Catalase from the Antarctic Fungus Aspergillus fumigatus I-9-Biosynthesis and Gene Characterization.

Extremely cold habitats are a serious challenge for the existing there organisms. Inhabitants of these conditions are mostly microorganisms and lower mycetae. The mechanisms of microbial adaptation to extreme conditions are still unclear. Low temperatures cause significant physiological and biochemical changes in cells. Recently, there has been increasing interest in the relationship between low-temperature exposure and oxidative stress events, as well as the importance of antioxidant enzymes for survival in such conditions. The catalase is involved in the first line of the cells' antioxidant defense. Published information supports the concept of a key role for catalase in antioxidant defense against cold stress in a wide range of organisms isolated from the Antarctic. Data on representatives of microscopic fungi, however, are rarely found. There is scarce information on the characterization of catalase synthesized by adapted to cold stress organisms. Overall, this study aimed to observe the role of catalase in the survival strategy of filamentous fungi in extremely cold habitats and to identify the gene encoded catalase enzyme. Our results clearly showed that catalase is the main part of antioxidant enzyme defense in fungal cells against oxidative stress caused by low temperature exposure.

Cold-active catalase from the psychrotolerant fungus Penicillium griseofulvum.

Cold-active catalase (CAT) elicits great interest because of its vast prospective at the medical, commercial, and biotechnological levels. The study paper reports the production of cold-active CAT by the strain Penicillium griseofulvum P29 isolated from Antarctic soil. Improved enzyme production was achieved by optimization of medium and culture conditions. Maximum CAT was demonstrated under low glucose content (2%), 10% inoculum size, temperature 20°C, and dissolved oxygen concentration (DO) 40%. An effective laboratory technology based on changing the oxidative stress level through an increase of DO in the bioreactor was developed. The used strategy resulted in a 1.7- and 1.4-fold enhanced total enzyme activity and maximum enzyme productivity. The enzyme was purified and characterized. P. griseofulvum P29 CAT was most active at approximately 20°C and pH 6.0. Its thermostability was in the range between 5°C and 40°C.

Study of helveticin gene in Lactobacillus crispatus strains and evaluation of its use as a phylogenetic marker.

Lactobacilli are a part of the human microbiome in healthy humans. Studies of their physiological and genetic characteristics are the basis for their use in probiotic preparations. This report is a brief description of the helveticin gene found in two Lactobacillus crispatus strains, which are a part of the human microbiome. Our analysis showed that the two variants of the gene are not solely characteristic of strains isolated from humans. In the phylogenetic analysis, we found that the studied sequence (this gene) showed a significant difference between the species of the genus Lactobacillus and could be used as a phylogenetic marker.

Bacteriocin production and gene sequencing analysis from vaginal Lactobacillus strains.

The human vagina is a complex and dynamic ecosystem containing an abundance of microorganisms. In women of childbearing age, this system is dominated by Lactobacillus spp. In the present work, seventeen newly isolated vaginal strains were identified by 16S rDNA sequencing and were investigated for their antimicrobial properties. Twelve of the isolated Lactobacillus strains showed activity against one or more microorganisms. Six and five of them produced substances that inhibited the growth of two different Klebsiella strains and Staphylococcus aureus, respectively. Two lactobacilli strains were active against an Escherichia coli strain, one isolate was active against an Enterococus faecalis strain and another lactobacilli strain showed antimicrobial activity against a Candida parapsilosis strain. The nature of the active compounds was additionally studied, and the presence of bacteriocin-like substances was proved. The genes related to the bacteriocin production in three of the newly isolated strains were identified and sequenced. The presence of gassericin A operon in the genome of the species Lactobacillus crispatus was described for the first time. The presence of antimicrobial activity contributes to their possible use as potential probiotic strains after further research.

Lysis of Antarctic algal strains by bacterial pathogen.

The present paper describes the isolation, physiological and genetic characteristic of a bacterial agent which inhibits the growth of algae and causes death of laboratory cultures of Antarctic microalgal strains: prokaryotic cyanobacteria Synechocystis salina and green eukaryotic microalga Choricistis minor. The bacterial strain LB1 was isolated from algal damaged laboratory cultures of S. salina. It was established that this bacterium is obligate aerobic, Gram-positive, non-spore-forming, immotile, irregular rods with dimensions 0.3-2 µm. Our results showed that LB1 has algicidal effect to S. salina as well as to C. minor. Transmission electron microscopy observations confirmed the destruction of S. salina by the bacterium. Biochemical analysis of LB1 revealed positive reaction to D-glucose, catalase, hydrolysis of gelatin, acid production from: lactose, L-arabinose, L-ramnose, esculin and ß-galactosidase. The partial sequence (1,404 bp) of the 16S rRNA gene of LB1 showed 99 % similarity with type strains of the genus Microbacterium. The results of the biochemical, antimicrobial and of 16S rRNA analysis of LB1 allowed us to identify LB1 as Microbacterium sp. Studying expression of pathogenicity of the bacteria to algal cultures will help to solve the problem of algal production for biotechnological purposes.

Exploring the mechanism underlying the antifungal activity of chitosan-based ZnO, CuO, and SiO2 nanocomposites as nanopesticides against Fusarium solani and Alternaria solani.

