Oxidized glutathione promotes association between eukaryotic translation elongation factor 1Bγ and Ure2p glutathione transferase from Phanerochaete chrysosporium.

The eukaryotic translation elongation factor 1Bγ (eEF1Bγ) is an atypical member of the glutathione transferase (GST) superfamily. Contrary to more classical GSTs having a role in toxic compound detoxification, eEF1Bγ is suggested to act as a scaffold protein, anchoring the elongation factor complex EF1B to the endoplasmic reticulum. In this study, we show that eEF1Bγ from the basidiomycete Phanerochaete chrysosporium is fully active as a glutathione transferase in vitro and undergoes conformational changes upon binding of oxidized glutathione. Using real-time analyses of biomolecular interactions, we show that GSSG allows eEF1Bγ to physically interact with other GSTs from the Ure2p class, opening new perspectives for a better understanding of the role of eEF1Bγ in cellular oxidative stress response.

Toxicity of nanodiamonds to white rot fungi Phanerochaete chrysosporium through oxidative stress.

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.

The cytotoxicities in prokaryote and eukaryote varied for CdSe and CdSe/ZnS quantum dots and differed from cadmium ions.

The present study focused on the bioaccumulation and cytotoxicities of Cd2+, CdSe quantum dots (QDs) and CdSe/ZnS QDs in Escherichia coli (E. coli, represents prokaryotic system) and Phanerochaete chrysosporium (P. chrysosporium, represents eukaryotic system), respectively. Two types of QDs were characterized by transmission electron microscopy (TEM) and dynamic light scattering. The inductively coupled plasma optical emission spectrometer results showed that the bioaccumulation amounts of CdSe QDs by E. coli and P. chrysosporium were larger than those of CdSe/ZnS QDs due to the smaller particle size and less negative surface charges of CdSe QDs. Confocal microscopy and TEM results showed that there was an interaction between QDs and cells, and QDs have entered into the cells eventually, leading to the change of cell morphology. Plasma membrane fluidities and membrane H+-ATPase activities of E. coli and P. chrysosporium decreased gradually with the increasing concentrations of Cd2+, CdSe and CdSe/ZnS QDs. Results of the cell viabilities and intracellular reactive oxygen species levels indicated that the induced cytotoxicities were decreased as follows: CdSe QDs > CdSe/ZnS QDs > Cd2+. These findings suggested that the cytotoxicity of QDs was not only attributed to their heavy metal components, but also related to their nanosize effects which could induce particle-specific toxicity. The above results offer valuable information for exploring the cytotoxicity mechanism of QDs in prokaryote and eukaryote.

Toxicity of carbon nanotubes to white rot fungus Phanerochaete chrysosporium.

Carbon nanotubes (CNTs) are widely used in diverse areas with increasing annual production, thus the environmental impact of CNTs needs thorough investigation. In this study, we evaluated the effect of pristine multi-walled CNTs (p-MWCNTs) and oxidized multi-walled CNTs (o-MWCNTs) on white rot fungus Phanerochaete chrysosporium, which is the decomposer in carbon cycle and also has many applications in environmental remediation. Both p-MWCNTs and o-MWCNTs had no influence on the dry weight increase of P. chrysosporium and the pH value of culture system. The fibrous structure of P. chrysosporium was disturbed by p-MWCNTs seriously, while o-MWCNTs had litter influence. The ultrastructural changes were more evident for P. chrysosporium exposed to p-MWCNTs and only p-MWCNTs could penetrate into the cell plasma. The chemical composition of P. chrysosporium was nearly unchanged according to the infrared spectra. The laccase activity was suppressed by p-MWCNTs, while o-MWCNTs showed stimulating effect. The decoloration of reactive brilliant red X-3B was not affected by both CNT samples. However, serious inhibition of wood degradation was observed in the p-MWCNTs exposed groups, suggesting the potential threat of CNTs to the decomposition of carbon cycle. The implication to the environmental risks and safe applications of carbon nanomaterials is discussed.

Toxicity of graphene oxide to white rot fungus Phanerochaete chrysosporium.

With the wide production and applications of graphene and its derivatives, their toxicity to the environment has received much attention nowadays. In this study, we investigated the toxicity of graphene oxide (GO) to white rot fungus (Phanerochaete chrysosporium). GO was prepared by modified Hummers method and well characterized before use. P. chrysosporium was exposed to GO at the concentrations of 0-4 mg/mL for 7 d. The fresh and dry weights, pH values of culture media, structures, ultrastructures, IR spectra and activities of the decomposition of pollutants were measured to reveal the hazards of GO to P. chrysosporium. Our results indicated that low concentrations of GO stimulated the growth of P. chrysosporium. The exposure to GO induced more acidic pH values of the culture media after 7 d. GO induced the disruption of the fiber structure of P. chrysosporium, while at 4 mg/mL some very long and thick fibers were formed. Such changes were reflected in the scanning electron microscopy investigations, where the disruption of fibers was observed. In the ultrastructural investigations, the shape of P. chrysosporium cells changed and more vesicles were found upon the exposure to GO. The infrared spectroscopy analyses suggested that the chemical compositions of mycelia were not changed qualitatively. Beyond the toxicity, GO did not alter the activities of P. chrysosporium at low concentrations, but led to the complete loss of activity at high concentrations. The implication to the ecological safety of graphene is discussed.

