Cultures Of Phanerochaete Chrysosporium Research Articles

Removal of perfluorooctanoic acid (PFOA) in the liquid culture of Phanerochaete chrysosporium

Perfluorooctanoic acid (PFOA) is becoming a concern due to its persistence, bioaccumulation, and potential harmful effects on humans and the environment. In this study, the fungus Phanerochaete chrysosporium (P. chrysosporium) was used to remove the PFOA in liquid culture system. The results showed that the average activities of laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP) enzymes secreted by P. chrysosporium were 0.0003 U/mL, 0.013 U/mL, and 0.0059 U/mL, respectively, during the incubation times of 0–75 days. The pH of 3 and incubation time of 45–55 days were the optimum parameters for the three enzymes activities. The enzyme activities in P. chrysosporium incubation system were firstly inhibited by adding PFOA and then they were enhanced after 14 days. The maximum removal efficiency of PFOA (69.23%) was achieved after 35 days in P. chrysosporium incubation system with an initial PFOA concentration of 0.002 mM and no veratryl alcohol (VA). Adsorption was not a main pathway for PFOA removal and the PFOA adsorbed in fungi mycelial mat accounted for merely 1.91%. The possible products of PFOA contained partially fluorinated aldehyde, alcohol, and aromatic ring. These partially fluorinated compounds might result from PFOA degradation via a combination of cross-coupling and rearrangement of free radicals.

Enzymatic degradation of carbonaceous matter in contrasting South African refractory gold ores using cell-free spent medium from Phanerochaete chrysosporium

Two carbonaceous gold ores (ZAFB and ZAFV) underwent sequential biotreatment to expose gold and minimize preg-robbing before cyanidation. These included comparable organic carbon contents (6.0%wt in ZAFB and 4.4%wt in ZAFV) but were contrasting in the graphitic degree of the carbonaceous matter. Based on Raman spectra, the carbonaceous matter in ZAFB was more physically and chemically disordered than the ZAFV. To reduce extraneous factors, ≥ 90% of sulfides in both samples were oxidatively dissolved by the thermophilic mixed culture. Afterwards, the secondarily-formed minerals (mainly jarosite) were dissolved by oxalic acid washing. The carbonaceous matter was then treated for 15 days with a cell-free spent medium (CFSM) with lignin-degrading enzymes produced from a Phanerochaete chrysosporium culture. The enzymatic treatment reduced the carbonaceous matter content in the ZAFB by 0.73%wt from an initial value of 7.56%wt, while the ZAFV was unchanged. Raman and 3D-fluorescence analysis of the carbonaceous matter showed that the CFSM treatment was much more effective on the ZAFB sample. This indicates that the graphitic degree of the carbonaceous matter is a significant factor when applying crude enzymes derived from P. chrysosporium. The lignin-degrading enzyme treatment improved the gold extraction of ZAFB from 88% to 93%. It was found that Raman spectroscopy could serve as a handy tool for determining carbonaceous gold ores that are suitable for enzymatic treatment.

Fungal transformation of graphene by white rot fungus Phanerochaete chrysosporium

The wide applications of graphene materials require the thorough investigation on their biosafety and environmental risks. Transformation of graphene materials is a fundamental issue in their environmental risk evaluations. The enzymatic degradation of graphene is widely reported using peroxidases, but the information on the fungal transformation of graphene is still unavailable. Herein, we incubated reduced graphene oxide (RGO) in the white rot fungus Phanerochaete chrysosporium culture system for 4 weeks and investigated the transformation of RGO by multiple techniques. P. chrysosporium efficiently added oxygen to RGO and decreased the its carbon contents accordingly. The ID/IG ratios of RGO showed statistically increases upon the transformation by P. chrysosporium according to Raman spectroscopy, suggesting the increase of defects on carbon skeleton. The negatively charged oxygen containing groups exfoliated the graphene sheets as indicated by the larger layer distance according to the X-ray diffraction spectra and the increased roughness under scanning electron microscopy. The transformation was more obvious in the RGO separated from the fungal balls than the precipitates in the culture medium. The mechanism of transformation was attributed to the enzymatic degradation by P. chrysosporium. The environmental implication of the fungal transformation of graphene materials and the potential of using fungi to reduce the environmental risks of graphene materials are discussed.

