Prediction and optimization of the laccase-mediated synthesis of the antimicrobial compound iodine (I2).

An artificial neural network (ANN) and genetic algorithm (GA) were applied to improve the laccase-mediated oxidation of iodide (I(-)) to elemental iodine (I2). Biosynthesis of iodine (I2) was studied with a 5-level-4-factor central composite design (CCD). The generated ANN network was mathematically evaluated by several statistical indices and revealed better results than a classical quadratic response surface (RS) model. Determination of the relative significance of model input parameters, ranking the process parameters in order of importance (pH>laccase>mediator>iodide), was performed by sensitivity analysis. ANN-GA methodology was used to optimize the input space of the neural network model to find optimal settings for the laccase-mediated synthesis of iodine. ANN-GA optimized parameters resulted in a 9.9% increase in the conversion rate.

Agrobacterium-mediated transformation of the white-rot fungus Physisporinus vitreus.

The biotechnologically important white-rot fungus Physisporinus vitreus was co-cultivated with Agrobacterium tumefaciens AGL-1 carrying plasmids with nourseothricin resistance as the selectable marker gene and red fluorescence protein as a visual marker. Mitotically stable transformed isolates were obtained showing red fluorescence protein activity.

Protection of wood from microorganisms by laccase-catalyzed iodination.

In the present work, Norway spruce wood (Picea abies L.) was reacted with a commercial Trametes versicolor laccase in the presence of potassium iodide salt or the phenolic compounds thymol and isoeugenol to impart an antimicrobial property to the wood surface. In order to assess the efficacy of the wood treatment, a leaching of the iodinated and polymerized wood and two biotests including bacteria, a yeast, blue stain fungi, and wood decay fungi were performed. After laccase-catalyzed oxidation of the phenols, the antimicrobial effect was significantly reduced. In contrast, the enzymatic oxidation of iodide (I(-)) to iodine (I(2)) in the presence of wood led to an enhanced resistance of the wood surface against all microorganisms, even after exposure to leaching. The efficiency of the enzymatic wood iodination was comparable to that of a chemical wood preservative, VP 7/260a. The modification of the lignocellulose by the laccase-catalyzed iodination was assessed by the Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) technique. The intensities of the selected lignin-associated bands and carbohydrate reference bands were analyzed, and the results indicated a structural change in the lignin matrix. The results suggest that the laccase-catalyzed iodination of the wood surface presents an efficient and ecofriendly method for wood protection.

Modelling the effect of environmental factors on the hyphal growth of the basidiomycete Physisporinus vitreus.

The present work investigated the effects of environmental factors on the growth of fungal colonies of the white-rot basidiomycetes Physisporinus vitreus using a lattice-free discrete modeling approach called the fungal growth model (FGM), in which hyphae and nutrients are considered as discrete structures. A discrete modeling approach enables the underlying mechanistic rule concerning the basic architecture and dynamics of fungal networks to be studied on the scale of a single colony. By comparing simulations of the FGM with laboratory experiments of fungal colonies growing on malt extract agar we show that the combined effects of water activity, temperature and pH on the radial growth rate of fungal mycelia on the macroscopic scale may be explained by a power law for the costs of hyphal maintenance and expansion on the microscopic scale. Information about the response of the fungal mycelium at the micro- scopic level to environmental conditions is essential for simulating its behavior in complex structure substrates such as wood, where the effect of the fungus on the wood (i.e. the degradation of the cell wall) changes the local environmental condition (e.g. the permeability of the substrate and therefore the water activity in a colonized wood cell lumen). Using a combination of diffusion and moisture processes with the FGM may increase our understanding of the colonization strategy of P. vitreus and help to optimize its growth behavior for biotechnological applications such as bioincising.

Modelling the hyphal growth of the wood-decay fungus Physisporinus vitreus.

