Effect of fungal co-cultures on ligninolytic enzyme activities, H2O2 production, and orange G discoloration.

In this study, the effects of Aspergillus niger in coculture with the basidiomycetes, Trametes versicolor, T. maxima, and Ganoderma spp., were studied to assess H2O2 production and laccase (Lac), Lignin Peroxidase (LiP), and manganese peroxidase (MnP) activities. The results indicated that maximum discoloration was of 97%, in the T. maxima and A. niger coculture, where the concentration of H2O2 was 5 mg/L and 6.3 mg/L in cultures without and with dye, respectively. These concentrations of H2O2 were 1.6- and 1.8-fold higher than monocultures of T. maxima (3.37 mg/L) and A. niger (3.87 mg/L), respectively. In the same coculture, the LiP and MnP enzyme activities also increased 12-fold, (from 0.08 U/mg to 0.99 U/mg), and 67-fold, (from 0.11 U/mg to 7.4 U/mg), respectively. The Lac activity increased 1.7-fold (from 13.46 U/mg to 24 U/mg). Further, a Box-Behnken experimental design indicated a 1.8-fold increase of MnP activity (from 7.4 U/mg to 13.3 U/mg). In addition, dye discoloration regression model obtained from the Box-Behnken experimental design showed a positively correlation with H2O2, (R2 = 0.58) and a negatively correlation with Lac activity (R2 = -0.7).

Evaluation of bezafibrate, gemfibrozil, indomethacin, sulfamethoxazole, and diclofenac removal by ligninolytic enzymes.

The laccase (Lac), manganese peroxidases (MnP), and lignin peroxidase enzymes produced by basidiomycete have been studied due to their potential in bioremediation, therefore, in this study, degradation of diclofenac (DCF), sulfamethoxazole (SMX), indomethacin (IND), gemfibrozil (GFB), and bezafibrate (BZF) by enzymes produced by Trametes maxima, Pleurotus sp., and Pycnosporus sanguineus grown in culture was evaluated. The degradation of drugs can mainly be attributed to MnP because a correlation between the activity of this enzyme and the degree of removal was found. The specific activity of Lac did not show correlation with drug removal, while lignin peroxidase was not expressed. Trametes maxima showed the highest specific activity of MnP (387.6 ± 67.4 U/mg) and efficiency removal 90.2% of DCF, 72.62% of SMX, 60.76% of IND, 43.39% of GFB, and 32.59% of BZF) followed by Pleurotus sp. with specific activity of MnP of 55.9 ± 8.5 U/mg and 89.47% of DCF, 47.61% of GFB and 73% of IND were removed, P. sanguineus had the lowest specific activity of 18 ± 1.3 U/mg and was able to remove only 42% of SMX and 10.59% of IND. In order to prove that MnP remove drugs instead of Lac, the pure Lac was tested and only degraded DCF.

Fungal co-culture increases ligninolytic enzyme activities: statistical optimization using response surface methodology.

The optimization of ligninolytic enzyme (LE) activities in a novel fungal co-culture between Pycnoporus sanguineus and Beauveria brongniartii were studied using a Plackett-Burman experimental design (PBED) and a central composite design (CCD). In addition, H2O2 role was analyzed. Laccase (EC. 1.10.3.2) and MnP (EC 1.11.1.14) activities of P. sanguineus increased 6.0- and 2.3-fold, respectively, in the co-culture with B. brongniartii. The H2O2 content was higher in the co-culture (0.33-7.12-fold) than in the P. sanguineus monoculture. The PBED revealed that yeast extract (YE), FeSO4, and inoculum amount were significant factors for laccase and MnP activities and H2O2 production in the co-culture, which increased by 8.2-, 5.2- and 1.03-fold, respectively. The YE and FeSO4 were studied using a CCD to optimize the studied response variables. Laccase activity was enhanced 1.5-fold by CCD, the optimal amount of YE was 0.366 g L-1. Quadratic term of FeSO4 modulated MnP activity and was associated with a 4.28-fold increase compared to the PBED. Both YE and its quadratic term significantly affected H2O2 production; however, the CCD did not enable an increase in H2O2 production. Pearson correlation indicated an increase in laccase (r2=0.4411, p = 0.0436) and MnP (r2=0.5186, p = 0.0198) activities following increases in H2O2 in the co-culture system.