Protein improvement of banana peel through sequential solid state fermentation using mixed-culture of Phanerochaete chrysosporium and Candida utilis.

Banana peel (BP) is a major waste produced by fruit processing industries. Pre-treatment of BP at different temperatures led to 40% reduction in saponin at 100°C (from 9.5 to 5.7mg/g). Sequential mixed culture of Phanerochaete chrysosporium (P. chrysosporium) and Candida utilis (C. utilis) gave highest protein enrichment (88.93mg/g). There is 26% increase in protein synthesis (from 88.93 to 111.78mg/g) after media screening. Inclusion of KH2PO4, FeSO4·7H2O, wheat flour and sucrose in the media contributed positively to protein synthesis, while elevated concentration of urea, peptone, K2HPO4, KCl, NH4H2PO4, and MgSO4.7H2O are required to reach optimum protein synthesis. Total soluble sugar (TSS), total reducing sugar (TRS) and total carbohydrate (CHO) consumption varied with respect to protein synthesis in all experimental runs. Optimum protein synthesis required 6 days and inclusion of 5% sucrose, 0.6% NH4H2PO4, 0.4% KCl, and 0.5% MgSO4·7H2O as concentration media constituents to reach 140.95mg/g protein synthesis equivalent to 300% increase over the raw banana peel protein content (35.0mg/g).

Application of Fenton pretreatment on the degradation of rice straw by mixed culture of Phanerochaete chrysosporium and Aspergillus niger

The rice straw (RS) is a kind of recalcitrant lignocellulosic material. The Fenton reaction has been widely used for the degradation of organic compounds and toxic chemicals. Therefore, applying it to pretreatment straw seems to have good potential. This study was to investigate the degradation of the pretreated RS by the mixed solid-state culture with Phanerochaete chrysosporium and Aspergillus nigert. The results showed that the degree of delignifition was 15.01% by using an optimized Fenton reagent (6 g/L of FeSO4·7H2O and 15% concentration of H2O2) at a solids loading of 5% (w/v) and a moderate temperature for 15 h. The analysis of the Fenton pretreated RS by Scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) showed significant changes in physicochemical structure, favoring the subsequently susceptibility of ligninolytic enzymes. The volatile organic compounds collected from the mixed solid-state culture implied the degradation degree of the lignocellulose to some extent. In conclusion, Fenton pretreatment could effectively disrupt the recalcitrant structure of RS and accelerate its biodegradation, which has high potential application in the energy conversion and utilization of lignocellulose.

Effect of glucose oxidase on decolorization efficiency of crystal violet by Phanerochaete chrysosporium cultures

An enzymatic reaction using glucose oxidase (GOx) was applied for continues production of hydrogen peroxide and organic acid in Phanerochaete chrysosporium cultures for use simultaneously in catalytic cycle of peroxidases. Decolorization efficiency of crystal violet (CV) as a model pollutant was investigated in 16 d old cultures which overproduced manganese peroxidase (MnP) in response to daily GOx addition and control cultures (i.e. no GOx was added). However, the ability of overproduced cultures in decolorization of CV was not increased significantly, through addition of GOx (300 U/L) + glucose (10 Mm) to the culture medium at the start of decolorization, the time needed to obtain 87 ± 0.5% removal of CV was reduced 10.7-fold in compared with the control culture. The best GOx concentration in culture medium for more efficient decolorization was obtained to be 300 U/L. These findings indicated that GOx in the presence of glucose could increase the degradation of CV not only by inducing ligninolytic activity in cultures but also as a subsidiary source for in situ H2O2 and organic acid production for catalytic activity of peroxidases in P. chrysosporium cultures.

Improvement in ligninolytic activity of Phanerochaete chrysosporium cultures by glucose oxidase

The effects of glucose oxidase (GOx) addition on enzymatic activity of manganese peroxidase (MnP) and lignin peroxidase (LiP) and protein production of Phanerochaete chrysosporium cultures were studied for the first time. In cultures supplemented daily with 300U/L GOx after beginning of ligninolysis, MnP activity was increased 4-fold (6144U/L) on day 15, LiP activity was induced (maximum activity was 220U/L.) and protein content of culture filtrates was increased significantly as compared to the control culture (i.e., no GOx-glucose was added.). In addition, remarkable increase in stability of MnP productivity by fungi, in cultures supplemented daily with GOx-glucose (more than 32 days), as compared with control cultures (22 days) was observed. Neither H2O2 nor gluconic acid produced by GOx was responsible for improvement of peroxidase activity in nitrogen-limited cultures. The new findings could be useful for improving the yield of peroxidase enzymes for degradation of a broad range of man-made aromatic compounds.

