Accelerated PAH Transformation in the Presence of Dye Industry Landfill Leachate Combined with Fungal Membrane Lipid Changes.

The ascomycete fungus Nectriella pironii, previously isolated from soil continuously contaminated by dye industry waste, was used for the biodegradation of phenanthrene (PHE), benz[a]anthracene (B[a]A), and benz[a]pyrene (B[a]P). The degradation of polycyclic aromatic hydrocarbons (PAHs) by N. pironii was accelerated in the presence of landfill leachate (LL) collected from the area of fungus isolation. The rate of cometabolic elimination of PHE and B[a]P in the presence of LL was, respectively, 75% and 94% higher than in its absence. LC-MS/MS analysis revealed that PAHs were converted to less-toxic derivatives. The parallel lipidomic study showed changes in membrane lipids, including a significant increase in the content of phosphatidylcholine (PC) (almost double) and saturated phospholipid fatty acids (PLFAs) and a simultaneous reduction (twofold) in the content of phosphatidylethanolamine (PE) and unsaturated PLFAs, which may have promoted the fungus to PHE + LL adaptation. In the presence of PHE, an intense lipid peroxidation (fivefold) was observed, confirming the stabilization of the cell membrane and its extended integrity. Determining the course of elimination and adaptation to harmful pollutants is essential for the design of efficient bioremediation systems in the future.

Ecotoxicological Estimation of 4-Cumylphenol, 4-t-Octylphenol, Nonylphenol, and Volatile Leachate Phenol Degradation by the Microscopic Fungus Umbelopsis isabellina Using a Battery of Biotests.

The phenolic xenobiotics nonylphenol (NP), 4-tert-octylphenol (4-t-OP), and 4-cumylphenol (4-CP) have the potential to seriously disrupt the endocrine system. Volatile phenols (VPs), especially those present in landfill leachate, also adversely affect the health of numerous organisms. Microbial degradation of xenobiotics can result in the formation of intermediates with higher toxicity than the precursor substrates. Therefore, the main aim of this study was to assess the changes in environmental ecotoxicity during the biotransformation of nonylphenol, 4-tert-octylphenol, 4-cumylphenol and volatile phenols by Umbelopsis isabellina using a battery of biotests. The application of bioindicators belonging to different taxonomic groups and diverse trophic levels (producers, consumers, and reducers) indicated a significant reduction in toxicity during the cultivation of fungus cultures both for nonylphenol, 4-tert-octylphenol, 4-cumylphenol and volatile phenols. The rate of toxicity decline was correlated with the degree of xenobiotic biotransformation. Removal of 4-cumylphenol and 4-tert-octylphenol also led to a decrease in the anti-androgenic potential. Moreover, this is the first report demonstrating the anti-androgenic properties of 4-cumylphenol. The results showed that U. isabellina is an attractive tool for the bioremediation and detoxification of contaminated environments.

Environmental and molecular approach to dye industry waste degradation by the ascomycete fungus Nectriella pironii.

Textile industry effluents and landfill leachate contain chemicals such as dyes, heavy metals and aromatic amines characterized by their mutagenicity, cytotoxicity and carcinogenicity. The aim of the present study was investigation of the ascomycete fungus N. pironii isolated from urban postindustrial textile green space for its ability to grow and retain metabolic activity in the presence of the dye industry waste. Research focused mainly on dyes, heavy metals and aromatic amines, which had been detected in landfill leachate via HPLC-MS/MS analysis. Presence of all tested compounds as well as leachate in the growth medium clearly favored the growth of fungal biomass. Only slight growth limitation was observed in the presence of 50 mg L-1 o-tolidine. The fungus eliminated o-tolidine as well as dyes at all tested concentrations. The presence of metals slightly influenced the decolorization of the azo dyes; however, it was still similar to 90%. During fungal growth, o-tolidine was hydroxylated and/or converted to toluidine and its derivatives. Laccase and cytochrome P450 involvement in this process has been revealed. The results presented in the paper provide a valuable background for the development of a fungus-based system for the elimination of toxic pollutants generated by the textile industry.

