Improvement of bilirubin oxidase productivity of Myrothecium verrucaria and studies on the enzyme overproduced by the mutant strain in the solid-state fermentation.

Bilirubin oxidase has applications in the health and environmental sectors. Hence, several attempts have been made to increase enzyme yields. However, improvements were not very high. We report here the development of a mutant strain of Myrothecium verrucaria by using UV-rays, which produced 28.8 times more enzyme compared with the parent and was higher than the yields reported in earlier submerged cultures. The mutant strain produced 35.6 times more enzyme than the parent in solid-state fermentation, which is better than that previously reported for a solid-state fermentation process. The specific activity of the enzyme produced by the mutant was higher than that of the parental enzyme. Bilirubin oxidase from both strains showed an optimum activity at pH 7 and 40°C. However, the time required to inactivate half of the initial enzyme activity at 60°C was much higher in the case of the enzyme obtained from the mutant compared with the parental enzyme. The improved thermostability of the enzyme from the mutant strain could be due to the point mutations induced during the UV irradiation, since there was no change in the mass of the enzyme compared with the parental enzyme. The bilirubin oxidase of the mutant strain degraded the bilirubin faster than the enzyme obtained from the parent under similar conditions. Faster activity of the enzyme obtained from the mutant strain could be due to its lower Km (79.4 µM) compared with that of the parental enzyme (184 µM). Hence, the mutant enzyme showed a better functionality and thermostability, which will be beneficial for industrial applications.

Fungal Spores Viability on the International Space Station.

In this study we investigated the security of a spaceflight experiment from two points of view: spreading of dried fungal spores placed on the different wafers and their viability during short and long term missions on the International Space Station (ISS). Microscopic characteristics of spores from dried spores samples were investigated, as well as the morphology of the colonies obtained from spores that survived during mission. The selected fungal species were: Aspergillus niger, Cladosporium herbarum, Ulocladium chartarum, and Basipetospora halophila. They have been chosen mainly based on their involvement in the biodeterioration of different substrate in the ISS as well as their presence as possible contaminants of the ISS. From biological point of view, three of the selected species are black fungi, with high melanin content and therefore highly resistant to space radiation. The visual inspection and analysis of the images taken before and after the short and the long term experiments have shown that all biocontainers were returned to Earth without damages. Microscope images of the lids of the culture plates revealed that the spores of all species were actually not detached from the surface of the wafers and did not contaminate the lids. From the adhesion point of view all types of wafers can be used in space experiments, with a special comment on the viability in the particular case of iron wafers when used for spores that belong to B. halophila (halophilic strain). This is encouraging in performing experiments with fungi without risking contamination. The spore viability was lower in the experiment for long time to ISS conditions than that of the short experiment. From the observations, it is suggested that the environment of the enclosed biocontainer, as well as the species'specific behaviour have an important effect, reducing the viability in time. Even the spores were not detached from the surface of the wafers, it was observed that spores used in the long term experiment lost the outer layer of their coat without affecting the viability since they were still protected by the middle and the inner layer of the coating. This research highlights a new protocol to perform spaceflight experiments inside the ISS with fungal spores in microgravity conditions, under the additional effect of possible cosmic radiation. According to this protocol the results are expressed in terms of viability, microscopic and morphological changes.

[Influence of different types of ionizing radiation on fatty acid profiles of cell lipids in microscopic fungi with radioadaptive properties].

The fatty acid profiles of strains of dark-pigmented (melanin-containing) species Cladosporium cladosporioides and Hormoconis resinae, and light-pigmented Aspergillus versicolor and Paecilomyces lilacinus, showing radioadaptive properties in comparison with control strains of the same species, which did not have such properties and the influence on them of two (121Sn; 137Cs) types of ionizing radiation have been studied. It was established that the most important fatty acids were hexadecanoic acid (C16:0), octadecanoic acid (C18:0), octadecenoic acid (C18:1) and octadecadienoic acid (C18:2). The strains, showing radioadaptive properties in comparison with control differed in the relative concentrations of saturated and unsaturated fatty acids and some minor components. The two types of radiation had different influence on the fatty acid profiles of the investigated strains. At dark-pigmented species C. cladosportoides, H. resinae the fatty acid unsaturation rate was higher, at strains showing radioadaptive properties (0.98) as against control ones (0.73; 0.9), and at light-pigmented species A. versicolor and P lilacinus--it was lower at strains with radioadaptive properties (1.00; 0.83) as against control ones (1.08; 0.92). The paper is presented in Ukrainian.

