Enzymatic and non-enzymatic removal of organic micropollutants with spent mushroom substrate of Agaricus bisporus.

Water bodies are increasingly contaminated with a diversity of organic micropollutants (OMPs). This impacts the quality of ecosystems due to their recalcitrant nature. In this study, we assessed the removal of OMPs by spent mushroom substrate (SMS) of the white button mushroom (Agaricus bisporus) and by its aqueous tea extract. Removal of acesulfame K, antipyrine, bentazon, caffeine, carbamazepine, chloridazon, clofibric acid, and N, N-diethyl-meta-toluamide (DEET) by SMS and its tea was between 10 and 90% and 0-26%, respectively, in a 7-day period. Sorption to SMS particles was between 0 and 29%, which can thus not explain the removal difference between SMS and its tea, the latter lacking these particles. Carbamazepine was removed most efficiently by both SMS and its tea. Removal of OMPs (except caffeine) by SMS tea was not affected by heat treatment. By contrast, heat-treatment of SMS reduced OMP removal to < 10% except for carbamazepine with a removal of 90%. These results indicate that OMP removal by SMS and its tea is mediated by both enzymatic and non-enzymatic activities. The presence of copper, manganese, and iron (0.03, 0.88, and 0.33 µg L-1, respectively) as well as H2O2 (1.5 µM) in SMS tea indicated that the Fenton reaction represents (part of) the non-enzymatic activity. Indeed, the in vitro reconstituted Fenton reaction removed OMPs > 50% better than the teas. From these data it is concluded that spent mushroom substrate of the white button mushroom, which is widely available as a waste-stream, can be used to purify water from OMPs.

Binding of micro-nutrients to the cell wall of the fungus Schizophyllum commune.

The cell wall fulfils several functions in the biology of fungi. For instance, it provides mechanical strength, interacts with the (a)biotic environment, and acts as a molecular sieve. Recently, it was shown that proteins and ß-glucans in the cell wall of Schizophyllum commune bind Cu2+. We here show that the cell wall of this mushroom forming fungus also binds other (micro-)nutrients. Ca2+, Mg2+, Mn2+, NO3-, PO43-, and SO42- bound at levels > 1 mg per gram dry weight cell wall, while binding of BO3-, Cu2+, Zn2+ and MoO42- was lower. Sorption of Ca2+, Mn2+, Zn2+ and PO43- was promoted at alkaline pH. These compounds as well as BO33-, Cu2+, Mg2+, NO3-, and SO42- that had bound at pH 4, 6, or 8 could be released from the cell wall at pH 4 with a maximum efficiency of 46-93 %. Solid-state NMR spectroscopy showed that the metals had the same binding sites as Cu2+ when a low concentration of this ion is used. Moreover, data indicate that anions bind to the cell wall as well as to the metal ions. Together, it is shown that the cell wall of S. commune binds various (micro-)nutrients and that this binding is higher than the uptake by hyphae. The binding to the cell wall may be used as a storage mechanism or may reduce availability of these molecules to competitors or prevent toxic influx in the cytoplasm.

Heat resistance acquirement of the spoilage yeast Saccharomyces diastaticus during heat exposure.

The main fungal cause of spoilage of carbonated fermented beverages in the brewing industry is the amylolytic budding yeast Saccharomyces cerevisiae subsp. diastaticus (Saccharomyces diastaticus). Heat treatment is used to avoid microbial spoilage of the fermented beverages. Therefore, the spoilage capacity of S. diastaticus may be linked to its relative high heat resistance. Here, we assessed whether S. diastaticus can acquire heat resistance when exposed to heat stress. To this end, ascospores of S. diastaticus strain MB523 were treated at 60°C for 10 min followed by growing the surviving spores on a glucose-containing medium. The resulting vegetative cells were then allowed to sporulate again in sporulation medium. This cycle of heat treatment, vegetative growth, and sporulation was performed eight times in three independent lineages. After these eight cycles, the sporulation rate was similar to the start (∼75%) but the resulting ascospores were more heat resistant. The time needed to kill 90% of the population at 60°C (i.e. the D60-value) increased from 6.5 to 9.0 min (p = 0.005). The vegetative cells also showed a trend to increased heat resistance with an increase in the D52-value from 9.2 to 16.2 min (p = 0.1). In contrast, heat resistance of the vegetative cells that had not been exposed to heat during the eight cycles had been reduced with a D52-value of 4.2 min (p = 0.003). Together, these data show that S. diastaticus MB523 can easily acquire heat resistance by inbreeding while subjected to heat stress. Conversely, heat resistance can be easily lost in the absence of this stress condition, indicative of a trade-off for heat resistance.

Probing Cell-Surface Interactions in Fungal Cell Walls by High-Resolution 1 H-Detected Solid-State NMR Spectroscopy.

Solid-state NMR (ssNMR) spectroscopy facilitates the non-destructive characterization of structurally heterogeneous biomolecules in their native setting, for example, comprising proteins, lipids and polysaccharides. Here we demonstrate the utility of high and ultra-high field 1 H-detected fast MAS ssNMR spectroscopy, which exhibits increased sensitivity and spectral resolution, to further elucidate the atomic-level composition and structural arrangement of the cell wall of Schizophyllum commune, a mushroom-forming fungus from the Basidiomycota phylum. These advancements allowed us to reveal that Cu(II) ions and the antifungal peptide Cathelicidin-2 mainly bind to cell wall proteins at low concentrations while glucans are targeted at high metal ion concentrations. In addition, our data suggest the presence of polysaccharides containing N-acetyl galactosamine (GalNAc) and proteins, including the hydrophobin proteins SC3, shedding more light on the molecular make-up of cells wall as well as the positioning of the polypeptide layer. Obtaining such information may be of critical relevance for future research into fungi in material science and biomedical contexts.