Correction: Laccase activity of the ascomycete fungus Nectriella pironii and innovative strategies for its production on leaf litter of an urban park.

[This corrects the article DOI: 10.1371/journal.pone.0231453.].

Laccase activity of the ascomycete fungus Nectriella pironii and innovative strategies for its production on leaf litter of an urban park.

A laccase-producing ascomycete fungus was isolated from soil collected around the premises of a textile dye factory and identified as Nectriella pironii. Efficient laccase production was achieved via the synergistic action of 1 mM copper sulfate and ferulic acid. Extracts of rapeseed oil cake, grass hay, and leaf litter collected in a pocket urban park were used for enzyme production. The highest laccase activity (3,330 U/L) was observed in the culture grown on the leaf litter extract. This is the first report on biosynthesis of laccase by N. pironii. This is also the first study on utilization of naturally fallen park leaves as a substrate for fungal laccase production. The extracellular enzyme possessing laccase activity was purified to homogeneity by ion-exchange and gel filtration chromatographic techniques. The amino acid sequence of the protein revealed highest similarity to the laccase enzyme produced by Stachybotrys chartarum-and considerable homology to those produced by other fungal species. The purified laccase possessed a molecular mass of 50 kDa. The enzyme had an optimum pH of 2.0 or 6.0 and retained more than 50% of residual activity after 3 hours of incubation at pH 3.0-10.6 or 4.0-9.0 when 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid or 2,6-dimethoxyphenol, respectively, were used. Dithiothreitol, ß-mercaptoethanol, and sodium azide at 1 mM concentration strongly inhibited the laccase activity, while in the presence of 50 mM urea, the enzyme was found to retain 25% of its activity. The laccase was able to decolorize more than 80% of Indigo Carmine, Remazol Brilliant Blue R, Reactive Orange 16, and Acid Red 27 dyes within 1 h. The possibility of leaf litter use for the production of the laccase enzyme from N. pironii (IM 6443), exhibiting high pH stability and degradative potential, makes it a promising tool for use in different environmental and industrial operations.

A proteomic study of Cunninghamella echinulata recovery during exposure to tributyltin.

A proteomic study of Cunninghamella echinulata recovery during exposure to tributyltin was conducted with 2-D SDS-PAGE protein separation and profiling, MALDI-TOF/TOF protein identification, and PCA analysis. The presence of TBT resulted in an upregulation of enzymes related to energy production via cellular respiration. The unique overexpression of NADH dehydrogenase and mitochondrial malate dehydrogenase, together with an increased level of cytochrome c oxidase, ATP synthase subunits, and inorganic pyrophosphatase, indicates a strong energy deficit in the cells, leading to an increase in the ATP production. The overexpression of Prohibitin-1, a multifunctional protein associated with the proper functioning of mitochondria, was observed as well. The data also revealed oxidative stress condition. Among reactive oxygen species (ROS)-scavenging enzymes, only superoxide dismutase (SOD) showed active response against oxidative stress induced by the xenobiotic. The induction of a series of ROS-scavenging enzymes was supported by a microscopic analysis revealing a considerably large concentration of ROS in the hyphae. The overexpression of cytoskeleton-related proteins in the TBT presence was also noticed. The obtained results allow explaining the recovery strategy of the fungus in response to the energy depletion caused by TBT.

Analysis of decolorization potential of Myrothecium roridum in the light of its secretome and toxicological studies.

