rapor edilmiştir. Pleurotus ostreatus OBCC 6043 ve Lentinus tigrinus OBCC 3007 miselleri taşıyıcı olarak kullanılan naylon temizlik süngeri üzerine immobilize edilmiştir. Fungusların immobilize misellerinin ayrı ayrı ve birlikte kültür edilmeleri durumundaki lakkaz ve mangan peroksidaz aktiviteleri karşılaştırılmıştır. Saf Pleurotus ostreatus OBCC 6043 ve Lentinus tigrinus OBCC 3007 kültürlerinin maksimum lakkaz aktiviteleri, sırası ile, 53.73 ve 27.58 U/L iken maksimum mangan peroksidaz aktiviteleri 12.54 ve 52.02 U/L olarak belirlenmiştir. Kokültür koşullarında lakkaz ve mangan peroksidaz aktiviteleri belirgin bir artış ile 319.28 ve 554.33 U/L seviyesine yükselmiştir. Pleurotus ostreatus OBCC 6043 ve Lentinus tigrinus OBCC 3007 birlikte üretildiklerinde lakkaz aktivitesi ayrı ayrı kültür edilmelerine göre, sırası ile, 5.94 ve 11.58 kat fazla bulunmuştur. Diğer taraftan mangan peroksidaz aktivitesi aynı sıra ile 44.21 ve 10.66 kat gibi belirgin biçimde arttırılabilmiştir. Sunulan birlikte kültür çalışmasının sonuçları, hem lakkaz hem de mangan peroksidaz için literatürde sunulan değerlerin çoğuna göre önemli derecede yüksektir., DOI: 10.15318/Fungus.2017.46

This study investigated pretreatment of corn stover using solid substrate cultivation (SSC) of Phanerochaete chrysosporium (P. chrysosporium) to improve subsequent accessibility to cellulose. Thereafter, Clostridium thermocellum (C. thermocellum) was directly inoculated onto the pretreated biomass to accomplish hydrolysis, followed by solventogenesis by introducing Clostridium beijerinckii (C. beijerinckii). An enzyme suite containing laccase, lignin peroxidase and manganese peroxidase activity was detected during the cultivation of P. chrysosporium on corn stover within 288 h at an initial moisture content w H 2 O = 80% (mass of water/total mass). Incubation factors, such as substrate moisture content and cultivation temperature affected the percent of lignin removal which ranged from 14.4% to 36.4% of the original lignin. Lignin removal increased as the cultivation of P. chrysosporium continued but was accompanied by increased cellulose loss. The 7-day fungal cultivation sufficiently delignified the corn stover for the subsequent processing. Approximately 25% of the original lignin was removed; however 18% of the initial cellulose was also removed with the lignin. The investigations of the effect of fungal pretreatment were extended to miscanthus, wheat straw and switch grass. The yield of reducing sugar produced by C. thermocellum on pretreated biomass was doubled compared with non-pretreated biomass, demonstrating that pretreatment resulted in a more accessible carbon source for the solvent-producing bacterium. The final comprehensive comparison between the pretreated biomass and non-pretreated biomass on the three-stage SSC for butanol production showed pretreatment by P. chrysosporium improved microbial utilization of lignocellulosic materials for solvent production by approximate 4–7 folds.

Corn silage is used as high-energy forage for dairy cows and more recently for biogas production in a process of anaerobic co-digestion with cow manure. In this work, fresh corn silage after the harvest was used as a substrate in solid-state fermentations with T. versicolor with the aim of phenolic acid recovery and enzyme (laccase and manganese peroxidase) production. During 20 days of fermentation, 10.4-, 3.4-, 3.0-, and 1.8-fold increments in extraction yield of syringic acid, vanillic acid, p-hydroxybenzoic acid, and caffeic acid, respectively, were reached when compared to biologically untreated corn silage. Maximal laccase activity was gained on the 4th day of fermentation (V.A. = 180.2 U/dm3), and manganese peroxidase activity was obtained after the 3rd day of fermentation (V.A. = 30.1 U/dm3). The addition of copper(II) sulfate as inducer during solid state fermentation resulted in 8.5- and 7-fold enhancement of laccase and manganese peroxidase activities, respectively. Furthermore, the influence of pH and temperature on enzyme activities was investigated. Maximal activity of laccase was obtained at T = 50 °C and pH = 3.0, while manganese peroxidase is active at temperature range T = 45–70 °C with the maximal activity at pH = 4.5.

