Frank J. M. Verhagen

Natural production of chloroform by fungi

Abstract Chloroform production was detected in the headspace of pure cultures of the basidiomycetes Mycena metata and Peniophora pseudopini and the deuteromycete Caldariomyces fumago. The average production rates were in the range of 0.07–70 μg\l culture fluid\day for Caldariomyces fumago and 0.7–40 ng\l culture fluid\day for the basidiomycetes; they depended on the composition of the medium, pH and the initial concentration of oxygen. In incidental cases, chloroform was identified in the headspace of pure cultures of the basidiomycetes Agaricus arvensis, Bjerkandera sp. BOS55, and Phellinus pini. It is suggested that fungi are important sources of elevated concentrations of chloroform in soil air.

Natural organohalogen production by basidiomycetes

Basidiomycetes are ecologically important higher fungi that synthesize three families of organohalogen metabolites: halomethanes, halogenated aromatics and haloaliphatic compounds. To date, a total of 53 halogenated metabolites have been identified in 34 genera of basidiomycetes. These organohalogen metabolites have demonstrable physiological roles as antibiotics, as methyl donors and as subsstrates for H 2 O 2 -generating oxidases. The concentration of chlorinated aromatic metabolites encountered in natural environments associated with the widespread occurrence of basidiomycetes have been shown to exceed the hazardous-waste norms that are applied to analogous anthropogenic chlorophenols in soil.

The ubiquity of natural adsorbable organic halogen production among basidiomycetes

Abstract Recently, several species of basidiomycetes were shown to produce de novo high concentrations of chloroaromatic metabolites. Since these lignocellulose-degrading fungi play a major role in the ecosphere, the purpose of this study was to determine the ubiquity of organohalogen production among basidiomycetes. A total of 191 fungal strains were monitored for adsorbable organic halogen (AOX) production when grown on defined liquid media. Approximately 50% of the strains tested and 55% of the genera tested produced AOX. A low production of 0.1–0.5 mg AOX/l was observed among 25% of the strains, a moderate production of 0.5–5.0 mg AOX/l was observed among 16% of the strains and 9% of the strains produced high levels (5–67 mg AOX/l). The latter group was dominated by species belonging to the genera Hypholoma, Mycena and Bjerkandera, showing specific AOX productions in the range 1074–30893 mg AOX/kg dry weight of mycelial biomass. Many highly ecologically significant fungal species were identified among the moderate to high producers. These species were also able to produce AOX when cultivated on natural lignocellulosic substrates. Hypholoma fasciculare and Mycena metata respectively produced up to 132 mg and 193 mg AOX/kg dry weight of forest litter substrate in 6 weeks.

Novel chlorometabolites produced by Bjerkandera species

The EtOAc extract from the extracellular fluid of the mycelium of Bjerkandera sp. BOS55 contained four novel chlorinated benzoic acid derivatives, i.e. 3-chloro-4-hydroxybenzoic acid, 3,5-dichloro-4-hydroxybenzoic acid, methyl 3,5-dichloro-4-hydroxybenzoate and methyl 3,5-dichloro-p-anisate. 3-Chloro-4-hydroxybenzoic acid was also produced by B. adusta.

Trichlorinated phenols from hypholoma elongatum

Three trichlorinated phenols, 2,4,6-trichloro-3-methoxyphenol, 3,5,6-trichloro-2,4-dimethoxyphenol and 3,4,6-trichloro-2,5-dimethoxyphenol, were detected as novel metabolites in the ethyl acetate extract from the culture medium of the Basidiomycete, Hypholoma elongatum (strain WIJS94-28).

Organohalogen production is a ubiquitous capacity among basidiomycetes

Several species of basidiomycetes are capable of producing de novo high concentrations of chloroaromatic metabolites. However, the extent to which basidiomycetes contribute to the natural pool of adsorbable organic halogen (AOX) found in the environment is unknown yet. The purpose of this study was to determine the ubiquity of organohalogen production among basidiomycetes and to determine maximal specific organohalogen production rates. Finally, the fate of the fungal chloroaromatic compounds in the environment was studied. A total of 191 fungal strains were tested for AOX production when grown on defined liquid medium. Approximately 50% of the strains tested and 55% of the genera tested produced AOX. Organohalogen production seemed to be a ubiquitous capacity among basidiomycetes. Many highly ecologically significant fungal species were identified among the moderate and high producers. Although it was found that the final AOX concentrations produced by Hypholoma fasciculare was strongly influenced by the substrate used, all maximal specific AOX production rates on different substrates were in the same order of magnitude. Seven new species and four new genera of basidiomycetes could be added to the list of known chlorinated anisyl metabolites (CAM) producing basidiomycetes. In degradation studies of the major fungal metabolite 3,5-dichloro-anisyl alcohol, it was found that in forest soils there seems to be ubiquitous mineralizing capacity for this chlorinated aromatic compound. It was found that Burkholderia cepacia was responsible for the fast degradation of the fungal compound in the oak forest soils.

Chlorinated anisyl metabolites produced by basidiomycetes

Chlorinated anisyl metabolites (CAM) were detected in ethyl acetate extracts from the culture medium of species of seven basidiomycete genera including, for the first time, Mycena, Peniophora, Phellinus and Phylloporia . Extension of CAM biosynthesis in Bjerkandera (B. fumosa), Hypholoma (H. elongatum) and Pholiota (P. adiposa) is a new finding. The selective and high-yield production of 3,5-dichloro- p anisyl alcohol by H. elongatum is remarkable.

Chemical bromination of phenol red by hydrogen peroxide is possible in the absence of haloperoxidases

Abstract The dye phenol red (phenolsulphonphthalein) was chemically brominated to bromophenol blue (3′,3″,5′,5″-tetrabromophenolsulphonphthalein) directly by hydrogen peroxide (20 mM or higher) under acidic pH (0.5–4.5) and moderate temperature (30°C). Since this bromination reaction takes place in the absence of any chemical or biological catalyst, the results represent an important warning concerning a commonly used assay for detecting or screening haloperoxidases. Bromophenol red (5′,5″-dibromophenolsulphonphthalein) and another unidentified compound were observed as temporary intermediates. Incubation of the reaction mixture with KCl instead of NaBr did not yield any chlorinated products under the same conditions.

The formation of veratryl chloride by Bjerkandera sp. Strain BOS55.

In the ethyl acetate extract from the extracellular fluid of the mycelium of Bjerkandera sp. BOS55 the presence of veratryl chloride was detected. Addition of deuterated benzoic acid or 4-hydroxybenzoic acid to the culture resulted in the formation of deuterated veratryl chloride. The detection of veratryl chloride constitutes the first report of a biologically derived natural product which contains a halogenated benzylic carbon. Since veratryl chloride is not very stable in aqueous medium, its detection also suggests a continuous production.