Mutagenesis and Adaptation of the Psychrotrophic Fungus Chrysosporium pannorum A-1 as a Method for Improving ß-pinene Bioconversion.

Mutagenesis and adaptation of the psychrotrophic fungus Chrysosporium pannorum A-1 to the toxic substrate ß-pinene were used to obtain a biocatalyst with increased resistance to this terpene and improved bioconversion properties. Mutants of the parental strain were induced with UV light and N-methyl-N'-nitro-N-nitrosoguanidine. Mutants resistant to ß-pinene were isolated using agar plates with a linear gradient of substrate concentrations. Active mutants were selected based on their general metabolic activity (GMA) expressed as oxygen consumption rate. Compared to the parental strain, the most active mutant showed an enhanced biotransformation ability to convert ß-pinene to trans-pinocarveol (315 mg per g of dry mycelium), a 4.3-fold greater biocatalytic activity, and a higher resistance to H2O2-induced oxidative stress. Biotransformation using adapted mutants yielded twice as much trans-pinocarveol as the reaction catalyzed by non-adapted mutants. The results indicate that mutagenesis and adaptation of C. pannorum A-1 is an effective method of enhancing ß-bioconversion of terpenes.

Adaptation of Rhizobium leguminosarum sv. trifolii strains to low temperature stress in both free-living stage and during symbiosis with clover.

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. This process occurs within special new structures, called nodules, formed mainly on legume roots. Soil bacteria, commonly known as rhizobia, fix atmospheric dinitrogen, converting it into a form that can be assimilated by plants. Various environmental factors, including a low temperature, have an impact on the symbiotic efficiency. Nevertheless, the effect of temperature on the phenotypic and symbiotic traits of rhizobia has not been determined in detail to date. Therefore, in this study, the influence of temperature on different cell surface and symbiotic properties of rhizobia was estimated. In total, 31 Rhizobium leguminosarum sv. trifolii strains isolated from root nodules of red clover plants growing in the subpolar and temperate climate regions, which essentially differ in year and day temperature profiles, were chosen for this analysis. Our results showed that temperature has a significant effect on several surface properties of rhizobial cells, such as hydrophobicity, aggregation, and motility. Low temperature also stimulated EPS synthesis and biofilm formation in R. leguminosarum sv. trifolii. This extracellular polysaccharide is known to play an important protective role against different environmental stresses. The strains produced large amounts of EPS under tested temperature conditions that facilitated adherence of rhizobial cells to different surfaces. The high adaptability of these strains to cold stress was also confirmed during symbiosis. Irrespective of their climatic origin, the strains proved to be highly effective in attachment to legume roots and were efficient microsymbionts of clover plants. However, some diversity in the response to low temperature stress was found among the strains. Among them, M16 and R137 proved to be highly competitive and efficient in nodule occupancy and biomass production; thus, they can be potential yield-enhancing inoculants of legumes.

Symbiotic efficiency of Rhizobium leguminosarum sv. trifolii strains originating from the subpolar and temperate climate regions.

Red clover (Trifolium pratense L.) is a forage legume cultivated worldwide. This plant is capable of establishing a nitrogen-fixing symbiosis with Rhizobium leguminosarum symbiovar trifolii strains. To date, no comparative analysis of the symbiotic properties and heterogeneity of T. pratense microsymbionts derived from two distinct geographic regions has been performed. In this study, the symbiotic properties of strains originating from the subpolar and temperate climate zones in a wide range of temperatures (10-25 °C) have been characterized. Our results indicate that all the studied T. pratense microsymbionts from two geographic regions were highly efficient in host plant nodulation and nitrogen fixation in a wide range of temperatures. However, some differences between the populations and between the strains within the individual population examined were observed. Based on the nodC and nifH sequences, the symbiotic diversity of the strains was estimated. In general, 13 alleles for nodC and for nifH were identified. Moreover, 21 and 61 polymorphic sites in the nodC and nifH sequences were found, respectively, indicating that the latter gene shows higher heterogeneity than the former one. Among the nodC and nifH alleles, three genotypes (I-III) were the most frequent, whereas the other alleles (IV-XIII) proved to be unique for the individual strains. Based on the nodC and nifH allele types, 20 nodC-nifH genotypes were identified. Among them, the most frequent were three genotypes marked as A (6 strains), B (5 strains), and C (3 strains). Type A was exclusively found in the temperate strains, whereas types B and C were identified in the subpolar strains. The remaining 17 genotypes were found in single strains. In conclusion, our data indicate that R. leguminosarum sv. trifolii strains derived from two climatic zones show a high diversity with respect to the symbiotic efficiency and heterogeneity. However, some of the R. leguminosarum sv. trifolii strains exhibit very good symbiotic potential in the wide range of the temperatures tested; hence, they may be used in the future for improvement of legume crop production.