The effect of altering the carbon source in the growing environment was investigated relative to the production of ligninolytic enzymes by Phanerochaete chrysosporium. Glucose, cellobiose, and cellulose (or mixtures thereof) were used as the carbon sources. Glucose oxidase and glyoxal oxidase activities in all carbon sources were produced during cultivation. High peak levels (0.17 to 0.24 IU/mL) of manganese peroxidase activity were observed only in mediums containing oligosaccharides. Lignin peroxidase activity was high in glucose medium (0.21 IU/mL of peak value); however, minimal amounts were formed in the cellulose medium (0.01 IU/mL of peak value). High amounts of cellobiose:quinone oxidoreductase (3.33-3.99 IU/mL of peak value) and cellobiose dehydrogenase (0.04-0.2 IU/mL of peak value) were measured when P. chrysosporium was grown on a medium containing cellulose. This work discovered that the mixture of glucose and cellulose as a carbon source favored high co-production of ligninolytic enzymes by P. chrysosporium.

Herbicides cause environmental concerns because they are toxic and accumulate in the environment, food products and water supplies. There is a need to develop safe, efficient and economical methods to remove them from the environment, often by biodegradation. Atrazine is such herbicide. White-rot fungi have the ability to degrade herbicides of potential utility. This study formulated a novel pelletized support to immobilize the white-rot fungus Anthracophyllum discolor to improve its capability to degrade the atrazine using a biopurification system (BS). Different proportions of sawdust, starch, corn meal and flaxseed were used to generate three pelletized supports (F1, F2 and F3). In addition, immobilization with coated and uncoated pelletized supports (CPS and UPS, respectively) was assessed. UPS-F1 was determined as the most effective system as it provided high level of manganese peroxidase activity and fungal viability. The half-life (t1/2) of atrazine decreased from 14 to 6Â days for the control and inoculated samples respectively. Inoculation with immobilized A. discolor produced an increase in the fungal taxa assessed by DGGE and on phenoloxidase activity determined. The treatment improves atrazine degradation and reduces migration to surface and groundwater., Grant CONICYT/FONDAP/15130015, Grant FONDECYT 1120963

The white-rot fungus Anthracophyllum discolor immobilized on wheat grains was evaluated for chlorophenol (2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol) degradation in allophanic soil columns activated by acidification. Columns without inoculation were used as the control to evaluate the adsorption capacity of the soil columns. The chlorophenols were removed efficiently in soil columns by both adsorption and degradation processes. In inoculated soil columns, 2,4-dichlorophenol was highly degraded and this degradation is associated with a high production of manganese peroxidase. 2,4,6-trichlorophenol was degraded to a lesser extent compared with 2,4-dichlorophenol. Pentachlorophenol was first removed by adsorption and then through degradation by the fungus. Manganese peroxidase activity was lowest when the column was fed with pentachlorophenol and highest when the column was fed with 2,4-dichlorophenol. Laccase was also produced by the fungus but to a lesser degree.

Filamentous fungi derived from marine environments are well known as a potential genetic resource for various biotechnological applications. Although terrestrial fungi have been reported to be highly efficient in the remediation of xenobiotic pollutants, fungi isolated from the marine environment may possess biological advantages over terrestrial fungi because of their adaptations to high salinity and pH extremes. The present study describes the production of ligninolytic enzymes under saline and non-saline conditions and the decolorization of Remazol Brilliant Blue R (RBBR) dye by three basidiomycetes recovered from marine sponges (Tinctoporellus sp. CBMAI 1061, Marasmiellus sp. CBMAI 1062, and Peniophora sp. CBMAI 1063). Ligninolytic enzymes were primarily produced by these fungi in a salt-free malt extract and malt extract formulated with artificial seawater (saline condition). CuSO4 and wheat bran were the best inducers of lignin peroxidase and manganese peroxidase activity. RBBR was decolorized up to 100% by the three fungi, and Tinctoporellus sp. CBMAI 1061 was the most efficient. Our results revealed the biotechnological potential of marine-derived basidiomycetes for dye decolorization and the treatment of colored effluent as well as for the degradation of other organopollutants by ligninolytic enzymes in non-saline and saline conditions that resemble the marine environment.

Wood decaying fungi were isolated from several forests in Andhra Pradesh, India and screened for their lignolytic activity. Eighty-five species were isolated and screened qualitatively by guaiacol plate assay and spectrophotometric enzyme assays were carried out to examine the production of laccase, manganese peroxidase and lignin peroxidase quantitatively. Eighty-three species formed the circular zones of reddish brown color while all the species showed positive result for lignolytic activities in quantitative estimation. Russula sp. showed the highest zone in guaiacol plate assay and also given highest laccase and manganese peroxidase activity followed by Schizophyllum sp. Fomes fomentarius showed highest lignin peroxidase activity. The importance of this study is to screen and select the potential wood decaying fungi for large scale lignolytic enzyme production, which will be very useful in biobleaching and biopulping of paper and rayon industries.