[Influence of light of different spectral composition on growth characteristics of microscopic fungi].

The features of radial growth of three microscopic fungi species were investigated at illumination by yellow, blue, green and red light. The studied species of fungi differed by isolation site (some of them were isolated from the places of high radioactive pollution, others--from the places with background level of radioactivity) and pigmentation degree (melanin-containing and light-colored). The parameters of radial growth, which are the integral indexes of the fungal organism physiology state were investigated: radial growth rate, branching degree and summarizing index--the intensity of substrate consumption. It was shown that the melanin-containing fungi Cladosporium cladosporioides growth was twice more rapid in response to blue and yellow light. Whereas such light depressed growth of non-pigmented strain of the same species as compared to control. A light response of other pigment-containing species Hormoconis resinae was far less expressed, and the light-colored species Paecilomyces lilacinus had no response to any offered conditions of illumination.

Inactivation of food spoilage fungi by ultra violet (UVC) irradiation.

The effect of ultraviolet irradiation (254 nm, UVC) on Aspergillus flavus, Aspergillus niger, Penicillium corylophilum and Eurotium rubrum was investigated using three different exposure techniques. Survival was determined for spores suspended in liquid medium after 1, 2 and 3 min UVC exposure at 4644 J/m(2)/min. The same UVC dose was applied to spores on the surface of agar plates for 5, 10, 15, 30, 60 and 120 s. Spores of A. niger were dried onto a membrane filter, then exposed to UVC treatment. In the liquid medium, treatments from 1-3 min significantly (P<0.001) reduce the number of viable spores. On the surface of agar plates, after a 15 s exposure, a 80-99% reduction of viable spores was observed for all species except A. niger, for which the reduction was only 62%. For spores dried onto filter membranes, a 3.5 log(10) reduction was achieved for A. niger after 180 s exposure. These observations suggest that UVC irradiation can effectively inactivate spores of A. flavus, P. corylophilum, E. rubrum and A. niger but the efficacy of UVC radiation against fungal spores varies significantly according to methods of exposure to the irradiation, and among genera.

[Development of radioadaptive properties for microscopic fungi, long time located on terrains with a heightened background radiation after emergency on Chernobyl NPP].

In 7 species of micromycetes which were isolated from radioactive contaminated areas of Chernobyl NPP zone under exposure of two artificial sources: y-low energy (121Sn) and radiation mix type gamma + beta (137Cs). Two new earlier unknown radioadaptive properties for fungi--radiotropism and radiostimulation were established. The strains, which were isolated from clean areas, did not have such features. All investigated strains, which have shown positive radiotropism at the same time, have shown stimulation of conidia germination and of length of the emergent hyphum under exposure to one of sources of radiation. It was shown that micro fungi having radioadaptive properties, the adaptive response to high (100-1000 Gy) ionizing radiation doses was found that is evidence of that these strains have high radio resistance level. Nevertheless fungal strains have shown varied response to presence of ionizing radiation depending on its type and absorbed dose.

Effect of germicidal UVC light on fungi isolated from grapes and raisins.