Selection and adaptation of Saccharomyces cerevisae to increased ethanol tolerance and production.

A total of 24 yeast strains were tested for their capacity to produce ethanol, and of these, 8 were characterized by the best ethanol yields (73.11-8 1.78%). The most active mutant Saccharomyce s cerevisiae ER-A, resistant to ethanol stress, was characterized by high resistance to acidic (pH 1.0 and 2.0), oxidative (1 and 2% of H2O2), and high temperature (45 and 52 degrees C) stresses. During cultivation under all stress conditions, the mutants showed a considerably increased viability ranging widely from about 1.04 to 3.94-fold in comparison with the parent strain S. cerevisiae ER. At an initial sucrose concentration of 150 g/l in basal medium A containing yeast extract and mineral salts, at 300C and within 72 h, the most active strain, S. cerevisiae ER-A, reached an ethanol concentration of 80 g/1, ethanol productivity of 1.1 g/Il/h, and an ethanol yield (% of theoretical) of 99.13. Those values were significantly higher in comparison with parent strain (ethanol concentration 71 g/1 and productivity of 0,99 g/l/h). The present study seems to confirm the high effectiveness of selection of ethanol-resistant yeast strains by adaptation to high ethanol concentrations, for increased ethanol production.

Isoglucose production from raw starchy materials based on a two-stage enzymatic system.

A new low-cost glucoamylase preparation for liquefaction and saccharification of starchy raw materials in a one-stage system was developed and characterized. A non-purified biocatalyst with a glucoamylase activity of 3.11 U/mg, an alpha-amylase activity of 0.12 WU/mg and a protein content of 0.04 mg protein/mg was obtained from a shaken-flask culture of the strain Aspergillus niger C-IV-4. Factors influencing the enzymatic hydrolysis of starchy materials such as reaction time, temperature and enzyme and substrate concentration were standardized to maximize the yield of glucose syrup. Thus, a 90% conversion of 5% starch, a 67.5% conversion of 5% potato flour and a 55% conversion of 5% wheat flour to sweet syrups containing up to 87% glucose was reached in 3 h using 1.24 glucoamylase U/mg hydrolyzed substrate. The application of such glucoamylase preparation and a commercially immobilized glucose isomerase for the production of glucose-fructose syrup in a two-stage system resulted in high production of stable glucose/fructose blends with a fructose content of 50%. A high concentration of fructose in obtained sweet syrups was achieved when isomerization was performed both in a batch and repeated batch process.

Catalase activity in arctic microfungi grown at different temperatures.

The total of 98 strains of moulds were isolated from soils collected in arctic tundra (Spitzbergen). Among these strains Penicillium cyclopium 1, the most effective for production of catalase, was selected by the method of test-tube microculture. The time course of growth and catalase production by this strain showed the intracellular activity of this enzyme to be about 3-fold higher than its extracellular level Some properties of crude catalase preparation, isolated from postculture liquids by lyophilization, were also examined. Catalase activity showed its maximum at 15 degrees C, indicating adaptation of the enzyme to lower temperatures of the arctic environment.