The white-rot fungus, Physisporinus vitreus, degrades the membranes of bordered pits in tracheids and consequently increases the permeability of wood, which is a process that can be used by the wood industry to improve the uptake of wood preservatives and environmentally benign wood modification substances to enhance the use and sustainability of native conifer wood species. To understand and apply this process requires an understanding of how a complex system (fungus-wood) interacts under defined conditions. We present a three-dimensional fungal growth model (FGM) of the hyphal growth of P. vitreus in the heartwood of Norway spruce. The model considers hyphae and nutrients as discrete structures and links the microscopic interactions between fungus and wood (e.g. degradation rate and degree of opening of pits) with macroscopic system properties, such penetration depth of the fungus, biomass, and distribution of destroyed pits in early- and latewood. Simulations were compared with experimental data. The growth of P. vitreus is characterized by a stepwise capture of the substrate and the effect of this on wood according to different model parameters is discussed.

Efficient production of Al(OH)3-immobilized laccase with a Heterobasidion annosum strain selected by microplate screening.

AIMS: Wild-type white rot fungi are the most important production organisms for laccase, a promising oxidative biocatalyst with numerous applications. This study aimed at identifying novel highly productive strains, finding optimal cultivation conditions for laccase production and establishing a simple immobilization procedure. METHODS AND RESULTS: By using a newly developed 96-well microplate cultivation method, 23 species of white rot fungi, represented by 29 strains, were directly compared with regard to the amount of secreted laccase. Both, with glucose and spruce saw dust as growth substrate a Heterobasidion annosum strain and a Physisporinus vitreus strain were the most productive (730­2200 U l−1 of secreted laccase). Cultivation conditions for laccase production with H. annosum were optimized in larger-scale liquid cultures. Aeration with a sparger lead to a 3·8-fold increase in laccase activity when compared to nonaerated flask cultures. More than 3000 U l−1 laccase was produced in glucose medium supplemented with yeast extract and the inducer veratryl alcohol. Culture supernatant was incubated with short-range ordered Al(OH)3 particles to directly immobilize and concentrate laccase by adsorption. Active laccase was recovered in 40% yield and the Al(OH)3-adsorbed laccase was suitable for repeated decolourization of indigo carmine. CONCLUSIONS: Microplate cultivation allowed a large-scale comparison of the capacity of different fungal species for laccase production. Laccase secretion of a highly productive H. annosum strain was found to vary strongly with different cultivation conditions. Adsorption to Al(OH)3 proved to be suitable as direct immobilization technique.

Evaluation of the interspecific competitive ability of the bioincising fungus Physisporinus vitreus.

A hierarchical set of assays were used to evaluate the interspecific competitive ability of the bioincising fungus Physiporinus vitreus (response species). The competitiveness and growth mode of P. vitreus against a range of blue stain fungi and Trichoderma species (challenge species) was investigated in dual culture tests (a) and a spatially heterogeneous system of tessellation agar (b) on different media. In addition, the robustness of the bioincising process against biotic influences was determined by controlled interaction tests with different inocula of the challenge species (colony-forming units: 10(2), 10(4), 10(6) ml(-1)) on Norway spruce (Picea abies Karst.) heartwood. The results of the dual culture and tessellation agar tests revealed a significant (P ≤ 0.001) antagonistic potential (lethal effect) of the T. atroviride strain (15603.1), whereas no or only a slight effect of the blue stain fungi on P. vitreus was detectable. The interaction tests on Norway spruce heartwood confirmed that an increase in inoculum size of the challenge fungi correlated with an increased negative effect (Spearman's rho coefficient (ρ) = -0.521). Among the challenge species, T-15603.1 revealed the most negative effect on the homogeneous development of P. vitreus on wood and thus on the outcome of the bioincising process. To improve the uniformity of colonization and to reduce the rate of contamination, the incubation of wood in a sealed system (bioreactor) after sterilization is suggested.

Statistical approach to determine the effect of combined environmental parameters on conidial development of Trichoderma atroviride (T-15603.1).