Rhizoctonia solani causes worldwide losses in numerous crops. Sclerotia of R. solani remain viable for several years in soil and are an important source of primary infection. In this study the effect of soil incorporation of Kraft pine lignin, a side product of the paper industry, on viability of R. solani AG1-1B sclerotia was investigated. The efficacy of lignin was assessed in a sandy loam (Oppuurs) and a silt loam soil (Leest) collected from commercial fields in Belgium. Evaluating sclerotial viability after 4 weeks incubation in the two soils amended with 1% (w/w) Kraft pine lignin demonstrated a soil-dependent effect. In Leest soil the addition of lignin resulted in a significantly reduced sclerotial viability, together with an increased mycoparasitism by Trichoderma spp.; in Oppuurs soil, on the other hand, only a slight and insignificant reduction in sclerotial viability was observed. Based on phospholipid fatty acid analysis, different changes in microbial community structure upon lignin amendment were detected in the two soils. Both amended soils showed a significant increase in Gram negative bacteria. In Leest soil this increase was accompanied with a significantly higher increase in fungi and actinomycetes compared with Oppuurs soil. In addition, Kraft pine lignin resulted in both soils in a small but significant increase in manganese peroxidase activity and this increase tended to be higher in Leest soil. Manganese peroxidase produced by lignin-degrading basidiomycetes has previously been shown to degrade melanin, which protects the sclerotia against biotic and abiotic stress. We hypothesize that lignin-degrading fungi increased the susceptibility of the sclerotia to sclerotial antagonists such as Trichoderma, Gram negative bacteria and actinomycetes. Clearly, the effect observed here did not rely on the stimulation of one microbial group, but is the result of an interaction of different groups.

The effect of different carbon, nitrogen sources and inducers on growth and ligninolytic activity by Morel mushroom Morchella crassipes was investigated. The maximum growth was observed in mineral salts broth containing glucose as the carbon source and sodium nitrate as the nitrogen source. Among the inducers, chemical inducers inhibited the growth whereas in natural substrates, growth was not affected much. Manganese peroxidase and lignin peroxidase activity were not detected in the medium with different carbon and nitrogen sources, whereas laccase activity varied depending on carbon source (0.7–3.48 U/ml). Among the inducers, natural inducers resulted in an increase in the enzyme activities. Maximum laccase activity was observed in rice straw (12. 6 U/ml) followed by ABTS (11.6 U/ml); Manganese peroxidase activity was maximum in rice straw (14.32 U/l) wheat straw (12.16 U/l) and phenol red (15 U/l) as the inducers, whereas for Lignin peroxidase activity, rice straw (22 U/l), wheat straw (16 U/l) and veratrylalcohol (20 U/l) served as the best inducers.

To investigate biodegradability by Trametes versicolor, five structurally different direct azo-dyes--Direct Black 38, Direct Blue 15 (DB 15), Direct Orange 26, Direct Green 6, and Direct Yellow 12--were studied. The DB 15 was determined as the best biodegradable dye by this white-rot fungus. Laccase and manganese peroxidase activities were monitored with the biodegradation process; it was observed that laccase played an important role in the dye degradation, while manganese peroxidase activity could not be detected. Possible degradation products also were examined by gas chromatography-mass spectrometry, but no metabolite was detected after the degradation and/or decolorization process. To enhance performance of the fungi during the degradation, Trametes versicolor cells were immobilized in alginate beads. Then, DB 15 decolorization by immobilized Trametes versicolor was studied in a small-scale packed-bed reactor. The color removal efficiency in repeated batches was found to be 98 and 93% for 50 mg/L DB 15.

We analyzed the effect of fungal infection on dye decolorization in continuous and sequential white rot fungus bioreactors. Biological samples of biofilm were collected to determine the ability of these bioreactors to degrade dye in synthetic wastewater. Polymerase chain reaction–denaturing gradient gel electrophoresis was used to analyze the taxonomy of the fungal community. Decolorization rate and manganese peroxidase activity were analyzed. Scanning electron microscopy was used to examine the physical structure of the biofilm. At an influent concentration of 50 mg/L, decolorization rates and chemical oxygen demand (COD) removal rates were as high as 88.7% and 53.7% in the sequential reactor and 85.5% and 54.6% in the continuous reactor on day 6 of the studies, respectively. The quantity of foreign fungi in the reactors increased over time and negatively affected Phanerochaete chrysosporium (PC) growth by competitively absorbing nutrients from the culture medium, resulting in a decline in PC-me...

The presence of cytochrome P450 and P450-mediated phenanthrene oxidation in the white rot fungus Phanerochaete chrysosporium under ligninolytic condition was first demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (130 pmol mg(-1) in the microsomal fraction) by phenanthrene. The microsomal P450 degraded phenanthrene with a NADPH-dependent activity of 0.44 ± 0.02 min(-1). One of major detectable metabolites of phenanthrene in the ligninolytic cultures and microsomal fractions was identified as phenanthrene trans-9,10-dihydrodiol. Piperonyl butoxide, a P450 inhibitor which had no effect on manganese peroxidase activity, significantly inhibited phenanthrene degradation and the trans-9,10-dihydrodiol formation in both intact cultures and microsomal fractions. Furthermore, phenanthrene was also efficiently degraded by the extracellular fraction with high manganese peroxidase activity. These results indicate important roles of both manganese peroxidase and cytochrome P450 in phenanthrene metabolism by ligninolytic P. chrysosporium.