Transport, fate, and stimulating impact of silver nanoparticles on the removal of Cd(II) by Phanerochaete chrysosporium in aqueous solutions.

Despite the knowledge about increasing discharge of silver nanoparticles (AgNPs) into wastewater and its potential toxicity to microorganisms, the interaction of AgNPs with heavy metals in the biological removal process remains poorly understood. This study focused on the effect of AgNPs (hydrodynamic diameter about 24.3±0.37 nm) on the removal of cadmium (Cd(II)) by using a model white rot fungus species, Phanerochaete chrysosporium. Results showed that the biological removal capacity of Cd(II) increased with the concentration of AgNPs increasing from 0.1 mg/L to 1 mg/L. The maximum removal capacity (4.67 mg/g) was located at 1 mg/L AgNPs, and then decreased with further increasing AgNPs concentration, suggesting that an appropriate concentration of AgNPs has a stimulating effect on the removal of Cd(II) by P. chrysosporium instead of an inhibitory effect. Results of Ag(+) and total Ag concentrations in the solutions together with those of SEM and XRD demonstrated that added AgNPs had undergone oxidative dissolution and transported from the solution to the surface of fungal mycelia (up to 94%). FTIR spectra confirmed that amino, carboxyl, hydroxyl, and other reducing functional groups were involved in Cd(II) removal, AgNPs transportation, and the reduction of Ag(+) to AgNPs.

Visualization of the mycelia of wood-rotting fungi by fluorescence in situ hybridization using a peptide nucleic acid probe.

White rot fungus, Phanerochaete chrysosporium, and brown rot fungus, Postia placenta, grown on agar plates, were visualized by fluorescence in situ hybridization (FISH) using a peptide nucleic acid (PNA) probe. Mycelia grown on wood chips were also clearly detected by PNA-FISH following blocking treatment. To the best of our knowledge, this is the first report on the visualization of fungi in wood by FISH.

Comparative study of the kinetics and equilibrium of phenol biosorption on immobilized white-rot fungus Phanerochaete chrysosporium from aqueous solution.

In this study the kinetics and equilibrium of phenol biosorption were studied from aqueous solution using batch technique at an initial pH of 5.5. The biosorption was studied on Ca-alginate beads, on non-living mycelial pellets of Phanerochaete chrysosporium immobilized on Ca-alginate, and on free fungal biomass. Ph. chrysosporium was grown in a liquid medium containing mineral and vitamin materials with complex composition. The biosorption process followed pseudo second-order kinetics on all bioadsorbents. The bioadsorption-equilibrium on blank Ca-alginate, free and immobilized fungal biomass can be described by Langmuir, anti-Langmuir and Freundlich isotherm models using nonlinear least-squares estimation.

Degradation of lead-contaminated lignocellulosic waste by Phanerochaete chrysosporium and the reduction of lead toxicity.

Lead, as one of the most hazardous heavy metals to the environment interferes with lignocellulosic biomass bioconversion and carbon cycles in nature. The degradation of lead-polluted lignocellulosic waste and the restrain of lead hazards by solid-state fermentation with Phanerochaete chrysosporium were studied. Phanerochaete chrysosporium effectively degraded lignocellulose, formed humus and reduced active lead ions, even at the concentration of 400 mg/kg dry mass of lead. The highest lignocellulose degradation (56.8%) and organic matter loss (64.0%) were found at the concentration of 30 mg/kg of lead, and at low concentration of lead the capability of selective lignin biodegradation was enhanced. Microbial growth was delayed in polluted substrate at the initial stage of fermentation, and organic matter loss is correlated positively with microbial biomass after 12 day fermentation. It might be because Phanerochaete chrysosporium developed active defense mechanism to alleviate the lead toxicity. Scanning electron micrographs with energy spectra showed that lead was immobilized via two possible routes: adsorption and cation exchange on hypha, and the chelation by fungal metabolite. The present findings will improve the understandings about the degradation process and the lead immobilization pathway, which could be used as references for developing a fungi-based treatment technology for metal-contaminated lignocellulosic waste.

Biosorption of Reactive Blue 4 dye by native and treated fungus Phanerocheate chrysosporium: Batch and continuous flow system studies.

The native and treated fungal biomass of Phanerocheate chrysosporium was used for the biosorption of a textile dye (i.e., Reactive Blue 4). In the batch system, the biosorption equilibrium time was about 4 h and the maximum dye uptake on all the tested fungal biomass preparations was observed at pH 3.0. The dye uptake capacities of the biosorbents at 600 mg L(-1) dye concentration were found to be 132.5, 156.9, 147.6 and 81.1 mg g(-1) for native and heat-, acid- and base-treated dry fungal preparations, respectively. The dye uptake capacity order of the fungal preparations was found as heat-treated>acid-treated>native>base-treated. The Langmuir, Freundlich and Temkin adsorption models were used for the mathematical description of the biosorption equilibrium. The Freundlich and Temkin models were able to describe the biosorption equilibrium of Reactive Blue 4 on native and treated fungal preparations. The dye biosorption on the fungal biomass preparations followed Ritchie kinetic model. Biosorption of the dye from aqueous solution was also investigated in a continuous flow system. The maximum biosorption capacity of the heat-treated fungal biomass P. chrysosporium was 211.6 mg (g dry biomass)(-1) at an initial dye concentration of 600 mg L(-1) and at a flow rate of 20 mL h(-1).