Chitosan-based nanocomposites (CS NCs) are gaining considerable attention as multifaceted antifungal agents. This study investigated the antifungal activity of NCs against two phytopathogenic strains: Fusarium solani (F. solani) and Alternaria solani (A. solani). Moreover, it sheds light on their underlying mechanisms of action. The NCs, CS-ZnO, CS-CuO, and CS-SiO2, were characterized using advanced methods. Dynamic and electrophoretic light scattering techniques revealed their size range (60-170 nm) and cationic nature, as indicated by the positive zeta potential values (from +16 to +22 mV). Transmission electron microscopy revealed the morphology of the NCs as agglomerates formed between the chitosan and oxide components. X-ray diffraction patterns confirmed crystalline structures with specific peaks indicating their constituents. Antifungal assessments using the agar diffusion technique demonstrated significant inhibitory effects of the NCs on both fungal strains (1.5 to 4-fold), surpassing the performance of the positive control, nystatin. Notably, the NCs exhibited superior antifungal potency, with CS-ZnO NCs being the most effective. A. solani was the most sensitive strain to the studied agents. Furthermore, the tested NCs induced oxidative stress in fungal cells, which elevated stress biomarker levels, such as superoxide dismutase (SOD) activity and protein carbonyl content (PCC), 2.5 and 6-fold for the most active CS-CuO in F. solani respectively. Additionally, they triggered membrane lipid peroxidation up to 3-fold higher compared to control, a process that potentially compromises membrane integrity. Laurdan fluorescence spectroscopy highlighted alterations in the molecular organization of fungal cell membranes induced by the NCs. CS-CuO NCs induced a membrane rigidifying effect, while CS-SiO2 and CS-ZnO could rigidify membranes in A. solani and fluidize them in F. solani. In summary, this study provides an in-depth understanding of the interactions of CS-based NCs with two fungal strains, showing their antifungal activity and offering insights into their mechanisms of action. These findings emphasize the potential of these NCs as effective and versatile antifungal agents.

Metagenomic Analysis of Bacterial, Archaeal and Fungal Diversity in Two-Stage Anaerobic Biodegradation for Production of Hydrogen and Methane from Corn Steep Liquor.

The main purpose of this study was to identify the microbial communities (bacterial, archaeal and fungal) in a two-stage system of anaerobic bioreactors for the production of hydrogen and methane from the waste substrate-corn steep liquor. Wastes from the food industry are valuable resources with potential in biotechnological production because of their high organic matter contents. In addition, the production of hydrogen and methane, volatile fatty acids, reducing sugars and cellulose content was monitored. Two-stage anaerobic biodegradation processes were performed by microbial populations in the first hydrogen generating bioreactor (working volume of 3 dm3) and in the second methane-generating reactor (working volume of 15 dm3). Cumulative hydrogen yield reached 2000 cm3 or 670 cm3/L a day, while the methane production reached a maximum quantity of 3300 cm3 or 220 cm3/L a day. Microbial consortia in anaerobic digestion systems play an essential role for process optimization and biofuel production enhancement. The obtained results showed the possibility of conducting two separate processes-the hydrogenic (hydrolysis and acidogenesis) and methanogenic (acetogenesis and methanogenesis)-as two stages of anaerobic digestion to favor energy production under controlled conditions with corn steep liquor. The diversity of microorganisms as main participants in the processes in the bioreactors of the two-stage system was followed using metagenome sequencing and bioinformatics analysis. The obtained metagenomic data showed that the most abundant phylum in both bacterial communities was Firmicutes-58.61% and 36.49% in bioreactors 1 and 2, respectively. Phylum Actinobacteria were found in significant quantities (22.91%) in the microbial community in Bioreactor 1, whereas in Bioreactor 2, they were 2.1%. Bacteroidetes are present in both bioreactors. Phylum Euryarchaeota made up 0.4% of the contents in the first bioreactor and 11.4% in the second. As the dominant genera among methanogenic archaea are Methanothrix (8.03%) and Methanosarcina (3.39%), the main fungal representatives were Saccharomyces cerevisiae. New knowledge of anaerobic digestion mediated by novel microbial consortia could be widely used to convert different wastes to green energy.

Distribution of a novel enzyme of sialidase family among native filamentous fungi.

Sialidases (neuraminidases, EC 3.2.1.18) are widely distributed in biological systems but there are only scarce data on its production by filamentous fungi. The aim of this study was to obtain information about sialidase distribution in filamentous fungi from non-clinical isolates, to determine availability of sialidase gene, and to select a perspective producer. A total of 113 fungal strains belonging to Ascomycota and Zygomycota compassing 21 genera and 51 species were screened. Among them, 77 strains (11 orders, 14 families and 16 genera) were able to synthesize sialidase. Present data showed a habitat-dependent variation of sialidase activity between species and within species, depending on location. Sialidase gene was identified in sialidase-positive and sialidase-negative strains. . Among three perspective strains, the best producer was chosen based on their sialidase production depending on type of cultivation, medium composition, and growth temperature. The selected P. griseofulvum Р29 was cultivated in 3L bioreactor at 20 °C on medium supplemented with 0.5% milk whey. The results demonstrated better growth and 2.3-fold higher maximum enzyme activity compared to the shaken flask cultures. Moreover, the early occurring maximum (48 h) is an important prerequisite for future up scaling of the process.

Potential of ligninolytic enzymatic complex produced by white-rot fungi from genus Trametes isolated from Bulgarian forest soil.

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn-dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.

Molecular identification of vaginal lactobacilli isolated from Bulgarian women.

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in comparison with a set of 21 reference strains based on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of them to be classified as belonging to one of the following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.