The production of lignocellulolytic enzymes by eleven basidiomycetes species isolated from two ecosystems of Georgia was investigated for the first time under submerged (SF) and solid-state fermentation (SSF) of lignocellulosic by-products. Notable intergeneric and intrageneric differences were revealed with regard to the extent of hydrolase and oxidase activity. Several fungi produced laccase along with hydrolases in parallel with growth during the trophophase, showing that the synthesis of this enzyme is not connected with secondary metabolism. The lignocellulosic substrate type had the greatest impact on enzyme secretion. Some of the substrates significantly stimulated lignocellulolytic enzyme synthesis without supplementation of the culture medium with specific inducers. Exceptionally high carboxymethyl cellulase (CMCase, 122 U ml(-1)) and xylanase (195 U ml(-1)) activities were revealed in SF of mandarin peelings by Pseudotremella gibbosa IBB 22 and of residue after ethanol production (REP) by Fomes fomentarius IBB 38, respectively. The SSF of REP by T. pubescens IBB 11 ensured the highest level of laccase activity (24,690 U l(-1)), whereas the SSF of wheat bran and SF of mandarin peels provided the highest manganese peroxidase activity (570-620 U l(-1)) of Trichaptum biforme IBB 117. Moreover, the variation of lignocellulosic growth substrate provides an opportunity to obtain enzyme preparations containing different ratios of individual enzymes.

Some conditions in media composition for laccases production, such as different sources of carbon and organic nitrogen, antifoams and a surfactant, were studied in liquid cultures of Pleurotus sajor-caju strain PS-2001. Cultivation with fructose or glucose as carbon sources produced maximum enzyme activities of 37 and 36 U mL(-1), respectively. When sucrose was present in the medium, the best results were obtained using 5 g L(-1) of this carbohydrate, on the 11th day of the process, attaining laccase titres of 13 U mL(-1). In a medium without casein, practically no enzyme was produced during the experiments; among the sources of nitrogen studied, pure casein led to the highest titres of laccase activity. Different concentrations of pure casein and sucrose were also tested. As to the different concentrations of casein, the addition of 1.5 g L(-1) resulted in the highest titres of laccase activity. Negligible levels of manganese peroxidase activity were also detected in the culture medium. In low concentrations, polypropylene glycol or silicon-based antifoams and the surfactant Tween 80 have no significant influence on the formation of laccases by P. sajor-caju. However, enhanced concentration of polypropylene glycol negatively affected the production of laccases but favored the titres in total peroxidases, lignin peroxidase and veratryl alcohol oxidase.

The two-component system SenS–SenR from Streptomyces reticuli has been shown to influence the production of the redox regulator FurS, the mycelium-associated enzyme CpeB, which displays heme-dependent catalase and peroxidase activity as well as heme-independent manganese peroxidase activity, and the extracellular heme-binding protein HbpS. In addition, it was suggested to participate in the sensing of redox changes. In this work, the tagged cytoplasmic domain of SenS (SenSc), as well as the full-length differently tagged SenR, and corresponding mutant proteins carrying specific amino acid exchanges were purified after heterologous expression in Escherichia coli. In vitro, SenSc is autophosphorylated to SenSc∼P at the histidine residue at position 199, transfers the phosphate group to the aspartic acid residue at position 65 in SenR, and acts as a phosphatase for SenR∼P. Bandshift and footprinting assays in combination with competition and mutational analyses revealed that only unphosphorylated SenR binds to specific sites upstream of the furS–cpeB operon. Further specific sites within the regulatory region, common to the oppositely orientated senS and hbpS genes, were recognized by SenR. Upon its phosphorylation, the DNA-binding affinity of this area was enhanced. These data, together with previous in vivo studies using mutants lacking functional senS and senR, indicate that the two-component SenS–SenR system governs the transcription of the furS–cpeB operon, senS–senR and the hbpS gene. Comparative analyses reveal that only the genomes of a few actinobacteria encode two-component systems that are closely related to SenS–SenR.

The evaluation of eighteen strains of basidiomycetes laccase and manganese peroxidase (MnP) activity in submerged fermentation of mandarin peelings and ethanol production waste showed that the expression of enzyme activity is species- and strain-dependent. While all species of the genus Trametes expressed comparatively high laccase activity, the activity of this enzyme among species of the genus Ganoderma varied from 192 to 61,488 U l−1. Phellinus robustus 250 appeared to be a promising producer of MnP, accumulating more than 4000 U l−1 of enzyme activity. It has been shown for the first time that Omphalotus olearius 174 is capable of producing high levels of laccase and MnP, while Hypsizygus marmoreus produces only laccase. Laccase and MnP production proved to be very much dependent on the lignocellulosic growth substrate. Of eight complex substrates examined in submerged fermentation by Ganoderma lucidum 447, wheat bran and soy bran gave the highest laccase activity with a maximum value of 93–97 U ml−1. Proof that both the titre and time of maximal enzyme activity are influenced by nutrient nitrogen is presented.