Atrazine degradation by fungal co-culture enzyme extracts under different soil conditions.

This investigation was undertaken to determine the atrazine degradation by fungal enzyme extracts (FEEs) in a clay-loam soil microcosm contaminated at field application rate (5 µg g(-1)) and to study the influence of different soil microcosm conditions, including the effect of soil sterilization, water holding capacity, soil pH and type of FEEs used in atrazine degradation through a 2(4) factorial experimental design. The Trametes maxima-Paecilomyces carneus co-culture extract contained more laccase activity and hydrogen peroxide (H2O2) content (laccase = 18956.0 U mg protein(-1), H2O2 = 6.2 mg L(-1)) than the T. maxima monoculture extract (laccase = 12866.7 U mg protein(-1), H2O2 = 4.0 mg L(-1)). Both extracts were able to degrade atrazine at 100%; however, the T. maxima monoculture extract (0.32 h) achieved a lower half-degradation time than its co-culture with P. carneus (1.2 h). The FEE type (p = 0.03) and soil pH (p = 0.01) significantly affected atrazine degradation. The best degradation rate was achieved by the T. maxima monoculture extract in an acid soil (pH = 4.86). This study demonstrated that both the monoculture extracts of the native strain T. maxima and its co-culture with P. carneus can efficiently and quickly degrade atrazine in clay-loam soils.

Effect of Cd⁺² on phosphate solubilizing abilities and hydrogen peroxide production of soil-borne micromycetes isolated from Phragmites australis-rhizosphere.

The aims of this work were to evaluate the phosphate-solubilization and hydrogen peroxide (H2O2) production by the soil-borne micromycetes, Aspergillus japonicus, Penicillium italicum and Penicillium dipodomyicola, isolated from Phragmites australis rhizosphere and to study the effect of several concentrations of Cadmium (Cd(2+)) on both variables. Our results showed that P. italicum achieved a higher P-solubilization and H2O2 production than A. japonicus and P. dipodomyicola, as only P. italicum showed a positive correlation (R(2) = 0.71) between P-solubilization and H2O2 production. In dose-response assays, P. italicum was also more tolerant to Cd(2+) (0.31 mM) in comparison to A. japonicus (0.26 mM). Analysis of the 2(4) factorial experimental design showed that P-solubilization by P. italicum was negatively affected by increases in Cd(2+) (p = 0.04) and yeast extract (p = 0.02) in the culture medium. The production of H2O2 was positively affected only by glucose (p = 0.002). Fungal biomass production was reduced significantly (p = 0.0009) by Cd(2+) and increased (p = 0.0003) by high glucose concentration in the culture medium. The tolerance and correlation between P-solubilization and H2O2 production in the presence of Cd(2+) was strain and species dependent. The effects of Cd(2+), glucose, ammonium sulfate and yeast extract on those variables were evaluated through a two-level factorial design. P. italicum is promising for P-solubilization in soils contaminated with Cd(2+) and may be an alternative for manufacture of biofertilizers to replace chemical fertilizers.