Biosoftening of coir fiber using selected microorganisms.

Coir fiber belongs to the group of hard structural fibers obtained from coconut husk. As lignin is the main constituent of coir responsible for its stiffness, microbes that selectively remove lignin without loss of appreciable amounts of cellulose are extremely attractive in biosoftening. Five isolated strains were compared with known strains of bacteria and fungi. The raw fiber treated with Pseudomonas putida and Phanerocheate chrysosporium produced better softened fiber at 30+/-2 degrees C and neutral pH. FeSO4 and humic acid were found to be the best inducers for P. chrysosporium and P. putida, respectively, while sucrose and dextrose were the best C-sources for both. Biosoftening of unretted coir fibers was more advantageous than the retted fibers. Unlike the weak chemically softened fiber, microbial treatment produced soft, whiter fibers having better tensile strength and elongation (44.6-44.8%) properties. Scanning electron microscopy photos showed the mycelia penetrating the pores of the fiber, removing the tylose plug and degrading lignin.

A scalable membrane gradostat reactor for enzyme production using Phanerochaete chrysosporium.

This is the first demonstration of process scale-up of a membrane gradostat reactor for continuous enzyme production using Phanerochaete chrysosporium ME446. The fungus was immobilised by reverse filtration on to externally unskinned, ultrafiltration capillary membranes and then nutrient gradients were induced across the biofilm. A 10-fold scale-up from a single capillary bioreactor to a 2.4 l multi-capillary unit resulted in a 7-fold increase in enzyme productivity with a peak at 209 U l(-1) d(-1). Subsequent scale effects on the spore distribution, continuous manganese peroxidase production profile and biofilm development are discussed.

Direct characterization of the physicochemical properties of fungal spores using functionalized AFM probes.

A new method is described for characterizing the physicochemical properties of native microbial cells by using atomic force microscopy (AFM) with chemically functionalized probes. Adhesion forces were measured, under deionized water, between probes and model substrata functionalized with alkanethiol self-assembled monolayers terminated with OH and CH(3) groups. These were found to be 6 +/- 2 nN (n = 1024), 0.9 +/- 0.4 nN, and approximately 0 nN, for CH(3)/CH(3), CH(3)/OH, and OH/OH surfaces, respectively, and were not significantly influenced by changes of ionic strength (0.1 M NaCl versus deionized water). This shows that functionalized probes are very sensitive to changes of surface hydrophobicity. Using OH- and CH(3)-terminated probes, patterns of rodlets, approximately 10 nm in diameter, were visualized, under physiological conditions, at the surface of spores of Phanerochaete chrysosporium. Multiple (1024) force-distance curves recorded over 500 x 500-nm areas at the spore surface, either in deionized water or in 0.1 M NaCl solutions, always showed no adhesion for both OH- and CH(3)-terminated probes. Control experiments indicated that the lack of adhesion is not due to transfer of cellular material onto the probe, but to the hydrophilic nature of the spore surface.

Disordered ultrastructure in lignin-peroxidase-secreting hyphae of the white-rot fungus Phanerochaete chrysosporium.

The practice of exposing liquid cultures of the white-rot fungus Phanerochaete chrysosporium to a pure oxygen atmosphere under conditions of nutrient starvation has been widely adopted to induce lignin peroxidase (LiP) synthesis. Transmission electron microscopy was used to examine hyphal cells of carbon-limited cultures that had been exposed to an atmosphere of pure oxygen, and revealed evidence of a major loss in organization of cellular ultrastructure, which may be attributed to oxygen toxicity. Under some conditions (continuous agitation in air with cellulose as the carbon source) cultures will produce LiP without needing to be exposed to a pure oxygen atmosphere. A similar major loss of cellular ultrastructure was found in hyphal cells from such cultures upon examination. Investigation of the levels of H2O2, catalase and carbonyl content of intracellular proteins suggests that the latter cultures developed a hyperoxidant state because the rate of supply of carbon from cellulose hydrolysis was insufficient for oxygen homeostasis. The association of LiP with these cultures and with those exposed to an atmosphere of pure oxygen infers that LiP may be triggered in response to oxidant stress.

Direct probing of the surface ultrastructure and molecular interactions of dormant and germinating spores of Phanerochaete chrysosporium.

Atomic force microscopy (AFM) has been used to probe, under physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus Phanerochaete chrysosporium. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 +/- 1 nm, which is in agreement with earlier freeze-etching measurements. In contrast, germinating spores had a very smooth surface partially covered with rough granular structures. Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination are correlated with profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 +/- 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale and offers new prospects for mapping microbial cell surface properties under native conditions.