The aim of this work was to investigate the poorly understood effects of co-culturing of two white rot fungi on the production of lignin-degrading enzyme activities. Four species, Ceriporiopsis subvermispora, Physisporinus rivulosus, Phanerochaete chrysosporium and Pleurotus ostreatus were cultured in pairs to study the degradation of aspen wood and the production of lignin-degrading enzymes. Potential of co-culturing for biopulping was evaluated. Chemical analysis of decayed aspen wood blocks showed that co-culturing of C. subvermispora with P. ostreatus could significantly stimulate wood decay, when compared to monocultures. Based on the fungi tested here, however, this effect is species-specific. Other combinations of fungi were slightly stimulating or not stimulatory. The pattern of lignin degradation was altered towards the acid insoluble part of lignin especially in co-cultures where P. ostreatus was included as a partner. The use of agar plates containing the polymeric dye Poly R-478 showed elevated dye decolourization at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. ostreatus with C. subvermispora. Manganese peroxidase activity was stimulated in co-cultures of P. ostreatus with C. subvermispora or with P. rivulosus. Immunoblotting indicated changes in lignin-degrading enzymes and/or their isoform composition in response to co-culturing. This is the first report on the effects of co-culturing of potential biopulping fungi on wood degradation, and gives basic knowledge on fungal interactions during wood decay that can be utilized in practical applications.

Hydrogen peroxide is suspected of being highly implicated in mushroom nutrition and in substrate bleaching during cultivation. The parameters for measuring H2O2 in compost samples were examined and the methodology was applied to samples from both compost colonized by cultivars and wild isolates of Agaricus bisporus, and wheat straw or coffee pulp colonized by Pleurotus spp. Laccase and peroxidase activities were also measured. H2O2 concentration measured after heating at 80 °C for inactivating laccases and peroxidases was probably both H2O2 pre-existing in the compost and H2O2 generated from quinones and active oxygen species. This potential H2O2 concentration increased during the vegetative growth for all the strains, in agreement with a direct relationship between H2O2 concentration and active biomass of A. bisporus or Pleurotus spp. in their cultivation substrates. Correlations were observed between H2O2 concentration and manganese peroxidase activity in cultivation substrates at the stage of primordia formation. At this stage of development, H2O2 generation via biotic or abiotic mechanisms should be an important physiological trait of mushrooms. Copyright © 2007 Society of Chemical Industry

Background The fungal genus Phlebia consists of a number of species that are significant in wood decay. Biotechnological potential of a few species for enzyme production and degradation of lignin and pollutants has been previously studied, when most of the species of this genus are unknown. Therefore, we carried out a wider study on biochemistry and systematics of Phlebia species. Methods Isolates belonging to the genus Phlebia were subjected to four-gene sequence analysis in order to clarify their phylogenetic placement at species level and evolutionary relationships of the genus among phlebioid Polyporales. rRNA-encoding (5.8S, partial LSU) and two protein-encoding gene (gapdh, rpb2) sequences were adopted for the evolutionary analysis, and ITS sequences (ITS1 + 5.8S + ITS2) were aligned for in-depth species-level phylogeny. The 49 fungal isolates were cultivated on semi-solid milled spruce wood medium for 21 days in order to follow their production of extracellular lignocellulose-converting oxidoreductases and carbohydrate active enzymes. Results Four-gene phylogenetic analysis confirmed the polyphyletic nature of the genus Phlebia. Ten species-level subgroups were formed, and their lignocellulose-converting enzyme activity profiles coincided with the phylogenetic grouping. The highest enzyme activities for lignin modification (manganese peroxidase activity) were obtained for Phlebia radiata group, which supports our previous studies on the enzymology and gene expression of this species on lignocellulosic substrates. Conclusions Our study implies that there is a species-level connection of molecular systematics (genotype) to the efficiency in production of both lignocellulose-converting carbohydrate active enzymes and oxidoreductases (enzyme phenotype) on spruce wood. Thus, we may propose a similar phylogrouping approach for prediction of lignocellulose-converting enzyme phenotypes in new fungal species or genetically and biochemically less-studied isolates of the wood-decay Polyporales. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0538-x) contains supplementary material, which is available to authorized users.

