Ed de Jong

Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics

Woody tissues are composed mainly of three biopoly- mers: cellulose; hemicellulose; and lignin. Lignin, a highly irregular aromatic polymer which serves to pro- vide strength and structure to the tissue, is synthesized in plants by a random peroxidase-catalysed polym- erization of substituted p-hydroxy-cinnamyl alcohols. Only a few groups of microorganisms are capable of degrading complex lignin polymers, and they are best exemplified by the white-rot fungi, which cause the greatest degree of mineralization. The white-rot fungus

Isolation and screening of basidiomycetes with high peroxidative activity

Sixty-seven Poly R-478 decolorizing basidiomycetes were isolated with a selective medium (containing hemp ( Cannabis sativa ) stem wood, guaiacol and benomyl). Several of the new isolates seemed to be promising manganese peroxidase-containing white-rot fungi. Enzyme assays indicated that either glyoxal or veratryl alcohol oxidase were present in the culture fluids of peroxidative strains. In contrast, lignin peroxidase was only detected in Phanerochaete chrysosporium , despite attempts to induce this enzyme in other strains with oxygen and oxygen/veratryl alcohol additions. A highly significant correlation was found between two ligninolytic indicators: ethene formation from α-keto-γ-methylthiolbutyric acid and the decolorization of a polymeric dye, Poly R. Three of the new isolates had significantly higher Poly R decolorizing activities compared to P. chrysosporium . The Poly R decolorization rate is a good assay when trying to optimize culture conditions for peroxidase/H 2 O 2 production.

Evidence for a new extracellular peroxidase. Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55.

A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp, BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6‐dimethoxyphenol (DMP), Poly R‐478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named ‘Manganese‐Inhibited Peroxidase’.

Analysis of retted and non retted flax fibres by chemical and enzymatic means

Flax fibres (Linum usitatissimum L.) were subjected to chemical and enzymatic analysis in order to determine the compositional changes brought about by the retting process and also to determine the accessibility of the fibre polymers to enzymatic treatment. Chemical analysis involved subjecting both retted and non retted fibres to a series of sequential chemical extractions with 1% ammonium oxalate, 0.05 M KOH, 1 M KOH and 4 M KOH. Retting was shown to cause minimal weight loss from the fibres but caused significant changes to the pectic polymers present. Retted fibres were shown to have significantly lower amounts of rhamnogalacturonan as well as arabinan and xylan. In addition the average molecular mass of the pectic extracts was considerably lowered. Enzyme treatment of the 1 M KOH extracts with two different enzymes demonstrated that the non retted extract contained a relatively high molecular weight xylan not found in the retted extract. Treatment of the 1 M KOH extracts and the fibres with Endoglucanase V from Trichoderma viride demonstrated that while this enzyme solubilised cellulose as well as xylan and xyloglucan oligomers from the extract, it had limited access to these polymers on the fibre. MALDI-TOF MS analysis of the material solubilised from the extract suggested that the xylan was randomly substituted with 4-O-methyl glucuronic acid moieties. The xyloglucan was shown to be of the XXXG type and was substituted with galactose and fucose units. The enzyme treatments of the fibres demonstrated that the xylan and xyloglucan polymers in the fibres were not accessible to the enzyme but that material which was entrapped by the cellulose could be released by the hydrolysis of this cellulose.

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.

Physical characterization of enzymatically modified kraft pulp fibers

Abstract Douglas-fir kraft pulps were treated with an enzyme preparation containing both cellulase and xylanase activities. Treatments resulted in the solubilization of 21.1% the xylan and 1.8% of the cellulose. Changes in fiber and paper properties were observed. Monitoring pore volume, degree of polymerization, crystallinity, FT-IR spectra, and scanning electron microscopy helped elucidate changes in fiber composition and morphology. Data indicate a decline in intrinsic fiber strength due to an erosion of the fiber surface. Reduction in paper strength resulted from the collective effects of decreased intrinsic fiber strength and the reduction in the degree of polymerization of a large portion of the hemicellulose component of the fibers, as well as fiber defibrillation and fines hydrolysis.