To identify the enzymes potentially useful for the decolorization of azo dyes, the secretome of the ascomycetous fungus Myrothecium roridum IM6482 was studied by using a bottom-up proteomic approach. Among the identified proteins, the most promising for dye removal was laccase, which decolorized respectively, 66, 91, 79, and 80% of Acid Blue 113 (AB 113), Acid Red 27 (AR 27), Direct Blue 14 (DB 14), and Acid Orange 7 (AO 7). The degradation of dyes was enhanced at the wide range of pH from 4 to 8. The addition of redox mediators allowed eliminating AB 113 in concentrations up to 400 mg/L and decolorization of the simulated textile effluent. Microbial toxicity and phytotoxicity tests indicated that dyes are converted into low-toxicity metabolites. This is the first insight into the M. roridum secretome, its identification and its application for removal of select azo dyes. Obtained results extended knowledge concerning biodegradative potential of ascomycetous, ligninolytic fungi and will contribute to the improvement of dye removal by fungi.

Poly-Saturated Dolichols from Filamentous Fungi Modulate Activity of Dolichol-Dependent Glycosyltransferase and Physical Properties of Membranes.

 Mono-saturated polyprenols (dolichols) have been found in almost all Eukaryotic cells, however, dolichols containing additional saturated bonds at the ω-end, have been identified in A. fumigatus and A. niger. Here we confirm using an LC-ESI-QTOF-MS analysis, that poly-saturated dolichols are abundant in other filamentous fungi, Trichoderma reesei, A. nidulans and Neurospora crassa, while the yeast Saccharomyces cerevisiae only contains the typical mono-saturated dolichols. We also show, using differential scanning calorimetry (DSC) and fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) that the structure of dolichols modulates the properties of membranes and affects the functioning of dolichyl diphosphate mannose synthase (DPMS). The activity of this enzyme from T. reesei and S. cerevisiae was strongly affected by the structure of dolichols. Additionally, the structure of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) model membranes was more strongly disturbed by the poly-saturated dolichols from Trichoderma than by the mono-saturated dolichols from yeast. By comparing the lipidome of filamentous fungi with that from S. cerevisiae, we revealed significant differences in the PC/PE ratio and fatty acids composition. Filamentous fungi differ from S. cerevisiae in the lipid composition of their membranes and the structure of dolichols. The structure of dolichols profoundly affects the functioning of dolichol-dependent enzyme, DPMS.

Detoxification and simultaneous removal of phenolic xenobiotics and heavy metals with endocrine-disrupting activity by the non-ligninolytic fungus Umbelopsis isabellina.

Organic and inorganic pollutants well known to interfere with the major functions of the endocrine system co-occur widely in contaminated ecosystems. The aim of the study was to evaluate the ability of Umbelopsis isabellina fungus to simultaneously remove and detoxify multiple environmentally significant endocrine disruptors: the heavy metals Cd(II), Zn(II), Mn(II), Pb(II) and Ni(II) and the phenolic xenobiotics nonylphenol (t-NP), 4-cumylphenol (CP) and 4-tert-octylphenol (4-t-OP). The effects of the metals on fungal growth and efficiency of single-metal uptake were also investigated. U. isabellina exhibited considerable tolerance to Zn(II), Mn(II), Pb(II) and Ni(II), with IC50/24 values ranging from 5.08 for Ni(II) to 13.1 mM for Zn(II). In the presence of CP, the maximum efficiency of Pb(II) removal increased 25% relative to that of the control. Supplementation with Mn(II) or Zn(II) enhanced the 4-t-OP degradation by 18 or 9%, respectively, after 6 h of cultivation. Ecotoxicological assays monitoring bioindicators from different aquatic ecosystems revealed detoxification coinciding with the removal of metals and organic xenobiotics from binary mixtures. This work indicates the potential of a single microorganism, U. isabellina, to remove both heavy metals and organic xenobiotics from co-contaminated sites, making it a suitable candidate for the development of bioremediation strategies.

Novel laccase-like multicopper oxidases from the Myrothecium roridum fungus - production enhancement, identification and application in the dye removal process.

