In this paper we report a study of laccase immobilisation on different kinds of carrier particles. The immobilisation of enzyme on the particle surface with respect to the immobilisation efficiency and the properties of the immobilised enzymes is discussed. The immobilisation of laccase on polystyrene particles bearing reactive beta-diketone groups is characterised by high efficiency, but grafting of the enzyme increases the stability of the colloidal system, which makes the separation/purification procedure difficult. Additionally, the extreme colloidal stability of the immobilisates hinders the application of such particles with immobilised enzymes in some applications where the recycling of the enzyme should be performed. It has been found that hybrid PS-AAEM particles equipped with maghemite show similar immobilisation efficiency to that of their analogues without maghemite and can additionally be manipulated in magnetic fields. The activity of the immobilised laccase is much higher in the pH region 5-7 and the temperature range 50-70 degrees C as compared with that of the free enzyme. Immobilised enzymes also exhibit much better storage stability.

Trametes hirsuta, a filamentous basidiomycete, was successfully cultivated in solid‐state fermentation (SSF) on a mixture of pine wood chips and orange peel in a novel bioreactor that allows mixing of the inoculated solid material during the fermentation. Copper sulfate or xylidine (2,5‐dimethylaniline) were added to enhance the production of enzymes, especially laccases. For comparison, Trametes hirsuta was also cultivated in submerged conditions. The effect of additives in SSF was low, whereas the choice of the solid material and the rotation of the reactor vessel showed a significant influence on the enzyme production. The space‐time yields for the fermentations were calculated and showed that SSF on low‐cost substrates can effectively produce extracellular enzymes at a sufficient rate by a cultivation method that requires only low technology equipment.

Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX V R Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by

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