Influence of aeration and agitation modes on solid‐state fermentation of apple pomace waste by Phanerochaete chrysosporium to produce ligninolytic enzymes and co‐extract polyphenols

SummaryThe effect of different types of agitation (i) continuous agitation (C); (ii) continuous, discontinuous, continuous agitation (C/D/C); (iii) discontinuous, continuous, discontinuous agitation (D/C/D) and aeration (0.87, 1.25, 1.66 vvm) on ligninolytic enzyme production and polyphenolic compounds extraction by solid‐state cultures of Phanerochaete chrysosporium BKM‐F‐1767 was investigated. Higher production of manganese peroxidase (MnP) (1690.3 ± 87.6), lignin peroxidase (LiP) (387.9 ± 14.3) and laccase (898.9 ± 53.3 U gds−1) and liberation of total polyphenolics (ranging from 12.22 ± 1.1 to 30.12 ± 0.88 mg gallic acid equivalents per gram DW) was obtained after 195, 147, 219 and 204 h of fermentation, respectively, using 1.66 vvm as airflow and (C/D/C) agitation mode. Maximal enzyme production and total polyphenolic content were influenced by aeration, and higher values were obtained using 1.66 vvm as airflow rate, followed by 1.25 and 0.83 vvm, respectively. They were also influenced by agitation, and maximal values were obtained using C/D/C, followed by D/C/D and continuous agitation, respectively. The agitation modes influenced the production of ligninolytic enzymes and simultaneous extraction of polyphenols.

Degradation of Chrysanthemum (Dendranthema grandiflora) wastes by Pleurotus ostreatus for the production of reducing sugars

In Colombia, a great amount of waste is generated during the cut-off and harvest stages in flowers culture. This study examines the possibility of degrading Chrysanthemum wastes by using Pleurotus ostreatus, Trametes versicolor, and Phanerochaete chrysosporium cultures; this has not been studied previously. The initial effect of fungi on the degradation of Chrysanthemum wastes were studied individually and in co-cultures. The highest degradation was by P. ostreatus. After that, the influence of pH and waste, copper, and manganese concentrations on reducing sugars concentration were determined in a submerged culture in 100 mL Erlenmeyer flasks. There was a significant effect of manganese and waste concentrations on sugar concentration, while the effect of copper concentration and pH were not significant. Following, the process was carried out in a 1.5 L reactor at the optimal values of the variables studied in Erlenmeyer flask but varying air injection from 0 to 2 vvm. The highest concentration of sugars was 21.2 g/L with 78% of glucose content at 6.3% w/v of waste, 7.5 mM of Mn and Cu and 2 vvm of air injection. Finally, laccase, Manganese peroxidase, endo-1,4-β-glucanase, exo-1,4-β-glucanase and 1,4-β-glucosidase were detected in the extract obtained under these conditions. The highest activities were obtained for laccase (4,694 U/L) and 1,4-β-glucosidase (9,513 U/L).

Ethanol induced response in Phanerochaete chrysosporium and its role in the decolorization of triarylmethane dye

Ethanol was added to submerged cultures of Phanerochaete chrysosporium to enhance the production of laccase and improve the decolorization of triarylmethane dye. The addition of 10 g/l ethanol resulted in a 4.7-fold increase in laccase production, while higher ethanol concentrations caused a sharp decrease in activity. The protein content increased with the increase in ethanol concentrations; on the other hand, pellet diameter and fungal biomass wet weight decreased. The addition of ethanol to the cultivation media affected both the mycelial morphology and fungal wall permeability. Although catalase assay indicated a stress response behavior, the lipid peroxidation decreased, suggesting the presence of a protective protein compound which appears in the presence of ethanol. The partial purification of the extracellular fluid (ECF) of ethanol-amended cultures resulted in a low molecular weight fraction (<5 kDa), UV–visible spectrum for this fraction showed a single sharp peak at 356 nm under ethanol stress; this peak represents glutathione, a reductive peptide which increased 2-fold after the fungus was exposed to ethanol stress. The glutathione assay, reductive activity, and the in vitro decolorization confirm that glutathione is responsible for dye reduction (65.1%) as compared to decolorization in in vivo conditions (23.8%). The use of ethanol could improve the performance of Phanerochaete chrysosporium through laccase induction for oxidation or reductive activity depending on the concentration and mode of addition of ethanol.