AIMS: To examine how UVC affects the different genera of fungi commonly isolated from grapes, with the aim of understanding changes in mycobiota during grape ripening and possible applications for preventing grape decay during storage. METHODS AND RESULTS: Spores of Aspergillus carbonarius, Aspergillus niger, Cladosporium herbarum, Penicillium janthinellum and Alternaria alternata (between 100-250 spores/plate agar) were UVC irradiated for 0 (control), 10, 20, 30, 60, 300 and 600 s. Plates were incubated at 25 degrees C and colonies were counted daily up to 7 days. Alternaria alternata and Aspergillus carbonarius were the most resistant fungi. Conidial germination in these species was reduced by approx. 25% after 10 s of exposure, compared with greater than 70% reduction for the remaining species tested. Penicillium janthinellum spores were the most susceptible at this wavelength. UVC exposures of 300 s prevented growth of all isolates studied, except for Alternaria alternata. CONCLUSIONS: UVC irradiation plays a major role in selecting for particular fungi that dominate the mycobiota of drying grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The UVC irradiation of harvested grapes could prevent germination of contaminant fungi during storage or further dehydration.

Viability of fungal and actinomycetal spores after microwave radiation of building materials.

The effects of microwave radiation on viability of fungal and actinomycetal spores growing on agar (medium optimal for growth) as well as on wooden panel and drywall (common building construction/finishing materials) were studied. All materials were incubated at high (97-99%) and low (32-33%) relative humidity to mimic "wet" and "dry" environmental conditions. Two microwave power densities (10 and 60 mW/cm2) and three times of exposure (5, 30, and 60 min) were tested to find the most effective parameters of radiation which could be applied to non-invasive reduction or cleaning of building materials from microbial contaminants. Additionally, a control of the surface temperature during the experiments allowed differentiation between thermal and microwave effect of such radiation. The results showed that the viability of studied microorganisms differed depending on their strains, growth conditions, power density of microwave radiation, time of exposure, and varied according to the applied combination of the two latter elements. The effect of radiation resulting in a decrease of spore viability on "wet" wooden panel and drywall was generally observed at 60 min exposure. Shorter exposure times decreased the viability of fungal spores only, while in actinomycetes colonizing the studied building materials, such radiation caused an opposite (supporting growth) effect.

[The effect of hydrogen peroxide on the growth of microscopic mycelial fungi isolated from habitats with different levels of radioactive contamination].

The effect of hydrogen peroxide ( 10(-9)-10(-1) M) on the mycelial growth of the fungi Alternaria alternata, Cladosporium cladosporioides, Mucor hiemalis, and Paecilomyces lilacinus has been studied. The growth of fungi isolated from habitats with a background level of radioactive contamination was stopped by H2O2 concentrations equal to 10(-3) and 10(-2) M, whereas the growth of fungi that were isolated from habitats with high levels of radioactive contamination was only arrested by 10(-1) M H2O2. The response of the different fungi to hydrogen peroxide was of three types: (1) a constant growth rate of fungal hyphae at H2O2 concentrations between 10(-9) and 10(-4) M and a decrease in this rate at 10(-3) M H2O2, (2) a gradual decrease in the growth rate as the H2O2 concentration was increased, and (3) an increase in the growth rate as the H2O2 concentration was increased from 10(-7) to 10(2)-5 M. The melanin-containing species A. alternata and C. cladosporioides exhibited all three types of growth response to hydrogen peroxide, whereas the light-pigmented species M. hiemalis and P. lilacinus showed only the first type of growth response. A concentration of hydrogen peroxide equal to 10(-1) M was found to be lethal to all of the fungi studied. The most resistant to hydrogen peroxide was found to be the strain A. alternata 56, isolated from the exclusion zone of the Chernobyl Nuclear Power Plant.

Variations in UV-B tolerance and germination speed of Metarhizium anisopliae conidia produced on insects and artificial substrates.

Solar ultraviolet radiation (UV-A and UV-B) is a major factor in failure of programs using the insect pathogenic fungus Metarhizium anisopliae as a biological control agent. Studies were conducted to determine if growth conditions, viz. artificial (agar media or rice grain) or natural (infected insects) substrates for conidial production affect two traits that directly influence performance of conidia after field application: tolerance to UV-B radiation and conidial germination speed. Conidia of two isolates (ARSEF 23 and ARSEF 2575) of M. anisopliae var. anisopliae produced on potato dextrose agar plus yeast extract (PDAY) or on fungus-killed larvae of two insect species, Galleria mellonella and Zophobas morio, were inactivated by exposure to UV-B radiation. Conidia of both isolates when produced on insect cadavers were significantly more sensitive to UV-B radiation than conidia produced on PDAY. Also, conidia from insect cadavers germinated slower than those from PDAY cultures. A comparison of conidia from artificial substrates showed that conidia produced on Czapek's and Emerson's YpSs agar media or rice grains had higher tolerance to UV-B radiation and germinated faster than conidia raised on PDA and PDAY. Accordingly, the growth substrate and nutritional environment in which conidia are produced influences M. anisopliae conidial UV-B tolerance and speed of germination; and manipulation of these variables could be used to obtain conidia with increased tolerance to UV-B radiation and shorter germination times.

