Aloys Hüttermann

Oxidation of Polycyclic Aromatic Hydrocarbons (PAH) by Laccase of Trametes Versicolor

Laccase of Trametes versicolor was able to oxidize in vitro most of the 14 polycyclic aromatic hydrocarbons (PAH) tested. Acenaphthylene was removed by 37% followed by anthracene and benzo[a]pyrene which were oxidized by 18 and 19%, respectively. Lower but significant oxidation of about 10% was found for eight additional PAH: acenaphthene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, and perylene. Naphthalene, fluorene, and phenanthrene were recovered unchanged after incubation for 72 h with laccase. Addition of 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of PAH: acenaphthylene, acenaphthene, fluorene, anthracene, benzo[a]pyrene, and perylene were almost completely removed from the reaction mixture. Oxidation of pyrene and benzo[a]anthracene increased from 8 and 6% without a mediator to 48 and 53% in the presence of HBT. Other PAH were not significantly influenced by the addition of this mediator. PAH-quinones as oxidation products were formed from all PAH to different extents. A part of PAH was polymerized in the laccase/mediator system to products of weight-average molecular weight (MW) of approximately 1,500 Da. The correlation of the ionization potentials of PAH with the oxidation of these compounds is limited to the alternating PAH.

Addition of hydrogels to soil for prolonging the survival of Pinus halepensis seedlings subjected to drought

The effect of an amendment of a sandy soil with superabsorbent hydrogels (Stockosorb K 400, a highly cross-linked polyacrylamide with about 40% of the amide group hydrolysed to carboxylic groups) on the survival of Pinus halepensis seedlings during water stress was studied, with the following concentrations added to the soils: 0.04%, 0.08%, 0.12%, 0.20%, and 0.40%. The water retention of the soil increased exponentially with increasing additions of hydrogel to the soil. The highest concentration used changed the water retention capacity and its change in water potential with regard to its water content from a typical sand to a loam or even silty clay. During desiccation under controlled conditions, the seedlings treated with 0.4% hydrogel survived twice as long in the soils amended with 0.4% hydrogel as in the control soils. During the drought, the seedlings exhibited a pronounced growth both of the shoot and the roots, which was about three-fold higher than the one of the plants in the control soils. The data indicate that an amendment of soils with this type of hydrogels at 0.4% (w/w) will greatly enhance the drought tolerance of the seedlings growing on this substrate. A comparison between the reaction of the plants in the hydrogel amended soils and the pF-curve measured with the plate test revealed that less water was actually available for the plants, as could be expected from the pF-curves.

Degradation of anthracene by laccase of Trametes versicolor in the presence of different mediator compounds

Abstract Laccase of Trametes versicolor was generally able to oxidize anthracene in vitro. After 72 h incubation about 35% of the anthracene was transformed stoichiometrically to 9,10-anthraquinone. Transformation of anthracene increased rapidly in the presence of different mediators that readily generate stable radicals: 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 1-hydroxybenzotriazole. For the reaction, the presence of both the laccase and the mediator was necessary. In the presence of 0.005 mM 1-hydroxybenzotriazole this conversion had removed 47% of the anthracene after 72 h; 75% of the substrate was oxidized during this period when ABTS (1 mM) was used as mediator. In contrast to reactions without or with only low concentrations of a mediator, there was a discrepancy between the disappearance of anthracene and the formation of 9,10-anthraquinone in mediator-forced reactions. Coupling-products of mediators with anthracene degradation products were found. Anthracene disappeared nearly completely after incubation for 72 h with laccase in a 0.1 mM solution of 1-hydroxybenzotriazole and was transformed to 9,10-anthraquinone in about 80% yield; 90% of the substrate was transformed in the presence of ABTS (2.0 mM) resulting again in 80% quinone. Phenothiazine was not effective in this system.