De-novo biosynthesis of chlorinated aromatics by the white-rot fungus Bjerkandera sp. BOS55 : formation of 3-chloro-anisaldehyde from glucose

The white‐rot fungus Bjerkandera sp. BOS55 produced de‐novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white‐rot fungi, other metabolites were formed. These included anisaldehyde, 3‐chloro‐anisaldehyde and a yet unknown compound containing two chlorine atoms. Additionally GC/MS analysis revealed the production of small amounts of anisyl alcohol and 3‐chloro‐anisyl alcohol. After 14 days, the extracellular fluid of Bjerkandera BOS55 contained 100 μM veratraldehyde and 50 μM 3‐chloro‐anisaldehyde. This is the first report of de‐novo biosynthesis of simple chlorinated aromatic compounds by a white‐rot fungus. Anisaldehyde and 3‐chloro‐anisaldehyde were also produced by Bjerkandera adusta but not by Phanerochaete chrysosporium.

Size-exclusion chromatography of lignin- and carbohydrate-containing samples using alkaline eluents

Size-exclusion chromatography of carbohydrate- and lignin-containing samples, prepared from wood and pulp samples, was carried out using alkaline eluents. The elution of carbohydrate and lignin macromolecules can be monitored with good selectivity using a pulsed amperometric and a UV absorbance detector, respectively. However, the stability of the signals from samples stored in alkaline solutions requires careful consideration in the analysis because marked changes are observed in many cases. Furthermore, the interactions between aromatic molecules and the separation column are also dependent on the concentration of alkali in the eluent. Since there seem to be no adsorptive interactions between carbohydrate and lignin macromolecules during size-exclusion chromatography, the described method can be used to monitor chemical and enzymic modifications of the apparent molecular mass of these two classes of compounds, whether they occur in mixtures or covalently linked complexes.

Low-load compression testing: a novel way of measuring biofilm thickness.

ABSTRACT Biofilms are complex and dynamic communities of microorganisms that are studied in many fields due to their abundance and economic impact. Biofilm thickness is an important parameter in biofilm characterization. Current methods of measuring biofilm thicknesses have several limitations, including application, availability, and costs. Here, we present low-load compression testing (LLCT) as a new method for measuring biofilm thickness. With LLCT, biofilm thicknesses are measured during compression by inducing small loads, up to 5 Pa, corresponding to 0.1% deformation, making LLCT essentially a nondestructive technique. Comparison of the thicknesses of various bacterial and yeasts biofilms obtained by LLCT and by using confocal laser scanning microscopy (CLSM) resulted in the conclusion that CLSM underestimates the biofilm thickness due to poor penetration of different fluorescent dyes, especially through the thicker biofilms, whereas LLCT does not suffer from this thickness limitation.

Bleach boosting and direct brightening by multiple xylanase treatments during peroxide bleaching of kraft pulps.

The effects of multiple xylanase treatments were assessed during the peroxide bleaching of three pulps: Douglas-fir (kraft); Western hemlock (oxygen delignified kraft); and trembling Aspen (kraft). The addition of a xylanase treatment stage, either before or after the peroxide bleaching stage(s), resulted in the enhanced brightening of all pulps. A higher brightness was achieved using two enzyme treatments, one before and one after the peroxide stage(s). Both bleach boosting and direct brightening seemed to contribute to the enhancement of the peroxide bleaching. Compared to xylanase prebleaching, xylanase posttreatment of peroxide bleached pulps solubilized less lignin and chromophores and made smaller amounts of these materials alkaline soluble. Nevertheless, the final brightness achieved by xylanase posttreatment was similar or superior to that achieved with xylanase prebleaching of the corresponding unbleached pulps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 312-318, 1997.