The aim of this study was to overproduce, identify and apply novel laccase-like multicopper oxidases (LMCOs) from Myrothecium roridum in a dye removal process. LMCOs' production was enhanced by modifying the medium and adding copper ions. After purification, two proteins, LMCO1 and LMCO2, with molecular masses of 46.7 and 66.3 kDa were discovered. Peptide analysis by mass spectrometry revealed that they belong to the cupredoxin superfamily. Characteristic peptide sequences were obtained for MCOs and bilirubin oxidases. Crude enzymes were applied in a dye decolorization process. Supplementation with 1 mM of vanillin allowed an almost complete elimination of the Indigo carmine within 3 hours. The dye was removed from a solution containing metals, surfactants and organic solvents. The in-gel assessment of the activity and decolorization ability of MCOs, followed by protein extraction and SDS-PAGE, confirmed that only LMCO2 was responsible for the dye removal. MCOs produced by Myrothecium sp. have been poorly studied before. The obtained results broaden knowledge on this subject and may contribute to the development of an eco-friendly method of dye elimination.

Degradation and toxicity reduction of the endocrine disruptors nonylphenol, 4-tert-octylphenol and 4-cumylphenol by the non-ligninolytic fungus Umbelopsis isabellina.

Nonylphenol (NP), 4-tert-octylphenol (4-t-OP) and 4-cumylphenol (4-CP) are pollutants that are known as endocrine disruptors mainly due to their estrogen-mimicking activity. These phenolic substances are used in a wide range of industrial and commercial applications. In the present study, biodegradation of tNP, 4-t-OP and 4-CP using the non-ligninolytic fungus Umbelopsis isabellina was investigated. After 12h of incubation, more than 90% of initially applied tNP, 4-t-OP and 4-CP (25mgL(-1)) were eliminated. GC-MS analysis revealed several derivatives mainly (hydroxyalkyl)phenols. Moreover, xenobiotic biotransformation led to the formation of intermediates with less harmful effects than the parent compounds. For all xenobiotics, a decrease in growth medium toxicity was observed, using Artemia franciscana and Daphnia magna as bioindicators. The results indicate that U. isabellina has potential in the degradation and detoxification of contaminants with endocrine activity. Moreover, this is the first report demonstrating that a microorganism is capable of effective 4-CP elimination.

Efficient alachlor degradation by the filamentous fungus Paecilomyces marquandii with simultaneous oxidative stress reduction.

The acceleration of alachlor degradation by Paecilomyces marquandii under controlled and optimized conditions of fungal cultivation in liquid batches was observed (by ca. 20% in comparison to the flask cultures). Acidic environment and oxygen limitation resulted in deterioration of herbicide elimination. Efficient xenobiotic degradation did not correlate with free radicals formation, but some conditions of bioreactor cultivation such as neutral pH and oxygen enriched atmosphere (pO2⩾30%) caused a decrease in the reactive oxygen species (ROS) accumulation in mycelia. The changes in the glutathione (GSH) and ascorbic acid (AA) levels, also in the dismutase (SOD) and catalase (CAT) activities showed active response of the tested fungus against alachlor induced oxidative stress. These results will contribute to the improvement of chloroacetanilides elimination by fungi and extend the knowledge concerning oxidative stress induction and fungal cellular defense.

Malachite green decolorization by the filamentous fungus Myrothecium roridum--Mechanistic study and process optimization.

The filamentous fungus Myrothecium roridum isolated from a dye-contaminated area was investigated in terms of its use for the treatment of Malachite green (MG). The mechanisms involved in this process were established. Peroxidases and cytochrome P-450 do not mediate MG elimination. The laccase of M. roridum IM 6482 was found to be responsible for the decolorization of 8-11% of MG. Thermostable low-molecular-weight factors (LMWF) resistant to sodium azide were found to be largely involved in dye decomposition. In addition, MG decolorization by M. roridum IM 6482 occurred in a non-toxic manner. Data from antimicrobial tests showed that MG toxicity decreased after decolorization. To optimize the MG decolorization process, the effects of operational parameters (such as the medium pH and composition, process temperature and culture agitation) were examined. The results demonstrate that M. roridum IM 6482 may be used effectively as an alternative to traditional decolorization agents.

