Hyaluronic Acid/Ellagic Acid as Materials for Potential Medical Application.

The aim of this work was to develop and characterize a thin films composed of hyaluronic acid/ellagic acid for potential medical application. Its principal novelty, distinct from the prior literature in terms of hyaluronic acid films supplemented with phenolic acids, resides in the predominant incorporation of ellagic acid-a distinguished compound-as the primary constituent of the films. Herein, ellagic acid was dissolved in two different solvents, i.e., acetic acid (AcOH) or sodium hydroxide (NaOH), and the surface properties of the resultant films were assessed using atomic force microscopy and contact angle measurements. Additionally, various physicochemical parameters were evaluated including moisture content, antioxidant activity, and release of ellagic acid in phosphate buffered saline. Furthermore, the evaluation of films' biocompatibility was conducted using human epidermal keratinocytes, dermal fibroblasts, and human amelanotic melanoma cells (A375 and G361), and the antimicrobial activity was elucidated accordingly against Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442. Our results showed that the films exhibited prominent antibacterial properties particularly against Staphylococcus aureus, with the 80HA/20EA/AcOH film indicating the strong biocidal activity against this strain leading to a significant reduction in viable cells. Comparatively, the 50HA/50EA/AcOH film also displayed biocidal activity against Staphylococcus aureus. This experimental approach could be a promising technique for future applications in regenerative dermatology or novel strategies in terms of bioengineering.

Consortium of plant growth-promoting rhizobacteria enhances oilseed rape (Brassica napus L.) growth under normal and saline conditions.

A preparation development, which stimulates plant growth under normal and saline conditions, and protects against fungal infections, would increase crop yields and reduce damage in agriculture. This study was conducted using bacterial isolates from rape rhizosphere as a plant growth promoter and an alternative to chemical fertilizers. Three from fifty bacterial isolates: B14 (Pseudomonas sp.), B16 (Sphingobacterium sp.), and B19 (Microbacterium sp.) showed the best in vitro plant growth-promoting (PGP) characteristics. B14 strain had the best antifungal activity against phytopathogens inhibiting growth of B. cinerea, C. acutatum, and P. lingam. Moreover, B14, B16 and B19 isolates coded for several genes involved in PGP activities, aimed at improving nutrient availability, resistance to abiotic stress, and fungal pathogen suppression. Microbial consortium (B14, B16, and B19) had the best effect on rape growth, significantly increasing number of live leaves, compared to the untreated control and single inoculant treatments. Moreover, the consortium induced significant increase in shoots length and chlorophyll content in comparison to Pseudomonas sp. B14 and Microbacterium sp. B19. The consortium also induced plants tolerance to salt stress. The genomic information as well as the observed traits, and beneficial attributes towards rape, make the rhizobacterial consortium an ideal candidate for further development as biofertilizers.

Microbial Diversity in Deep-Subsurface Hot Brines of Northwest Poland: from Community Structure to Isolate Characteristics.

Deep-subsurface hot brines in northwest Poland, extracted through boreholes reaching 1.6 and 2.6 km below the ground surface, were microbiologically investigated using culture-independent and culture-dependent methods. The high-throughput sequencing of 16S rRNA gene amplicons showed a very low diversity of bacterial communities, which were dominated by phyla Proteobacteria and Firmicutes Bacterial genera potentially involved in sulfur oxidation and nitrate reduction (Halothiobacillus and Methylobacterium) prevailed in both waters over the sulfate reducers ("Candidatus Desulforudis" and Desulfotomaculum). Only one archaeal taxon, affiliated with the order Thermoplasmatales, was detected in analyzed samples. Bacterial isolates obtained from these deep hot brines were closely related to Bacillus paralicheniformis based on the 16S rRNA sequence similarity. However, genomic and physiological analyses made for one of the isolates, Bacillus paralicheniformis strain TS6, revealed the existence of more diverse metabolic pathways than those of its moderate-temperature counterpart. These specific traits may be associated with the ecological adaptations to the extreme habitat, which suggest that some lineages of B. paralicheniformis are halothermophilic.IMPORTANCE Deep-subsurface aquifers, buried thousands of meters down the Earth's crust, belong to the most underexplored microbial habitats. Although a few studies revealed the existence of microbial life at the depths, the knowledge about the microbial life in the deep hydrosphere is still scarce due to the limited access to such environments. Studying the subsurface microbiome provides unique information on microbial diversity, community structure, and geomicrobiological processes occurring under extreme conditions of the deep subsurface. Our study shows that low-diversity microbial assemblages in subsurface hot brines were dominated by the bacteria involved in biogeochemical cycles of sulfur and nitrogen. Based on genomic and physiological analyses, we found that the Bacillus paralicheniformis isolate obtained from the brine under study differed from the mesophilic species in the presence of specific adaptations to harsh environmental conditions. We indicate that some lineages of B. paralicheniformis are halothermophilic, which was not previously reported.

Exploring the properties of chitinolytic Bacillus isolates for the pathogens biological control.

Plant fungal diseases generate serious losses in the agriculture. The bacteria producing biologically active substances that inhibit the growth of fungal pathogens can be an alternative to the chemicals. The chitinolytic bacteria were isolated from the rhizosphere of wheat (Triticum aestivum L.) and their physiological properties which may be useful in the promotion of plant growth have been investigated. Their chitinases and antifungal activity were studied. The isolates were also tested for indirect growth-promoting traits such as ammonia production, siderophore production, hydrogen cyanide production, and salicylic acid production. Two chitinolytic strains B3 and B5 were identified as Bacillus subtilis and Bacillus sp., respectively. They produced active chitinases on a medium containing shrimp shell powder. The purified chitinases having the molecular weight of 35-45 kDa inhibited the growth of important plant pathogens such as Alternaria alternata, and Fusarium oxysporum. Additionally, the isolates showed the ability to produce a broad range of biological substances promoting the growth of plants.