Response of ligninolytic macrofungi to the herbicide atrazine: dose-response bioassays / Respuesta de macrohongos ligninolíticos al herbicida atrazina: bioensayos dosis-respuesta

The effect of atrazine concentrations on mycelial growth and ligninolytic enzyme activities of eight native ligninolytic macrofungi isolated in Veracruz, México, were evaluated in a semi-solid culture medium. Inhibition of mycelial growth and growth rates were significantly affected (p = 0.05) by atrazine concentrations (468, 937, 1875, and 3750 mg/l). In accordance with the median effective concentration (EC50), Pleurotus sp. strain 1 proved to be the most tolerant isolate to atrazine (EC50 = 2281.0 mg/l), although its enzyme activity was not the highest. Pycnoporus sanguineus strain 2, Daedalea elegans and Trametes maxima showed high laccase activity (62.7, 31.9, 29.3 U mg/protein, respectively) without atrazine (control); however, this activity significantly increased (p < 0.05) (to 191.1, 83.5 and 120.6 U mg/protein, respectively) owing to the effect of atrazine (937 mg/l) in the culture medium. Pleurotus sp. strain 2 and Cymatoderma elegans significantly increased (p < 0.05) their manganese peroxidase (MnP) activities under atrazine stress at 468 mg/l. The isolates with high EC50 (Pleurotus sp. strain 1) and high enzymatic activity (P. sanguineus strain 2 and T. maxima) could be considered for future studies on atrazine mycodegradation. Furthermore, this study confirms that atrazine can increase laccase and MnP activities in ligninolytic macrofungi.

Se evaluó el efecto de diferentes concentraciones de atrazina sobre el crecimiento micelial y la actividad enzimática de ocho macrohongos ligninolíticos aislados en Veracruz, México. La inhibición del crecimiento micelial y la tasa de crecimiento diaria fueron significativamente (p < 0,05) afectadas por todas las dosis de atrazina (468, 937, 1875 y 3750 mg/l) adicionadas al medio de cultivo. De acuerdo con la concentración efectiva media (CE50), Pleurotus sp. cepa 1 fue el aislamiento más tolerante a la atrazina (CE50 = 2281 mg/l), aunque sus actividades enzimáticas no fueron altas. Pycnoporus sanguineus cepa 2, Daedalea elegans y Trametes maxima mostraron actividades altas de lacasa (62,7, 31,9 y 29,3 U mg/proteína, respectivamente) en ausencia de atrazina (control); estas actividades se incrementaron (p < 0,05) significativamente (191,1, 83,5 y 120,6 U mg/proteína, respectivamente) en presencia de atrazina (937 mg/l) en el medio de cultivo. Pleurotus sp. cepa 2 y Cymatoderma elegans incrementaron significativamente (p < 0,05) sus actividades de manganeso peroxidasa (MnP) bajo la concentración de 468 mg/l de atrazina. Los aislamientos con alta CE50 (Pleurotus sp. cepa 1) y alta actividad enzimática (P. sanguineus cepa 2 y T. maxima) podrían ser considerados para futuros estudios en la micodegradación de atrazina. Además, el presente estudio confirma que la atrazina puede incrementar las actividades lacasa y MnP en macrohongos ligninolíticos.

Response of ligninolytic macrofungi to the herbicide atrazine: dose-response bioassays / Respuesta de macrohongos ligninolíticos al herbicida atrazina: bioensayos dosis-respuesta

The effect of atrazine concentrations on mycelial growth and ligninolytic enzyme activities of eight native ligninolytic macrofungi isolated in Veracruz, México, were evaluated in a semi-solid culture medium. Inhibition of mycelial growth and growth rates were significantly affected (p = 0.05) by atrazine concentrations (468, 937, 1875, and 3750 mg/l). In accordance with the median effective concentration (EC50), Pleurotus sp. strain 1 proved to be the most tolerant isolate to atrazine (EC50 = 2281.0 mg/l), although its enzyme activity was not the highest. Pycnoporus sanguineus strain 2, Daedalea elegans and Trametes maxima showed high laccase activity (62.7, 31.9, 29.3 U mg/protein, respectively) without atrazine (control); however, this activity significantly increased (p < 0.05) (to 191.1, 83.5 and 120.6 U mg/protein, respectively) owing to the effect of atrazine (937 mg/l) in the culture medium. Pleurotus sp. strain 2 and Cymatoderma elegans significantly increased (p < 0.05) their manganese peroxidase (MnP) activities under atrazine stress at 468 mg/l. The isolates with high EC50 (Pleurotus sp. strain 1) and high enzymatic activity (P. sanguineus strain 2 and T. maxima) could be considered for future studies on atrazine mycodegradation. Furthermore, this study confirms that atrazine can increase laccase and MnP activities in ligninolytic macrofungi.(AU)