Involvement of protein kinase C in lignin peroxidase expression in oxygenated cultures of the white rot fungus Phanerochaete chrysosporium

Hydroxyl radicals (OH ) were previously hypothesized as the principal ROS triggering lip-H2 expression in liquid cultures of Phanerochaete chrysosporium, through signal transduction. We therefore focused on determining the relationship between protein kinase C (PKC), ROS and LIP-H2. Significantly lower PKC activity was measured in high-LIP-producing (oxygenated) vs. low-LIP-producing (aerated—grown with free exchange of atmospheric air) cultures. In oxygenated cultures, inactivation of PKC activity by calphostin C, staurosporin or H7 caused significant elevation in lip-H2 and also in MnSOD1 expression. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) caused the reverse effect. Significantly low levels of lip-H2 expression were detected when O 2 −, H 2O 2 or OH were scavenged by 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), pyruvate or dimethylsulfoxide (DMSO), whether the level of PKC was normal, stimulated or inactivated. In situ generation of OH , via addition of Fenton reagents to aerated cultures, reduced pkc expression. In contrast, OH scavenging stimulated pkc expression. This work suggests that due to high OH levels, fungal cells activate a complex defensive system which regulates the levels of Fenton components by repressing PKC and stimulating LIP, MnSOD1 and catalase.

Screening of agro-industrial wastes to produce ligninolytic enzymes by Phanerochaete chrysosporium

Ligninolytic enzyme production by solid-state cultures of Phanerochaete chrysosporium BKM-F-1767 was investigated by employing different agro-industrial wastes, such as fishery residues, brewery waste, apple waste (pomace) and pulp and paper industry sludge. Different enzyme inducers, such as veratryl alcohol, Tween-80 and CuSO 4 at concentrations of 2 mM, 0.5% (v/w) and 3 mmole/kg, respectively were also tested. Use of veratryl alcohol and Tween-80 resulted in maximum manganese peroxidase (MnP) activity of 17.36 ± 0.5, 540.2 ± 5.1, 631.25 ± 14, and 507.5 ± 26.87 U/gds (units/gram dry substrate), respectively, for different wastes. Maximum lignin peroxidase (LiP) activity of 141.38 ± 3.39 and 14.1 ± 0.5 U/gds was attained with pomace and pulp and paper, respectively. Laccase activities were found to be insignificant for all wastes. Addition of Tween-80 and CuSO 4 resulted in highest values of MnP activity of 17.4 ± 0.6, 291 ± 2.8, 213.5 ± 3, and 213.2 ± 3.2 U/gds for fishery waste, brewery waste, pomace and pulp and paper industry sludge, respectively. Addition of CuSO 4 to the culture medium enhanced laccase activity. Maximum laccase activities of 738.97 ± 9.2, 719.97 ± 14.6, 308.8 ± 12.1, and 94.44 ± 1.2 U/gds were obtained for brewery waste, pomace, pulp and paper industry sludge and fishery waste, respectively. Brewery wastes and pomace served as excellent sources for the production of MnP, LiP and laccases.

Enhancement of laccase production by brown rot fungi and its role in decolorization