Biodegradation of nonylphenol by a novel entomopathogenic Metarhizium robertsii strain.

The biodegradation of nonylphenol (NP) by a newly isolated form of the larva fungal strain Metarhizium robertsii IM 6519 was investigated in this study. This isolate was capable of degrading 4-n-NP, and multiple metabolites were detected. The coexistence of parallel degradation pathways with versatile hydroxylation in different positions of the alkyl chain is a unique feature of this strain. Moreover, several metabolites previously described only in higher eukaryotes were detected in the fungal cultures. The degradation process led to the mineralization of 4-n-NP (with an efficiency of 36%), a great advantage of this strain that results in complete removal of toxic substrate from the environment.

Mechanism study of alachlor biodegradation by Paecilomyces marquandii with proteomic and metabolomic methods.

Alachlor is an herbicide that is widely used worldwide to protect plant crops against broadleaf weeds and annual grasses. However, due to its endocrine-disrupting activity, its application had been banned in the European Union. As described in our earlier work, Paecilomyces marquandii is a microscopic fungus capable of alachlor removal by N-acetyl oxidation. Our current work uses proteomics and metabolomics to gain a better understanding of alachlor biodegradation by the microscopic fungus P. marquandii. The data revealed that the addition of alachlor reduced the culture growth and glucose consumption rates. Moreover, the rates of glycolysis and the tricarboxylic acids (TCA) cycle increased during the initial stage of growth, and there was a shift toward the formation of supplementary materials (UDP-glucose/galactose) and reactive oxygen species (ROS) scavengers (ascorbate). Proteomic analysis revealed that the presence of xenobiotics resulted in a strong upregulation of enzymes related to energy, sugar metabolism and ROS production. However, the unique overexpression of cyanide hydratase in alachlor-containing cultures may implicate this enzyme as the key protein involved in the alachlor biodegradation pathway. The characterization of P. marquandii-mediated alachlor removal in terms of cell structure and function provides a deeper insight into the strategies of microorganisms toward xenobiotic biodegradation.

Biodegradation and utilization of 4-n-nonylphenol by Aspergillus versicolor as a sole carbon and energy source.

4-n-Nonylphenol (4-n-NP) is an environmental pollutant with endocrine-disrupting activities that is formed during the degradation of nonylphenol polyethoxylates, which are widely used as surfactants. Utilization of 4-n-NP by the filamentous fungus Aspergillus versicolor as the sole carbon and energy source was investigated. By means of gas chromatography-mass spectrometry, we showed that in the absence of any carbon source other than 4-n-NP in the medium, A. versicolor completely removed the xenobiotic (100 mg L(-1)) after 3 d of cultivation. Moreover, mass spectrometric analysis of intracellular extracts led to the identification of eight intermediates. The mineralization of the xenobiotic in cultures supplemented with 4-n-NP [ring-(14)C(U)] as a growth substrate was also assessed. After 3 d of incubation, approximately 50% of the initially applied radioactivity was recovered in the form of (14)CO2, proving that this xenobiotic was completely metabolized and utilized by A. versicolor as a carbon source. Based on microscopic analysis, A. versicolor is capable of germinating spores under such conditions. To confirm these observations, a microcalorimetric method was used. The results show that even the highest amount of 4-n-NP initiates heat production in the fungal samples, proving that metabolic processes were affected by the use of 4-n-NP as an energetic substrate.

Phospholipids and protein adaptation of Pseudomonas sp. to the xenoestrogen tributyltin chloride (TBT).

