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Background: The Antarctic fungus Lecanicillium muscarium CCFEE 5003 is one of the most powerful chitinolytic organisms. It can produce high level of chitinolytic enzymes in a wide range of temperatures (5-30°C). Chitinolytic enzymes have lot of applications but their industrial production is still rather limited and no cold-active enzymes are produced. In view of massive production of L. muscarium chitinolytic enzymes, its cultivation in bioreactors is mandatory. Microbial cultivation and/or their metabolite production in bioreactors are sometime not possible and must be verified and optimized for possible exploitation. Agitation and aeration are the most important parameters in order to allow process up-scaling to the industrial level., Results: In this study, submerged cultures of L. muscarium CCFEE 5003 were carried out in a 2-L bench-top CSTR bioreactor in order to optimise the production of chitinolytic enzymes. The effect of stirrer speed (range 200-500 rpm) and aeration rate (range 0.5-1.5 vvm) combination was studied, by Response Surface Methodology (RSM), in a medium containing 1.0% yeast nitrogen base and 1% colloidal chitin. Optimization was carried out, within a "quadratic D-optimal" model, using quantitative and quantitative-multilevel factors for aeration and agitation, respectively. The model showed very good correlation parameters (R2, 0.931; Q2, 0.869) and the maximum of activity (373.0 U/L) was predicted at ca. 327 rpm and 1.1 vvm. However, the experimental data showed that highest activity (383.7 ± 7.8 U/L) was recorded at 1 vvm and 300 rpm. Evident shear effect caused by stirrer speed and, partially, by high aeration rates were observed. Under optimized conditions in bioreactor the fungus was able to produce a higher number of chitinolytic enzymes than those released in shaken flasks. In addition, production was 23% higher., Conclusions: This work demonstrated the attitude of L. muscarium CCFEE 5003 to grow in bench-top bioreactor; outlined the strong influence of aeration and agitation on its growth and enzyme production and identified the optimal conditions for possible production at the industrial level.

Extracellular laccase from Panus tigrinus CBS 577.79 was produced in a bubble-column reactor using glucose-containing medium supplemented with 2,5-xylidine under conditions of nitrogen sufficiency. The main laccase isoenzyme was purified to apparent homogeneity by ultra-filtration, anion-exchange chromatography and gel filtration that led to a purified enzyme with a specific activity of 317 IU (mg protein)-1 and a final yield of 66%. Laccase was found to be a monomeric protein with a molecular mass of 69.1 kDa, pI of 3.15 and 6.9% N-glycosylation of the high mannose type. Temperature and pH optima were 55 degrees C and 3.75 (2,6-dimethoxyphenol as substrate). At 50 and 60 degrees C, the enzyme half-lives were 281 and 25 min, respectively. The P. tigrinus laccase oxidized a wide range of both naturally occurring and synthetic aromatic compounds: the highest catalytic efficiencies were for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid and 2,6-dimethoxyphenol (5.99x10(6) and 3.07x10(6) M-1 s-1, respectively). Catalytic rate constants for typical N-OH redox mediators, such as 1-hydroxybenzotriazole (2.6 s-1), violuric acid (8.4 s-1) and 2,2,6,6-tetramethylpiperidin-N-oxide radical (7.8 s-1), were found to be higher than those reported for other high redox potential fungal laccases.

The present study investigated the valorization of olive-mill wastewater (OMW) by its use as a possible growth medium for the microbial production of extra-cellular lipase. To this end, strains of Geotrichum candidum (NRRL Y-552 and Y-553), Rhizopus arrhizus (NRRL 2286 and ISRIM 383), Rhizopus oryzae (NRRL 6431), Aspergillus oryzae (NRRL 1988 and 495), Aspergillus niger (NRRL 334), Candida cylindracea (NRRL Y-17506) and Penicillium citrinum (NRRL 1841 and 3754, ISRIM 118) were screened. All strains were able to grow on the undiluted OMW, producing extra-cellular lipase activity. C. cylindracea NRRL Y-17506 showed the highest lipase activity on all the typologies of OMW used. Its lipase production on OMW was markedly affected by the type of nitrogen source and was induced by the addition of olive oil. The highest activity (9.23 IU ml(-1)) of the yeast was obtained on OMW supplemented with NH(4)Cl (2.4 g l(-1)) and olive oil (3.0 g l(-1)).

This report deals with the fundamental quantum physics behind two-photon excitation also providing a link to the experimental consequences exploited in microscopy. The optical sectioning effect is demonstrated as well as the distribution of excitation and of fluorescence emission., (Copyright 2003 Wiley-Liss, Inc.)

The possible use of olive-mill wastewater (OMW) as a growth medium for the production of extracellular laccase and manganese peroxidase (MnP) from the white-rot fungus Panus tigrinus (P. tigrinus) CBS 577.79 was studied using a properly formulated OMW-based medium (2-fold diluted OMW supplemented with 0.5% sucrose and 0.1% yeast extract) either in a stirred-tank or an air-lift reactor. Solid-state fermentation (SSF) was also performed in a rotary drum reactor using maize stalks moistened with the OMW-based medium. Highest levels of laccase and manganese peroxidase activity were obtained in the stirred-tank reactor (4600+/-98 U l(-1) on day 13) and in the air-lift reactor (410+/-22 on day 7), respectively. Based on total enzyme activities, SSF appears to be more suitable than LSF but the latter exhibits better volumetric productivities.

The effects of the refractive-index mismatch in confocal laser scanning microscopy were extensively studied. The axial aberration induced in the case of fluorescent microspheres was measured. The data were used to take into account the mismatch-induced aberrations and to consider object-size influence. Then we focused on the effect of refractive-index mismatch on the effective system's point-spread function under different mismatch conditions and on depth of focusing. We experimentally verified that the peak of the point-spread function intensity profile decreases and the point-spread function itself progressively broadens as a function of the combined effect of the refractive-index mismatch and of the penetration depth, leading to a worsening of the system's overall performances. We also performed these same measurements by embedding subresolution beads in an oocyte's cytoplasm, which can be considered a turbid medium. We found evidence consistent with the previously developed theoretical model; in particular we found a strong dependence of the intensity peak on the focusing depth.