Ionizing radiation attracts soil fungi.

During the last 15 years, about 2000 strains of 200 species of 98 genera of fungi have been isolated from around the Chernobyl Atomic Energy Station. Many of these microfungi are capable of growing into and decomposing 'hot particles'; carbon based radioactive graphite from the reactor and there are suggestions that some fungi actively direct their growth toward sources of radioactivity, possibly attracted to the carbon skeleton of these structures. In our experiments, we eliminated the confounding effects of carbon as a fungal resource, by developing experimental protocols that expose fungal spores and their germinating hyphae to directional sources of ionizing radiation allowing us to measure fungal response to ionizing radiation per se. We show that both beta and gamma radiation promote directional growth of hyphae towards the source of ionizing radiation.

[Growth characteristics of microscopic fungi capable of growing actively under the conditions of the 4th unit of the Chernobyl Nuclear Power Plant]. / Rostovye kharakteristiki mikroskopicheskikh gribov, aktivno rastushchikh v usloviiakh 4-go bloka ChAES.

Growth parameters of fungi species which, from the preliminary data, were brought to those growing actively under the conditions of the Chernobyl NPP 4th unit have been studied in the presented work. Radial growth velocity, hyphal growth unit and intensity of the substrate colonization were such growth indices. All the investigations were carried out on two media--the wort agar, being of full value for fungi, and deficient agar limited as to major sources of nutrition. Two main types of population of the 4th unit rooms with the studied fungi have been found. The first method was characteristic of fungi capable to form the great number of small conidia and, respectively, fungal colonies, and to grow with low growth velocity, which was considerably higher at the initial stages than during the following ones. The second method was characterized by sufficiently high averaged growth velocity of mycelium; by its slowing down at the initial stages and its considerable increase in the process of further ontogenesis. Weak conidium formation was characteristic of this type; sometimes sporulation was lost at all. It has been shown that fungal species ascribed to those growing actively in the rooms of the unit 4 of the Chernobyl NPP are such ones, and they play the major part in the population and biodestruction of the building structures of the 4th unit.

[Survivability of some black yeast-like fungal species under UV-irradiation]. / Vyzhivaemost' nekotorykh vidov chernykh drozhzhepodobnykh gribov pri vozdeistvii UF-luchei.

The survival of 4 species (14 strains) of black-pigmented yeast-like fungi, isolated from radioactive polluted ecotopes, after UV-irradiation has been studied. Their survival was different in the limits of 3 orders from 0.7 to 0.0001% and was described by the exponential composite exponential and composite sigmoid types of dose curves. Essential heterogeneity for the level and character of survival after UV-irradiation of conidia Aureobasidium pullulans, Hormonema dematioides, Exophiala cf. jeanselmei and Exophiala sp. has been presented.

[Comparative analysis of radial linear growth velocity of micromycetes isolated from different ecotopes]. / Sravnitel'nyi analiz skorosti radial'nogo rosta mikromitsetov, vydelennykh iz razlichnykh ékotopov.