The biodegradation of fluorene by immobilized Coprinus plicatilis was studied in pinewood and foam glass bead-packed reactors. The reactors were operated in a sequencing batch system. Removal efficiency increased over time and elevated influent fluorene concentration (85 mg/L) was removed 100% in 24–30 h batch cycles. Increased laccaseactivity was detected with the introduction of the compounds, and optimum activity corresponded to optimum removal periods. Significantly higher laccase activity (16.7–19 U/L) was detected in the glassbead-packed reactor compared to the pinewood-packed reactor (0.2–5 U/L). The presence of Mn2+ ions in the wood material possibly caused elevated manganese peroxidase activity (0.3–5.8U/L) compared to low to negligible activity in the glass bead reactor. Reactor performances are discussed in relation to sequencing batch operation and nutrient requirements necessary to induce and sustain fungal enzyme activity in inert-like organic material packed systems. Biodegradation metabolites were detected in samples via GC/MS.

El objetivo de este trabajo fue evaluar el potencial para producir enzimas ligninolíticas de diversas cepas de hongos de pudrición blanca, nativas de la Provincia de Misiones (Argentina). Coriolus versicolor v. antarcticus BAFC 266, Pycnoporus sanguineus BAFC 2126 y Phlebia brevispora BAFC 633 mostraron un gran potencial para producir fenoloxidasas. En Ganoderma applanatum cepa E, P. sanguineus BAFC 2126 y P. brevispora BAFC 633 se observó una marcada actividad lacasa y peroxidasa. C. versicolor v. antarcticus. G. applanatum (cepa A) y Trametes villosa, mostraron una reacción oxidativa altamente positiva sobre 2,6-dimetoxifenol a pH 4. C. versicolor v. antarcticus, G. applanatum (cepas D y F), T. elegans BAFC 2127 y T. villosa, mostraron una alta actividad manganeso peroxidasa y solo C. versicolor v. antarcticus mostró una alta actividad de lignino peroxidasa. La actividad tirosinasa fue evidente en las cepas D y F de G. applanatum y en Phanerochaete chrysosporium HHB 11741. La decoloración del licor negro Kraft reveló una actividad ligninolítica variable para las diferentes cepas estudiadas dependiendo de la concentración utilizada. Algunos hongos mostraron una moderada actividad ligninolítica con grandes halos de decoloración (p.e. Pleurotus sajor-caju y Steccherinium sp. BAFC 1171) indicando un significativo potencial para el aprovechamiento biotecnológico de estas cepas. This research aimed to evaluate the potential of several native white rot fungi (WRF) isolated from subtropical environments of Misiones (Argentina) to produce different ligninolytic enzymes. Coriolus versicolor f. antarcticus BAFC 266, Pycnoporus sanguineus BAFC 2126 and Phlebia brevispora BAFC 633 showed the highest phenoloxidase activity. Ganoderma applanatum strain E, P. sanguineus BAFC 2126 and P. brevispora BAFC 633 revealed marked laccase and peroxidase activity. C. versicolor f. antarcticus, G. applanatum (strain A) and Trametes villosa, gave high positive reactions with 2,6-dimethoxyphenol oxidation at the lowest tested pH. C. versicolor f. antarcticus, G. applanatum strains D and F, T. elegans BAFC 2127and T. villosa, showed the highest manganese peroxidase activity. C. versicolor f. antarcticus also produced the highest lignin peroxidase activity. Tyrosinase activity was mostly evident in G. applanatum strains (D and F) and Phanerochaete chrysosporium HHB 11741. Kraft liquor decolorization results were variable and depended on the fungus and the liquor concentration. Some fungi with moderate ligninolytic activity showed high decolorization rates (e.g. Pleurotus sajor-caju and Steccherinium sp. BAFC 1171) indicating the significance of additional approach to evaluate a potential biotechnological application. Keywords: oxidative enzymes, screening, white-rot fungi. Fil: Fonseca, Maria Isabel. Universidad Nacional de Misiones. Facultad de Cs.exactas Quimicas y Naturales. Departamento de Bioquimica Clinica. Laboratorio de Biotecnologia Molecular; Argentina Fil: Zapata, Pedro Dario. Universidad Nacional de Misiones. Facultad de Cs.exactas Quimicas y Naturales. Departamento de Bioquimica Clinica. Laboratorio de Biotecnologia Molecular; Argentina Fil: Villalba, Laura. Universidad Nacional de Misiones. Facultad de Cs.exactas Quimicas y Naturales. Departamento de Bioquimica Clinica. Laboratorio de Biotecnologia Molecular; Argentina Fil: Fariña, Julia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; Argentina

The degree of decolorization of Poly R-478, a highly recalcitrant anthraquinone dye, by three basidiomycetes belonging to Polyporales isolated from compost was investigated together with the enzymes involved. Decolorization tests in two liquid cultures, one with a simple mycelium inoculum, the other with an inoculum of mycelium grown on straw, resulted in 70% and 87% decolorization respectively in 7 days. However, the efficiency did not increase significantly in the presence of the lignocellulose substrate. The three strains produced laccase and/or manganese peroxidase activity during the decolorization, whereas lignin peroxidase activity was not observed. Previous growth on straw enhanced the synthesis of ligninolytic enzymes, though there was no correlation between enzyme activity and decolorization. The three fungi can be proposed as promising candidates for the treatment of colored industrial effluents and probably for soils contaminated by complex polymers, such as polycyclic aromatic hydrocarbons.