Ligninolytic enzyme production by solid-state cultures of Phanerochaete chrysosporium BKM-F-1767 was investigated by employing different agro-industrial wastes, such as fishery residues, brewery waste, apple waste (pomace) and pulp and paper industry sludge. Different enzyme inducers, such as veratryl alcohol, Tween-80 and CuSO4 at concentrations of 2 mM, 0.5% (v/w) and 3 mmole/kg, respectively were also tested. Use of veratryl alcohol and Tween-80 resulted in maximum manganese peroxidase (MnP) activity of 17.36 ± 0.5, 540.2 ± 5.1, 631.25 ± 14, and 507.5 ± 26.87 U/gds (units/gram dry substrate), respectively, for different wastes. Maximum lignin peroxidase (LiP) activity of 141.38 ± 3.39 and 14.1 ± 0.5 U/gds was attained with pomace and pulp and paper, respectively. Laccase activities were found to be insignificant for all wastes. Addition of Tween-80 and CuSO4 resulted in highest values of MnP activity of 17.4 ± 0.6, 291 ± 2.8, 213.5 ± 3, and 213.2 ± 3.2 U/gds for fishery waste, brewery waste, pomace and pulp and paper industry sludge, respectively. Addition of CuSO4 to the culture medium enhanced laccase activity. Maximum laccase activities of 738.97 ± 9.2, 719.97 ± 14.6, 308.8 ± 12.1, and 94.44 ± 1.2 U/gds were obtained for brewery waste, pomace, pulp and paper industry sludge and fishery waste, respectively. Brewery wastes and pomace served as excellent sources for the production of MnP, LiP and laccases.

Changes in the Gene Expression of the White Rot FungusPhanerochaete chrysosporiumDue to the Addition of Atropine

We constructed a LongSAGE (Long Serial Analysis of Gene Expression) library from a 3-d culture of Phanerochaete chrysosporium supplemented with atropine, which inhibits the production of lignin-degrading enzymes. The library (the atropine library) contains 13,108 LongSAGE tags and 6,783 unique tags. The gene expression profile represented by the tags was compared with those of two previously constructed libraries, one of which was constructed using 2-d cultures in which the fungus had not yet produced ligninolytic enzymes (the 2-d library) and the other was constructed using 3-d cultures in which the fungus had just started to produce the enzymes (the 3-d library). We found a total of 595 genes that were at least twice more highly or at least twice less highly expressed in the 3-d library than in the 2-d library or the atropine library, and the fluctuations were statistically significant. The relationships among these 595 genes were considered using cluster analysis. Of the 595 genes, 164 showed expression patterns similar to those of four ligninolytic enzyme genes, which were more expressed on day 3 than under any other conditions. Many of these 164 genes comprised genes possibly involved in lignin degradation, lipid metabolism, xenobiotic degradation, stress response, or signal transduction pathways.

Adsorption of Congo Red dye on hazelnut shells and degradation with Phanerochaete chrysosporium

The present work concerns the experimental evaluation of hazelnut shells as a low cost natural biosorbent. Adsorption of the direct azo dye Congo Red was performed within a concentrations range of 50-5000 mg/L. Hazelnut shells were employed as organic support for Phanerochaete chrysosporium cultures to study the best cultural medium composition for the MnP production. The capability of Phanerochaete chrysosporium to take macronutrients as carbon and nitrogen from hazelnut shells was demonstrated. Cultures of Phanerochaete chrysosporium were carried out with hazelnut shells coming from Congo Red adsorption tests, showing that 43% of the adsorbed dye was degraded.

A biodegradabilidade da blenda de poli(β-Hidroxibutirato-co-Valerato)/amido anfótero na presença de microrganismos

O crescimento do consumo de plásticos vem gerando grandes problemas ambientais, pois um polímero, uma vez descartado no ambiente, necessita de mais de cem anos para se degradar. O plástico ideal deve apresentar propriedades industriais desejáveis e, ao mesmo tempo ser degradável num período considerado satisfatório. Busca-se desenvolver plásticos com boas propriedades para embalagens e que possam ser biodegradados quando descartados ao ambiente. Neste trabalho avaliamos a biodegradação da blenda do copolímero poli(β-hidroxibutirato-co-valerato), PHB-HV, que é um termoplástico natural, biodegradável e biocompatível, e do amido anfótero, na proporção de 75 e 25% m/m, respectivamente. Os resultados foram obtidos através do teste de Sturm, uma metodologia para a avaliação da biodegradação na presença de uma cultura mista dos fungos Phanerochaete chrysosporium e Talaromyces wortmannii. Os resultados evidenciam a biodegradação da blenda em função do tempo, de acordo com os resultados do teste de Sturm, com o aparecimento de grupos carboxílicos terminais. Foi detectado também o aparecimento de nova simetria cristalina na estrutura polimérica.