The work aim was the discovering of peculiarities of microscopic fungi responding to radioactive pollution by growth velocity change. The linear growth velocity of a broad range of micromycetes was analyzed. They were chosen in such a way that every species, as far as possible, was presented by the strains isolated from radioactive polluted and clean regions. Most of the studied species were characterized by the absence of reliable growth velocity differences of strains of the same species growing on rich and starving media. Only two species--Aureobasidium pullulans and Penicillium spinulosum were the exception. Their growth velocities on malt-agar were much more then those on starving medium (1.7-3.6. times). Species Hormonema dematioides did not have reliable differences of growth velocity on optimal and starving media, but A. pullulans had this difference (2.3-3.6 times). It is supposed to use this observation to simplify identification of these species. Only strains within species Cladosporium sphaerospermum and especially P. funiculosum isolated from radioactive polluted ecotopes had growth velocity which was less then that of the strains from clean regions. The delay of growth velocity in such conditions can testify to adaptive change of metabolic processes of these strains. The strains which were characterized by delay of growth velocity existed at the substrates with high radioactive pollution for a long time.

Both solar UVA and UVB radiation impair conidial culturability and delay germination in the entomopathogenic fungus Metarhizium anisopliae.

The entomopathogenic hyphomycete Metarhizium anisopliae has been used in programs of agricultural pest and disease vector control in several countries. Exposure to simulated solar radiation for a few hours can completely inactivate the conidia of the fungus. In the present study we determined the effect of exposures to full-spectrum sunlight and to solar ultraviolet A radiation at 320-400 nm (UVA) on the conidial culturability and germination of three M. anisopliae strains. The exposures were performed in July and August 2000 in Logan, UT. The strains showed wide variation in tolerance when exposed to full-spectrum sunlight as well as to UVA sunlight. Four-hour exposures to full-spectrum sunlight reduced the relative culturability by approximately 30% for strain ARSEF 324 and by 100% for strains ARSEF 23 and 2575. The relative UV sensitivity of the two more sensitive strains was different under solar UV from that under ultraviolet B radiation at 280-320 nm (UVB) in the laboratory. Four-hour exposures to solar UVA reduced the relative culturability by 10% for strain ARSEF 324, 40% for strain ARSEF 23 and 60% for strain ARSEF 2575. Exposures to both full-spectrum sunlight and UVA sunlight delayed the germination of the surviving conidia of all three strains. These results, in addition to confirming the deleterious effects of UVB, clearly demonstrate the negative effects of UVA sunlight on the survival and germination of M. anisopliae conidia under natural conditions. The negative effects of UVA in sunlight also emphasize that the biological spectral weighting functions for this fungus must not neglect the UVA wavelengths.

Effects of UVB irradiance on conidia and germinants of the entomopathogenic Hyphomycete Metarhizium anisopliae: a study of reciprocity and recovery.

We tested the effects of irradiances of 920 and 1200 mW m-2 (weighted irradiance) on the conidia and germinants of the entomopathogenic Hyphomycete Metarhizium anisopliae. The conidia were exposed to the two irradiances for 1, 2, 4, 6, 7 or 8 h. Increased exposure decreased relative percent culturability. The inactivation provoked by the irradiance of 1200 mW m-2 was higher than for the 920 mW m-2, with a reduction in the 50% lethal time (LT50) from 6 h 40 min to 4 h 26 min. Reciprocity was not observed when conidia in water suspension and germinants in different stages of the germinative process were exposed to a 17.3 kJ m-2 total dose at both irradiance levels. Although nonreciprocity was observed in all situations, its magnitude varied as a function of metabolic state and/or cell-cycle phase in which the conidia were at the exposure time. The least difference between the effects of the two irradiance levels was observed when nongerminating conidia in suspension were exposed, and the greatest was observed when conidia were exposed during an advanced germination phase. Doses of 6.6 and 17.3 kJ m-2 supplied through the two irradiance levels delayed the germination of the surviving conidia. At both doses, delay was greater during exposure to the higher irradiance. Nonreciprocity was higher for the 17.3 kJ m-2 dose. Nonreciprocity magnitude, in addition to depending on the conidial physiological state, also depended on dose. The results demonstrate the importance of evaluating the impact of the increase in irradiance during the different stages of the fungal life cycle, especially during the stages which are more sensitive to UV, and not simply in dormant conidia.

Variability in response to UV-B among species and strains of Metarhizium isolated from sites at latitudes from 61 degrees N to 54 degrees S.