During the interaction of two tropical agaric fungi, Marasmius pallescens and Marasmiellus troyanus, on agar media, initial deadlock between the two mycelia was ultimately followed by take-over by M. troyanus. When shaken liquid cultures of these two fungi were mixed, a rapid increase in laccase and manganese peroxidase activity, but no lignin peroxidase, was detected in the culture supernatant. Even more rapid and elevated induction of laccase occurred when filter-sterilized supernatant of Marasmius pallescens was added to Marasmiellus troyanus cultures, but the reciprocal experiment (addition of M. troyanus supernatant to M. pallescens cultures), did not lead to any increase in laccase activity. Addition of autoclaved supernatant of M. pallescens also induced laccase activity from M. troyanus cultures, but over a period of days rather than hours. Although both M. troyanus, and to a lesser extent M. pallescens, are able to produce laccases in shaken liquid culture following addition of the inducer 2,5-dimethylalanine, these experiments suggest that the presence of heat-stable and heat-labile laccase inducers secreted by M. pallescens mycelia lead to induction of laccases by M. troyanus.

A fungus Cladosporium cladosporioides isolated from coal sample as a decolorizing microorganism. It decolorized five different azo and triphenylmethane dyes like acid blue 193, acid black 210, crystal violet, reactive black B(S) and reactive black BL/LPR both on solid and in liquid broth medium. Culture broth of this fungus decolorized completely 100 mg of acid blue 193 l−1 in 8 days. The extracellular enzyme of Cladosporium cladosporioides decolorized acid blue 193 on repeated addition to a total (out of 700 mg l−1) concentration of 564 mg l−1 within 168 h without significant decline in the activity, showing the resistant property of Cladosporium cladosporioides to a high concentration of the dye. The optimal temperature 40 °C, pH 5.6 and sugar concentration of 4% required for decolorization of acid blue 193. Cladosporium cladosporioides showed manganese peroxidase activity with 41 U l−1, laccase activity with 1413 U l−1 and lignin peroxidase activity was negligible after day 8 of incubation.

An amperometric enzyme sensor for rapid and simultaneous detection of the lignin-degrading peroxidase activities secreted by Phanerochaete chrysosporium was developed, using H 2 O 2 , hydroquinone and veratryl alcohol as substrates. In the amperometric measurement, samples of culture filtrate with different lignin-degrading peroxidase activities measured by spectrophotometry were placed into electrochemical cells. The slope of the current increase ( Δ current / Δ time ) upon the addition of H 2 O 2 into the culture filtrate solution containing hydroquinone was used as the index for total activity of lignin peroxidase and manganese peroxidase. Then a specific detection of lignin peroxidase was achieved by the addition of veratryl alcohol, which led to current decrease due to the redox competition between veratryl alcohol and hydroquinone. A good linear correlation was found between the electrochemical response and lignin peroxidase activity, manganese peroxidase activity in the range of 8.14–29.79 U l −1 and 0.085–1.37 U l −1 , respectively. A regression model was established describing the relationship. The amperometric sensor described here is more rapid, sensitive and precise than conventional spectrophotometric assays, free from interference of turbidity and UV–vis-light-absorbing substances. In this paper, it was also applied in the detection of lignin-degrading peroxidases in compost bioremediation using P. chrysosporium , showing considerable advantages.

An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus . This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase ( GPD ) gene of Schizophyllum commune. SC3 -driven expression resulted in a maximal laccase activity of 107 nkat ml −1 in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1 -driven expression strongly increased when 25 g of ethanol liter −1 was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml −1 . These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter −1 when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml −1 (i.e., 360 mg liter −1 ) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter −1 . In this case, maximal activities were 3,900 and 4,660 nkat ml −1 , respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.

The possible use of olive-mill wastewater (OMW) as a growth medium for the production of extracellular laccase and manganese peroxidase (MnP) from the white-rot fungus Panus tigrinus (P. tigrinus) CBS 577.79 was studied using a properly formulated OMW-based medium (2-fold diluted OMW supplemented with 0.5% sucrose and 0.1% yeast extract) either in a stirred-tank or an air-lift reactor. Solid-state fermentation (SSF) was also performed in a rotary drum reactor using maize stalks moistened with the OMW-based medium. Highest levels of laccase and manganese peroxidase activity were obtained in the stirred-tank reactor (4600±98 U l−1 on day 13) and in the air-lift reactor (410±22 on day 7), respectively. Based on total enzyme activities, SSF appears to be more suitable than LSF but the latter exhibits better volumetric productivities.