Se evaluó el efecto de diferentes concentraciones de atrazina sobre el crecimiento micelial y la actividad enzimática de ocho macrohongos ligninolíticos aislados en Veracruz, México. La inhibición del crecimiento micelial y la tasa de crecimiento diaria fueron significativamente (p < 0,05) afectadas por todas las dosis de atrazina (468, 937, 1875 y 3750 mg/l) adicionadas al medio de cultivo. De acuerdo con la concentración efectiva media (CE50), Pleurotus sp. cepa 1 fue el aislamiento más tolerante a la atrazina (CE50 = 2281 mg/l), aunque sus actividades enzimáticas no fueron altas. Pycnoporus sanguineus cepa 2, Daedalea elegans y Trametes maxima mostraron actividades altas de lacasa (62,7, 31,9 y 29,3 U mg/proteína, respectivamente) en ausencia de atrazina (control); estas actividades se incrementaron (p < 0,05) significativamente (191,1, 83,5 y 120,6 U mg/proteína, respectivamente) en presencia de atrazina (937 mg/l) en el medio de cultivo. Pleurotus sp. cepa 2 y Cymatoderma elegans incrementaron significativamente (p < 0,05) sus actividades de manganeso peroxidasa (MnP) bajo la concentración de 468 mg/l de atrazina. Los aislamientos con alta CE50 (Pleurotus sp. cepa 1) y alta actividad enzimática (P. sanguineus cepa 2 y T. maxima) podrían ser considerados para futuros estudios en la micodegradación de atrazina. Además, el presente estudio confirma que la atrazina puede incrementar las actividades lacasa y MnP en macrohongos ligninolíticos.(AU)

Enhancement of ligninolytic enzyme activities in a Trametes maxima-Paecilomyces carneus co-culture: Key factors revealed after screening using a Plackett-Burman experimental design

Background In the industrial biotechnology, ligninolytic enzymes are produced by single fungal strains. Experimental evidence suggests that co-culture of ligninolytic fungi and filamentous microfungi results in an increase laccase activity. In this topic, only the ascomycete Trichoderma spp. has been studied broadly. However, fungal ligninolytic-filamentous microfungi biodiversity interaction in nature is abundant and poorly studied. The enhancement of laccase and manganese peroxidase (MnP) activities of Trametes maxima as a function of time inoculation of Paecilomyces carneus and under several culture conditions using Plackett-Burman experimental design (PBED) were investigated. Results The highest increases of laccase (12,382.5 U/mg protein) and MnP (564.1 U/mg protein) activities were seen in co-cultures I3 and I5, respectively, both at 10 d after inoculation. This level of activity was significantly different from the enzyme activity in non-inoculated T. maxima (4881.0 U/mg protein and 291.8 U/mg protein for laccase and MnP, respectively). PBED results showed that laccase was increased (P < 0.05) by high levels of glucose, (NH4)2SO4 and MnSO4 and low levels of KH2PO4, FeSO4 and inoculum (P < 0.05). In addition, MnP activity was increased (P < 0.05) by high yeast extract, MgSO4, CaCl2 and MnSO4 concentrations. Conclusions Interaction between indigenous fungi: T. maxima-P. carneus improves laccase and MnP activities. The inoculation time of P. carneus on T. maxima plays an important role in the laccase and MnP enhancement. The nutritional requirements for enzyme improvement in a co-culture system are different from those required for a monoculture system.