The effects of irradiances of 920 and 1200 mW m(-2) (biologically effective weighted irradiance) were examined in 2 Metarhizium album strains, 26 M. anisopliae strains, 1 M. flavoviride strain, and 1 M. taii strain isolated from sites located at latitudes from 61 degrees N to 54 degrees S. Conidia were exposed to UV-B from 1 to 6 h and subsequently examined for relative percentage culturability. Total dosage received at the end of the exposure periods ranged from 3.3 to 19.9 kJ m(-2) for the lower irradiance and from 4.3 to 25.9 kJ m(-2) for the higher irradiance. Both the irradiance values and the doses are environmentally realistic and can be observed even in temperate regions. The relationships between latitude of origin and UV-B tolerance were compared for the two levels of irradiance for the data from 1 and 2 h exposure. Exposure to both irradiances drastically reduced the relative percentage culturability of all strains. Tolerance to UV-B varied widely among strains and high variation was observed for both irradiances after all periods of exposure. After 1 h of exposure, a difference between the two irradiance levels was detectable, and this difference was magnified at longer irradiations. A significant quadratic relationship of decreasing UV-B tolerance with increasing latitude was observed after exposure of 1 and 2 h. The shape of the relationship did not differ for the two levels of irradiance. Also, we studied the effect of 1200 mW m(-2) irradiance on conidial germination time in 1 M. album strain, 7 M. anisopliae strains, and 1 M. taii strain. Exposure to UV-B delayed the germination of surviving conidia of all strains. In general, the delay in germination was directly proportional to the dose.

[Peculiarities of linear growth of the melanin-containing fungi Cladosporium sphaerospermum Penz. and Alternaria alternata (Fr.) Keissler]. / Osobennosti lineinogo rosta melaninsoderzhashchikh gribov Cladosporium sphaerospermum Penz. i Alternaria alternata (Fr.) Keissler.

Results on determination of radial (linear) growth rate of melanin-containing fungi Cladosporium sphaerospermum Penz. and Alternaria alternata (Fr.) Keissler concerning place of their isolation and ecologic peculiarities are presented in the paper. Essential differences, as to all studied indices of the strains of C. sphaerospermum strains, isolated from premices of the ChNPP 4th unit from the control strains of this species have been established. Only strain 34 of C. sphaerospermum, isolated from the cable driving region of the "Shelter" object, which did not differ from the control strains as to all the studied indices, made the exception. The radial growth rate was 2-4 times as low and variability amplitude 10-30 times as high in strains 60 and 5-1 of C. sphaerospermum, (which suffered high radiation load), as in the control strains of this species. The radial growth rate of C. sphaerospermum studied strains was an order lower as a whole than that of A. alternata strains. All the above said evidence for implementation of K-type vital strategy by C. sphaerospermum species and in this connection one can suppose its active growth on the surface of walls and ferroconcrete structures of the ChNPP 4th unit. Absence of differences, as to the studied radial growth indices, between A. alternata strains isolated from the ChNPP 4th unit premices and control strains of the species can evidence for the existence of A. alternata species under the conditions of high radioactivity in the surviving state.

Two-photon autofluorescence microscopy and spectroscopy of Antarctic fungus: new approach for studying effects of UV-B irradiation.

We combined two-photon fluorescence microscopy and spectroscopy to provide functional images of UV-B (280-315 nm) induced stress on an Antarctic fungus. Two-photon excitation microscopy was used to characterize the distribution of autofluorescence inside the spore and the hyphae of the fungus. The imaging analysis clearly shows that the autofluorescence response of spores is higher than that of hyphae. The imaging analysis at different depths shows that, strikingly enough, the spore autofluorescence originates from the cell wall and membrane fluorophores. The spectroscopic results show moreover that the fluorescence spectra of spores are redshifted upon UV-B irradiation. Tentative identification of the chromophores involved in the autofluorescence response and their biological relevance are also discussed on the basis of a previous steady-state fluorescence spectroscopic study performed on both whole spore suspension and organic-soluble extracts.