Jerzy Długoński

Biodegradation of 4-n-nonylphenol by the non-ligninolytic filamentous fungus Gliocephalotrichum simplex: a proposal of a metabolic pathway.

4-Nonylphenols (NPs) are endocrine disrupting compounds (EDCs) which are known to interfere with the endocrine system of humans and animals. The aim of this study was to test the ability of non-ligninolytic filamentous fungus Gliocephalotrichum simplex to biodegrade 4-n-NP. The results revealed that during the first 24h of incubation, 4-n-NP at the concentration of 50 mg L(-1) was eliminated from the culture medium by 88%, whereas at the concentration of 100 mg L(-1) by 50%. In this paper, glucose utilization as a co-substrate during toxic compound degradation was also shown. It was found that the presence of 4-n-NP caused sugar metabolism retardation and this inhibition was dependent on NP concentration. The qualitative GC-MS analysis showed the presence of products of G. simplex 4-n-NP biodegradation. We proposed the metabolic pathway of 4-n-NP biodegradation, which is based on subsequent C1 removals from the alkyl chain followed by the aromatic ring cleavage. In further experiments with 4-n-NP [ring-(14)C(U)] we proved aromatic ring cleavage occurrence. After 72 h of incubation the evolution of (14)CO(2) was observed and the mineralization efficiency was on the level of 29%. The results suggest the existence of a novel mechanism of 4-n-NP degradation in fungi.

Concurrent corticosteroid and phenanthrene transformation by filamentous fungus Cunninghamella elegans

A filamentous fungus Cunninghamella elegans IM 1785/21Gp which displays ability of 17alpha,21-dihydroxy-4-pregnene-3,20-dione (cortexolone) 11-hydroxylation (yielding epihydrocortisone (eF) and hydrocortisone (F)) and polycyclic aromatic hydrocarbons (PAHs) degradation, was used as a microbial eucaryotic model to study the relationships between mammalian steroid hydroxylation and PAHs metabolization. The obtained results showed faster transformation of phenanthrene in Sabouraud medium supplemented with steroid substrate (cortexolone). Simultaneously phenanthrene stimulated epihydrocortisone production from cortexolone. In phenanthrene presence the ratio between cortexolone hydroxylation products (hydrocortisone and epihydrocortisone) was changed from 1:5.1-6.2 to 1:7.6-8.4 in the culture without phenanthrene. Cytochrome P-450 content significantly increased after the culture supplementation by the second substrate, phenanthrene or cortexolone, adequately. To confirm the involvement of cytochrome P-450 in phenanthrene metabolism, the inhibition studies were performed. The cytochrome P-450 inhibitors SKF 525-A (1.5mM) and 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) (2mM) inhibited phenanthrene transformation by 80 and 62%, respectively. 1-aminobenzotriazole (1mM) completely blocked phenanthrene metabolism. The obtained results suggest a presence of connections between steroid hydroxylases and enzymes involved in PAH degradation in C. elegans.

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.

Removal of anthracene and phenanthrene by filamentous fungi capable of cortexolone 11‐hydroxylation

Nine fungal strains showing ability of cortexolone hydroxylation to epicortisol and/or cortisol were screened in this work for anthracene and phenanthrene elimination (250 mg/l). All of the strains (Cylindrocladium simplex IM 2358, C. simplex IM 2358/650, Monosporium olivaceum IM 484, Curvularia lunata IM 2901, C. lunata IM 2901/366, C. tuberculata IM 4417, Cunninghamella elegans IM 1785, C. elegans IM 1785/21Gp, C. elegans IM 1785/10Gi) significantly removed anthracene and phenanthrene. During incubation with anthracene formation of intermediate products was observed. The amount of the main intermediate product, identified as 9,10‐anthraquinone, was not greater than 22.2% of the anthracene introduced to the fungal cultures. C. elegans IM 1785/21Gp was the best degrader of both anthracene and phenanthrene, removing 81.6 and 99.4% of these compounds after 7 days, respectively. Phenanthrene removal by C. elegans IM 1785/21Gp was preceded by PAHs accumulation in mycelium and growth inhibition. Elimination of phenanthrene started after one day of incubation and was related to the fungus growth.

Transformation of steroids by fungal protoplasts

SummaryProtoplasts of Cunninghamella elegans transformed cortexolone to the same products as did the mycelium. Transformation of the steroid by non-induced mycelium and by protoplasts released from it was almost completely inhibited by cycloheximide. However, hydroxylation of cortexolone was not affected by this antibiotic if mycelium grown in the presence of an enzyme inducer or protoplasts obtained from the “induced” mycelium were used. The transformation rate of protoplasts, on the basis of dry weight or protein units, was about four times higher than that of the mycelium, indicating that the mycelial cell wall was a serious rate-limiting factor in steroid bioconversion.

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.

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.

BACTERIAL ELIMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS AND HEAVY METALS

The elimination of polycyclic aromatic hydrocarbons (PAHs), in the presence and absence of heavy metals by bacteria isolated from a contaminated area of Poland was examined. Among fifty four isolates ten strains had an ability to utilize at least one of the following xenobiotic substrates: anthracene (250 mg/l), phenanthrene (250 mg/l) and pentachlorophenol (10 mg/l) at a rate of 50ń100%. The strains were also found to be able to grow at high concentration of lead added as a lead acetate. The isolated strain, most active in utilization of applied xenobiotics was identified as Rhodococcus equi. Although the degradation of anthracene was inhibited in the presence of lead acetate, protein synthesis and the xenobiotic uptake was observed (at lead concentration of 1000 mg/l). Acetate as a more readily metabolized organic substrate was found as a co‐metabolic substrate. The further experiments with cells entrapped on glass membrane filter indicated that Rhodococcus equi IM 6KB3 could be useful for the rapid removal of lead and anthracene from contaminated liquids.

Synthesis of silver nanoparticles from Metarhizium robertsii waste biomass extract after nonylphenol degradation, and their antimicrobial and catalytic potential

Microorganisms are commonly applied to remove toxic compounds from the contaminated environment. Decontamination processes result in the generation of large amounts of biomass waste, whose disposal still remains a problem. Biomass waste is a very good source of various organic and inorganic substances, and it can be further used in many processes. In this study, an innovative approach, synthesis of silver nanoparticles (AgNPs) from biomass waste obtained from the filamentous fungus Metarhizium robertsii after nonylphenol degradation, was applied using different biomass extract contents (25, 50, 75 and 100%). The highest reduction rates were noted for diluted samples (25 and 50%) and photoinduction allowed reduction of the time required for synthesis to 3 h. AgNP synthesis from waste biomass extracts was not only more efficient compared to the control biomass extract, but also the obtained nanoparticles were smaller, more homogenous in size and predominantly spherical, which makes them more applicable for medical or catalytic purposes. FT-IR and XPS analyses revealed the protein involvement in AgNP synthesis. The studies conducted with the application of enzyme inhibitors pointed to the enzyme being engaged in AgNP synthesis, while the amounts of capping agents present in nanoparticles were determined by thermogravimetric analysis. AgNPs formed from waste biomass extract express not only high biocatalytic activity in toxic compound degradation (e.g. nitrophenol and methylene blue), but also possess antimicrobial activity against Gram-positive and Gram-negative bacteria and can be applied as an effective biocatalyst or for medical purposes.