Oxidation of aromatic alcohols by laccase from Trametes versicolor mediated by the 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) cation radical and dication

Abstract Oxidation of aromatic alcohols, such as non-phenolic lignin model compounds, by oxidised species of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) has been investigated. The cation radical and dication formed from ABTS were both capable of oxidising aromatic alcohols to aldehydes. The reactions terminated at the level of the aldehyde and no acids were formed. The cation radical and dication worked in a cycle as an electron-transfer compound between an oxidant and alcohol. In addition to the oxidation of the primary benzyl-hydroxyl group, an oxidation of the secondary α-hydroxyl group to the ketone by the dication was possible. All distinguishing features of these reactions corresponded to the results of the oxidation performed by the laccase of Trametes versicolor in the presence of ABTS. The decomposition products from the dication alone and ABTS with laccase confirmed the supposition that the dication was involved in the laccase mediator system. A reaction mechanism based on deprotonation of the alcohol cation radical was predicted to play a key role in the irreversible followup reaction and to be the driving force of the process.

Sodium and chloride distribution in roots and transport in three poplar genotypes under increasing NaCl stress

Abstract We investigated the effects of increasing soil NaCl on growth, water flow, ion transport and intracellular compartmentation of Na + and Cl − in 1-year-old seedlings of Populus euphratica Oliv. and 1-year-old rooted cuttings of P .× euramericana cv. I-214 (cv. Italica) and P. ‘ popularis 35-44’ ( P. popularis ). Relative growth rates of leaf area (RGR A ) and shoot height (RGR H ) of cv. Italica and P. popularis were severely restricted by increasing salinity, whereas in P. euphratica both were not significantly inhibited . Salinised trees of P. euphratica experienced 10% leaf area loss during a 30-day study, however, cv. Italica and P. popularis shed over 50% of their initial surface area. Leaf necrosis of the two salt-sensitive genotypes (cv. Italica and P. popularis ) was attributed to excessive salt accumulation and reduced water loss. The rapid built-up of leaf salt in these two genotypes was mainly the result of high ion concentrations in the transpiration stream. An artificially generated lower shoot-to-root ratio, which was reached by removing approximately 50% of total leaf area from shoots of cv. Italica and P. popularis prior to the salt treatment did not enhance their salinity tolerance since root-to-shoot salt fluxes were largely independent of water flow. Compared with cv. Italica and P. popularis , P. euphratica maintained considerably higher unit leaf transpiration rates with lower salt concentrations in the transpiration stream during the period of salt stress. Therefore, salt tolerance of P. euphratica likely depends on its ability to restrict salt transport to leaves. X-ray microanalysis of root compartments showed that there was genotypic difference in the pattern of ion compartmentation. Cv. Italica exhibited a greater capacity to accumulate salt in cortical vacuoles compared with P. popularis , even though both were considered as salt-sensitive genotypes. P. euphratica was more effective than the other two genotypes to block apoplasmic ion transport and sequester Cl − in cortical vacuoles at high salinity. These limited ion loading into the xylem during radial transport, and thus contributed to the restriction of subsequent axial transport.

Photosynthetic response of Populus euphratica to salt stress

Abstract P. euphratica Olive and the hybrids (P. talassica Kom X (P. euphratica + Salix alba L)) seedlings were subjected to low (50 mM NaCl) and high salt (200 mM NaCl) treatments to determine their photosynthetic responses to salt stress. The photosynthetic pattern indicated that P. euphratica is a C3 plant with a high CO2 compensation point (150 μmol mol−1) and saturation point (900 μmol mol−1), but has some characteristics of C4 plants with a high light saturation point (2800 μmol m−2 s−1) in control conditions. CO2 compensation and saturation points increased with high salt treatment for both the hybrid and P. euphratica while light saturation point decreased with salt treatment. The net photosynthesis of P. euphratica with high salt treatment declined in the first 14 days and recovered to 70% that with control in day 21 when soil salt content was about 0.7% NaCl, while that of the hybrids did not recover when soil salt content was about 0.5% NaCl with hgih salt treatment. With increasing salt levels, Chlorophyll a contents and Chlorophyll a b ratio increased, while Chlorophyll b and carotenoid contents decreased by day 10, when photosynthesis was heavily depressed for both species. Fluorescence patterns confirmed that reduction of photosynthesis under high salt treatment was not due to damage of the photosynthetic apparatus, but more likely due to inhibition of the dark reaction.