Aims: The main problem that arises during the cultivation of Lentinula edodes, the Asian Shiitake mushroom, is that the logs on which the cultivation is performed are contaminated by competing micro-organisms, especially Trichoderma spp. The aim of this study was to examine the changes in activity of extracellular enzymes in dual cultures of Trichoderma spp. and L. edodes. Methods and Results: Extracellular enzyme activities were determined spectrophotometrically. Trichoderma enzymes important for the degradation of fungal cell walls (N-acetyl-β-glucosaminidase and laminarinase) were shown to be induced by inactive L. edodes mycelia in liquid culture. The changes that occurred in the extracellular enzyme activities of L. edodes and mycoparasitic Trichoderma spp. (T. aureoviride, T. harzianum and T. viride) were examined during antagonistic interactions on solid medium. The extracellular enzyme patterns of both partners proved to be altered. Trichoderma spp. were induced to produce N-acetyl-β-glucosaminidase and laminarinase in the presence of active L. edodes mycelia, similarly as observed in liquid culture. The activities of both laccase and manganese peroxidase of L. edodes decreased after physical contact with active Trichoderma mycelia, possibly in consequence of the beginning of degradation of L. edodes by the Trichoderma enzymes. However, besides a decrease in manganese peroxidase activity, an enhancement of L. edodes laccase activity was observed on solid media containing crude culture fluids from Trichoderma liquid cultures. The metabolites responsible for these effects proved to be heat stable. Conclusions: Induction and inhibition of several extracellular enzymes of both partners were shown in dual cultures of L. edodes and Trichoderma strains, indicating the important role of these enzymes in the antagonistic interaction between the two species. Significance and Impact of the Study: As the main problem during the large-scale cultivation of L. edodes is the contamination of the growth substrate by Trichoderma mycelia, the particular knowledge of the mechanism of this competition might be relevant.

Several species of white-rot fungi were investigated for their utility in prolonged decolouration of the recalcitrant sulfonated azo dye, amaranth. Trametes pubescens, T. multicolor, T. meyenii and T. versicolor decoloured amaranth azo-dye best on low-nitrogen agar-solidified media whereas Bjerkandera adusta and Phlebia radiata were most effective in low nitrogen medium supplemented with manganese. Trametes cotonea did not decolour effectively under any condition. The decolouring Trametes species were also effective in liquid culture whereas B. adusta and P. radiata were not. Trametes meyenii, T. pubescens and T. multicolor were equal to or better than commonly employed T. versicolor at decolouring amaranth. This is the first study to show the dye decolouration potential of T. meyenii, T. pubescens, and T. multicolor. Supplementing with Mn(II) increased assayable manganese peroxidase activity, but not long-term decolouration, indicating that laccase is the main decolourizing enzyme in these Trametes species. This appears to be because of inadequate Mn3+ chelation required by manganese peroxidase because adding relatively low amounts of malonate enhanced decolouration rates. The ability of Trametes meyenii to simultaneously decolour dye over prolonged periods of time while growing in relatively nutrient-rich medium appears to be unique amongst white-rot fungi, indicating its potential in wastewater bioremediation.

Aims: Four selected fungi were screened for their ability to decolourize a textile effluent and commercial reactive dyes in a solid medium. Methods and Results: Ligninolytic enzymes activities (lignin peroxidase, manganese peroxidase and laccase) and siderophores presence were monitored in decolourized plates. Results: The results showed low lignin peroxidase activity and no manganese peroxidase activity was detected for all fungi. Laccase activity was observed in Reactive Blue 19 decolourized plates by Trametes versicolor and Trametes villosa. Siderophores presence was observed in Trametes versicolor, Phanerochaete chrysosporium and Lentinus edodes decolourized plates. Conclusions: Lentinus edodes displayed the greatest decolourization ability both in terms of extent and rapidity of decolourization. Significance and Impact of the Study: The transformation observed for dyes open the possibility to study siderophores to treat dyes and textile effluents.

Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in selected sub-tropical white rot fungal species from Zimbabwe were determined. The enzyme activities were assayed at varying concentrations of C, N and Mn2+. Manganese peroxidase and laccase activities were the only expressed activities in the fungi under the culture conditions tested. Trametes species, T. cingulata, T. elegans and T. pocas produced the highest manganese peroxidase activities in a medium containing high carbon and low nitrogen conditions. High nitrogen conditions favoured high manganese peroxidase activity in DSPM95, L. velutinus and Irpex spp. High manganese peroxidase activity was notable for T. versicolor when both carbon and nitrogen in the medium were present at high levels. Laccase production by the isolates was highest under conditions of high nitrogen and those conditions with both nitrogen and carbon at high concentration. Mn2+ concentrations between 11–25 ppm gave the highest manganese peroxidase activity compared to a concentration of 40 ppm or when there was no Mn2+ added. Laccase activity was less influenced by Mn2+ levels. While some laccase activity was produced in the absence of Mn2+, the enzyme levels were higher when Mn2+ was added to the culture medium.