Optimized production of lignolytic manganese peroxidase in immobilized cultures of Phanerochaete chrysosporium

Manganese peroxidase (MnP) is a key enzyme involved in the lignolysis of white-rot fungi. The purpose of this study is to investigate the effect of immobilization and culture conditions on MnP production in cultures of Phanerochaete chrysosporium grown on polyurethane foam. Higher concentrations of foam and lower levels of spore inoculums resulted in the formation of scattered mycelial pellets, increased autolysis of chlamydospore-like cells (a reservoir of MnP), and a higher activity of MnP. Even though MnP was a secondary metabolite, the addition of 5 times more glucose and diammonium tartrate, as carbon and nitrogen sources, resulted in a 4 fold increase in the dry cell mass. However, MnP activity decreased under these conditions to less than half, due to the formation of increasingly dense pellets and the inhibited lysis of chlamydospore-like cells.

Polycyclic aromatic hydrocarbon biodegradation and extracellular enzyme secretion in agitated and stationary cultures of Phanerochaete chrysosporium

The extracellular enzyme secretion and biodegradation of polycyclic aromatic hydrocarbons (PAHs) were studied in agitated and shallow stationary liquid cultures of Phanerochaete chrysosporium. Veratryl alcohol and Tween80 were added to cultures as lignin peroxidase (LiP) and manganese peroxidase (MnP) inducer, respectively. Shallow stationary cultures were suitable for the production of enzyme, whereas agitated cultures enhanced overall biodegradation by facilitating interphase mass transfer of PAHs into aqueous phases. The use of a LiP stimulator, veratryl alcohol, did not increase PAH degradation but significantly enhanced LiP activity. In contrast, Tween80 increased both MnP secretion and PAH degradation in shallow stationary cultures. On the other hand, high PAH degradation was observed in agitated cultures in the absence of detectable LiP and MnP activities. The results suggested that extracellular peroxidase activities are not directly related to the PAH degradation, and the increased solubility rather than enzyme activity may be more important in the promotion of PAH degradation.

Evaluation of the types of starch for preparation of LDPE/starch blends

This study evaluated in relation the growth, and the amylolytic activity of mixed and isolated cultures of Phanerochaete chrysosporium and Talaromyces wortmanni on different types of starch. The thermal and mechanical properties in polyethylene/starch blends (proportion: 80/20 (w/w) before and after inoculation of the mixed cultures were evaluated. The regular starch Amidex 3 and the modified starch Fox5901 stood out in relation to the cellular growth and production of the amylase enzyme. In spite of the short time that the blends were exposed to the fungi, the microorganisms promoted physical and chemical changes in the structure of the blend, modifying its thermal and mechanical properties. The alteration of the degree of crystallinity and mechanical properties of the blends could be indications of the modification caused by the biodegradation process.

In vitro degradation of a reactive azo dye by crude ligninolytic enzymes from nonimmersed liquid culture of Phanerochaete chrysosporium

In vitro degradation of synthetic dyes by ligninolytic enzymes from white rot fungi is one potential way to deal with dye pollution compared with decolorization by conventional biological treatment. In this work, production of the ligninolytic enzymes was carried out at C/N ratios of 28/44 mM and 56/2.2 mM in nonimmersed liquid culture of Phanerochaete chrysosporium. In vitro decolorization of one industrial azo dye, Reactive brilliant red K-2BP, by crude lignin peroxidase (LiP) and manganese peroxidase (MnP) obtained under carbon and nitrogen limitation respectively, was examined and compared for their degradation characteristics. Both decolorization by LiP and MnP were sensitive to pH, peaking around pH 3.0, and improved at higher enzyme activities. Decolorization by LiP can be enhanced to the greatest degree (89%) with higher addition of H 2O 2 and veratryl alcohol, whereas decolorization by MnP was optimized only with a suitable dose of H 2O 2 (0.1 mM) and decreased by the addition of Mn 2+. Decolorization declined at high dye concentrations; LiP was able to decolorize a dye concentration of 60 mg/l and below to no less than 85%, and MnP of 10 mg/l to a maximum of 71%. Decolorization by LiP and MnP together was somewhat lower than that by LiP alone. It is suggested that the optimization of the H 2O 2 supply was mainly responsible for a high efficiency in continuous dye degradation by crude LiP and MnP.