Henk J. Swarts

Detoxification and partial mineralization of the azo dye mordant orange 1 in a continuous upflow anaerobic sludge-blanket reactor

Abstract In batch toxicity assays, azo dye compounds were found to be many times more toxic than their cleavage products (aromatic amines) towards methanogenic activity in anaerobic granular sludge. Considering the ability of anaerobic microorganisms to reduce azo groups, detoxification of azo compounds towards methanogens can be expected to occur during anaerobic wastewater treatment. In order to test this hypothesis, the anaerobic degradation of one azo dye compound, Mordant orange 1 (MO1), by granular sludge was investigated in three separate continuous upflow anaerobic sludge-blanket reactors. One reactor, receiving no cosubstrate, failed after 50 days presumably because of a lack of reducing equivalents. However, the two reactors receiving either glucose or a volatile fatty acids (acetate, propionate, butyrate) mixture, could eliminate the dye during operation for 217 days. The azo dye was reductively cleaved to less toxic aromatic amines (1,4-phenylenediamine and 5-aminosalicylic acid) making the treatment of MO1 feasible at influent concentrations that were over 25 times higher than their 50% inhibitory concentrations. In the reactor receiving glucose as cosubstrate, 5-aminosalicylic acid could only be detected at trace levels in the effluent after day 189 of operation. Batch biodegradability assays with the sludge sampled from this reactor confirmed the mineralization of 5-aminosalicylic acid to methane.

Substrate specificity and stereospecificity of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14; an enzyme showing sequential and enantioconvergent substrate conversion

Abstract Limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene degradation pathway of this microlorganism, has a narrow substrate specificity. Of the compounds tested, the natural substrate, limonene-1,2-epoxide, and several alicyclic and 2-methyl-1,2-epoxides (e.g. 1-methylcyclohexene oxide and indene oxide), were substrates for the enzyme. When LEH was incubated with a diastereomeric mixture of limonene-1,2-epoxide, the sequential hydrolysis of first the (1R,2S)- and then the (1S,2R)-isomer was observed. The hydrolysis of (4R)- and (4S)-limonene-1,2-epoxide resulted in, respectively, (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diol as the sole product with a diastereomeric excess of over 98%. With all other substrates, LEH showed moderate to low enantioselectivities (E ratios between 34 and 3).

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 monochlorinated metabolites with a 1-benzoxepin skeleton from Mycena galopus

Two novel monochlorinated 2,3-dihydro-1-benzoxepin derivatives were isolated from the ethyl acetate extracts of the stipes of Mycena galopus. The structures were established on the basis of their spectral data.

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).

Calculated ionisation potentials determine the oxidation of vanillin precursors by lignin peroxidase

In view of the biocatalytic production of vanillin, this research focused on the lignin peroxidase (LiP) catalysed oxidation of naturally occurring phenolic derivatives: O‐methyl ethers, O‐acetyl esters, and O‐glucosyl ethers. The ionisation potential (IP) of a series of model compounds was calculated and compared to their experimental conversion by LiP, defining a relative IP threshold of approximately 9.0 eV. Based on this threshold value only the O‐acetyl esters and glucosides of isoeugenol and coniferyl alcohol would be potential LiP substrates. Both O‐acetyl esters were tested and were shown to be converted to O‐acetyl vanillin in molar yields of 51.8 and 2.3%, respectively.

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.

Acid-catalyzed enzymatic hydrolysis of 1-methylcyclohexene oxide

Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene metabolism of this microorganism, catalyzes the enantioselective hydrolysis of 1-methylcyclohexene oxide. (1R,2S)-1- Methylcyclohexene oxide was the preferred substrate and it was mainly hydrolyzed to (1S,2S)-1-methylcyclohexane-1,2-diol, while (1S,2R)-1- methylcyclohexene oxide was converted more slowly and mainly yielded (1R,2R)- 1-methylcyclohexane-1,2-diol. The reaction proceeded with a high regioselectivity (C1:C2, 85:15). H218O-labelling experiments confirmed that the nucleophile was mainly incorporated at the most substituted carbon atom, suggesting that limonene-1,2-epoxide hydrolase uses an acid-catalyzed enzyme mechanism.

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.