Trichoderma atroviride (T-15603.1) is a promising fungal agent for biological control of wood decay fungi in urban tree wounds. The aim of this work was to determine the combined effects of water activity (a(w), 0.998-0.892), temperature (10-30 °C) and pH (3-7) on the development of conidia on low-nutrient agar (LNA). Lag phase prior to germination (h), germination rates (µ(m)) and germ-tube elongation were obtained at each set of conditions. The experimental data were used to fit a response surface model for predicting the germination rates of T-15603.1 and to analyze the effect of the abiotic parameters tested. The polynomial response surface model was mathematically evaluated using graphical plots and several statistical indices (RMSE, %SEP, A(f), B(f), pRE). Data analyses showed a highly significant effect on conidial development of a(w) and temperature (P < 0.001), whereas no significant effect of pH (P ≥ 0.05) was observed. The germination rate dropped and the lag prior to germination increased as the temperature and a(w) decreased, but T-15603.1 appeared to be more sensitive to a(w) reduction than to temperature. The minimum a(w) level for germination was 0.910 at 15-25 °C, and maximum germination rates were obtained at a(w) = 0.998, 25-30 °C and pH 5. The response surface model was useful and widely accurate (R² = 0.983) for predicting the germination rate of T-15603.1 and complemented the experimental results. These findings contribute to better understanding how combined environmental factors affect the environmental parameter tolerance levels of T-15603.1 and to the development of an adequate delivery system for optimized application of T-15603.

Determination of optimal growth parameters for the bioincising fungus Physisporinus vitreus by means of response surface methodology.

AIM: To evaluate the influence of water activity (a(w)), temperature and pH on the radial growth and lag phase of Physisporinus vitreus (E-642), a basidiomycete was used in the biotechnological process of bioincising. METHODS AND RESULTS: Radial growth was monitored for 20 days on malt extract agar medium. Five levels of a(w) (0.998, 0.982, 0.955, 0.928, 0.892) were combined with three incubation temperatures (10, 15, 20 degrees C) and three pH values (4, 5, 6). Data analyses showed a highly significant effect of a(w) and temperature (P < 0.0001) and a significant effect of pH (P < 0.05). The radial growth rate and lag phase of P. vitreus were very sensitive to a(w) reduction. Although P. vitreus was able to grow at all the selected temperatures and pH values, the lag phase increased with decreasing a(w) and growth became inhibited at a(w) = 0.955. Optimal conditions for growth of P. vitreus were a(w) = 0.998, 20 degrees C and pH 5. The response surface model provided reliable estimates of these growth parameters and confirmed a greater dependence on a(w) than on temperature or pH under in vitro conditions. CONCLUSIONS: Low levels of a(w) can prevent growth of P. vitreus, so wood moisture content should be adjusted accordingly. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of these results should contribute towards the optimization and efficiency of bioincising.

Large-leaved lime (Tilia platyphyllos) has a low ability to compartmentalize decay fungi via reaction zone formation.

• The growth of three basidiomycetes (Inonotus hispidus, Fomitopsis pinicola and Ganoderma adspersum) and one ascomycete (Ustulina deusta) was studied within the reaction zone (RZ) of large-leaved lime (Tilia platyphyllos) and host responses were compared with those formerly described in beech (Fagus sylvatica). The main objective of the study was to determine whether antimicrobial differences in host response and fungal growth within reaction zones exist. • The cell lumina in the RZ contained only sparse occlusions, which were easily degraded and did not prevent the passage of hyphae between adjacent cells via pits when RZs within excised wood blocks were challenged in vitro with the decay fungi. • In a comparison of extracts from the RZ of large-leaved lime and the stronger RZ of beech, differences were found in the concentrations and composition of induced antimicrobial compounds. The RZ of large-leaved lime was relatively weak, compared with the static RZ of beech, and migrated into the functional sapwood at an active microbial invasion front as a result of a dynamic interactive process. • These observations support previous interpretations that certain defence mechanisms in the xylem of trees are a means of maintaining hydraulic integrity, rather than physical and chemical barriers, which restrict or inhibit fungal growth.