Decomposition of 14C-labelled lignin, holocellulose and lignocellulose by mycorrhizal fungi

Five different species of known ecto-mycorrhizal fungi: Cenococcum geophilum, Amanita muscaria, Tricholoma aurantium, Rhizopogon luteolus and Rhizopogon roseolus were studied for their ability to metabolize the major components of plant cell walls. All strains were able to decompose 14C-labelled plant lignin, 14C-lignocellulose and 14C-DHP-lignin at a rate which was lower than the one observed for the known white rot fungi Heterobasidion annosum and Sporotrichum pulverulentum. Also 14C-(U)-holocellulose was relatively less degradable for the mycorrhizal fungi than for the white rotters. On the other hand, aromatic monomers like 14C-vanillic acid were decomposed to a much higher extent by two species of mycorrhizal fungi compared to the activity observed for Heterobasidion annosum. The results of the experiments reveal that these stains of mycorrhizal fungi are well able to utilize the major components of plant material and thus can contribute to litter decomposition in the forest floor.

Degradation of fluorene, anthracene, phenanthrene, fluoranthene, and pyrene lacks connection to the production of extracellular enzymes by Pleurotus ostreatus and Bjerkandera adusta

The degradation of polycyclic aromatic hydrocarbons (PAH) was studied in liquid cultures of Bjerkandera adusta and Pleurotus ostreatus during 7 weeks of cultivation. During only 3 days of incubation, B. adusta removed 56% and 38% of fluorene and anthracene, while P. ostreatus degraded 43% and 60% of these compounds; other PAH were degraded to a lower extent. Except for anthracene in cultures of P. ostreatus, all PAH were removed uniformly during the cultivation time but fluorene and anthracene were degraded faster than other PAH. Supplementation of liquid cultures with milled wood decreased the concentration of PAH in the solution and diminished the degradation of PAH. The fungi produced valuable activity of manganese-dependent peroxidase; laccase was secreted only by P. ostreatus and was strongly induced by the addition of milled wood. The production of the oxidative enzymes did not correlate directly to the metabolisation of PAH.

Chemo-enzymatic synthesis and characterization of graft copolymers from lignin and acrylic compounds

Chemo-enzymatic initiation of graft copolymerization of acrylic compounds onto different technical lignosulfonates (LS) was compared to a Fenton-like system (ferrous ion, t-BHP). The enzyme tested was a phenoloxidase laccase (EC 1.10.3.2) from the white rot basidomycete Trametes versicolor. Most applied lignins were successfully grafted, resulting in a polymer yield of more than 90%. The effect of initiator concentration and the lignin/monomer ratio on the yield and M(w) of enzymatically grafted polymers were studied. The homopolymer proportion in the enzymatically produced grafts of Ca-LS and acrylic acid was 5 to 6x lower than those initiated by the Fenton-like reagent; no such differences were observed for Na-LS.

Function of laccase in the white-rot fungus Fomes annosus.

Abstract1.Thioglycolic acid, a Cu-chelating agent, totally inhibited extracellular laccase activity without affecting growth and morphology of Fomes annosus.2.In the presence of thioglycolic acid Fomes annosus cleaved high molecular weight lignosulfonate with a molecular weight range of 2×106 to 1000. In the absence of thioglycolic acid the polymerizing activity of laccase prevented the detection of lignosulfonate breakdown products.3.Oxidative polymerization of a lignin monomer, coniferyl alcohol, occurred in the presence but not in the absence of laccase activity.4.Catechol and guaiacol added to the medium at a concentration of 2 mmol, are normally oxidized by fungal laccase and strongly inhibit growth. Presence of thioglycolic acid prevented the oxidation of these phenols and simultaneously permitted normal growth.