A tributyltin (TBT)-resistant strain of Pseudomonas sp. isolated from an overworked car filter was tested for its adaptation to TBT. The isolate was checked for organotin degradation ability, as well as membrane lipid and cellular protein composition in the presence of TBT. The phospholipid profiles of bacteria, grown with and without increased amounts of TBT, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. The strain reacted to the biocide by changing the composition of its phospholipids. TBT induced a twofold decline in the amounts of many molecular species of phosphatidylglycerol and an increase in the levels of phosphatidic acid (by 58%) and phosphatidylethanolamine (by 70%). An increase in the degree of saturation of phospholipid fatty acids of TBT exposed Pseudomonas sp. was observed. These changes in the phospholipid composition and concentration reflect the mechanisms which support optimal lipid ordering in the presence of toxic xenobiotic. In the presence of TBT the abundances of 16 proteins, including TonB-dependent receptors, porins and peroxidases were modified, which could indicate a contribution of some enzymes to TBT resistance.

Tributyltin (TBT) induces oxidative stress and modifies lipid profile in the filamentous fungus Cunninghamella elegans.

To investigate the response of the tributyltin-degrading fungal strain Cunninghamella elegans to the organotin, a comparative lipidomics strategy was employed using an LC/MS-MS technique. A total of 49 lipid species were identified. Individual phospholipids were then quantified using a multiple reaction monitoring method. Tributyltin (TBT) caused a decline in the amounts of many molecular species of phosphatidylethanolamine or phosphatidylserine and an increase in the levels of phosphatidic acid, phosphatidylinositol and phosphatidylcholine. In the presence of TBT, it was observed that overall unsaturation was lower than in the control. Lipidome data were analyzed using principal component analysis, which confirmed the compositional changes in membrane lipids in response to TBT. Additionally, treatment of fungal biomass with butyltin led to a significant increase in lipid peroxidation. It is suggested that modification of the phospholipids profile and lipids peroxidation may reflect damage to mycelium caused by TBT.

Alachlor oxidation by the filamentous fungus Paecilomyces marquandii.

Alachlor, a popular chloroacetanilide herbicide, can be a potential health risk factor. Soil microorganisms are primarily responsible for conversion and migration of alachlor in natural environment, but knowledge concerning alachlor biodegradation is not complete. Therefore, we studied the ability of Paecilomyces marquandii, soil fungus tolerant to heavy metals, to eliminate alachlor and proposed a new pathway of its transformation. After 7 days of incubation only 3.3% of alachlor was detected from an initial concentration 50 mg L(-1) and 20.1% from a concentration 100 mg L(-1). The qualitative IDA LC-MS analysis showed the presence of ten metabolites. All of them were dechlorinated mainly through oxidation, but also reductive dechlorination was observed. The main route of alachlor conversion progressed via N-acetyl oxidation resulting in the formation of mono-, di- and trihydroxylated byproducts. N-acetyl oxidation as a dominant route of alachlor metabolism by fungi has not been described so far. The toxicity of alachlor tested with Artemia franciscana did not increase after treatment with P. marquandii cultures. Paecilomyces marquandii strain seems to be an interesting model for the research on alachlor conversion by soil microscopic fungi, due to its dechlorination and hydroxylation ability as well as high tolerance to heavy metals.

Butyltins degradation by Cunninghamella elegans and Cochliobolus lunatus co-culture.

Organotin compounds are ubiquitous in environment. However, biodegradation of tributyltin (TBT) and dibutyltin (DBT) to non toxic metabolites by fungi has been seldom observed. In this study we constructed a fungal co-culture with an efficient ability of TBT and its metabolites removal. The microscopic fungus strain Cunninghamella elegans degraded TBT via hydroxybutyldibutyltin (OHBuDBT) to its metabolites: DBT and monobutyltin (MBT), which were then transformed by Cochliobolus lunatus. The sequential biodegradation resulted in a 10-fold decrease in samples toxicity to Artemia franciscana larvae. With an initial TBT concentration of 5 mg l(-1), the co-culture of both fungi almost completely eliminated butyltins during 12 days of incubation in synthetic medium. To our knowledge, this is the first report that the mixed fungal co-culture could efficiently degrade TBT. This process was associated with glucose utilization, and a cometabolic nature of butyltins removal by selected strains has been suggested.