Bacterial growth and biofilm formation in household-stored groundwater collected from public wells.

The research was aimed at assessing changes in the number of bacteria and evaluating biofilm formation in groundwater collected from public wells, both aspects directly related to the methods of household storage. In the research, water collected from Cretaceous aquifer wells in Torun (Poland) was stored in a refrigerator and at room temperature. Microbiological parameters of the water were measured immediately after the water collection, and then after 3 and 7 days of storage under specified conditions. The microbiological examination involved determining the number of heterotrophic bacteria capable of growth at 22 and 37 °C, the number of spore-forming bacteria, and the total number of bacteria on membrane filters. The storage may affect water quality to such an extent that the water, which initially met the microbiological criteria for water intended for human consumption, may pose a health risk. The repeated use of the same containers for water storage results in biofilm formation containing live and metabolically active bacterial cells.

Chitinolytic microorganisms and their possible application in environmental protection.

This paper provides a review of the latest research findings on the applications of microbial chitinases to biological control. Microorganisms producing these enzymes can inhibit the growth of many fungal diseases that pose a serious threat to global crop production. Currently, efforts are being made to discover producers of chitinolytic enzymes. The potential exists that natural biofungicides will replace chemical fungicides or will be used to supplement currently used fungicides, which would reduce the negative impact of chemicals on the environment and support the sustainable development of agriculture and forestry.

Biodegradability Study of Modified Chitosan Films with Cinnamic Acid and Ellagic Acid in Soil.

Currently, natural polymer materials with bactericidal properties are extremely popular. Unfortunately, although the biopolymer material itself is biodegradable, its enrichment with bactericidal compounds may affect the efficiency of biodegradation by natural soil microflora. Therefore, the primary objective of this study was to evaluate the utility of fungi belonging to the genus Trichoderma in facilitating the degradation of chitosan film modified with cinnamic acid and ellagic acid in the soil environment. Only two strains (T.07 and T.14) used chitosan films as a source of carbon and nitrogen. However, their respiratory activity decreased with the addition of tested phenolic acids, especially cinnamic acid. Addition of Trichoderma isolates to the soil increased oxygen consumption during the biodegradation process compared with native microorganisms, especially after application of the T.07 and T.14 consortium. Isolates T.07 and T.14 showed high lipolytic (55.78 U/h and 62.21 U/h) and chitinase (43.03 U/h and 41.27 U/h) activities. Chitinase activity after incorporation of the materials into the soil was higher for samples enriched with T.07, T.14 and the consortium. The isolates were classified as Trichoderma sp. and Trichoderma koningii. Considering the outcomes derived from our findings, it is our contention that the application of Trichoderma isolates holds promise for expediting the degradation process of chitosan materials containing bactericidal compounds.

Characterization and mass spectrometry analysis of aminopeptidase N from Pseudomononas putida Lup.

An intracellular aminopeptidase N synthesized by Pseudomonas putida Lup was purified and characterized. The approx. 150-fold purified enzyme showed highest activity against A-beta-naphthylamide at pH 7.5 and at temperature 40 degrees C and was 100% thermostable for 240 min at 40 degrees C. P putida lup aminopeptidase N is a monomer with molecular mass approx. 99 kDa determined by SDS-PAGE and gel permeation chromatography. The enzyme has broad substrate specificity, but is the most active against protein substrates with N-terminal alanine and arginine. The activity of P. putida Lup aminopeptidase N is strongly inhibited in the presence of specific metallopeptidase inhibitors and is partly recovered in the presence of Zn2+ and Co2+ ions. Co2+, Mg2+ and Ca2+ ions increased the activity of the enzyme. Moreover, the enzyme was inhibited by inhibitors of cysteine enzymes. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to PepN of Pseudomonas putida GB-1.

Bacillus paralicheniformis 2R5 and its impact on canola growth and N-cycle genes in the rhizosphere.

Chemical fertilization has a negative impact on the natural environment. Plant growth-promoting (PGP) rhizobacterial biofertilizers can be a safer alternative to synthetic agrochemicals. In this research, a culture-based method was used to assess the population size of rhizobacteria at the vegetative, flowering, and maturity stages of canola. Rhizobacteria were then isolated from each of the canola growth stages, and their seven PGP traits were determined. The highest abundance of culturable bacteria was found at the vegetative stage of the plants. Furthermore, four out of seven PGP traits were produced by the highest % of isolates at the vegetative stage. In the greenhouse experiment that included six rhizobacterial strains with best PGP traits, the greatest canola growth promotion ability under sterile conditions was observed after the introduction of Bacillus paralicheniformis 2R5. Moreover, under nonsterile conditions, 2R5 significantly increased canola growth. The presence of the trpA, B, C, D, E, F and pstA, and S genes in the 2R5 genome could be associated with canola growth promotion abilities. The chiA and mbtH genes could contribute to 2R5 antifungal activity against fungal pathogens. Moreover, the introduction of 2R5 significantly increased the abundance of the narG, nosZ, nifH, and nirS genes, which can prove that the 2R5 strain may be an important member of the soil bacterial community.

Pseudomonas sivasensis 2RO45 inoculation alters the taxonomic structure and functioning of the canola rhizosphere microbial community.

Inoculation with plant growth-promoting rhizobacteria (PGPR) is an eco-friendly sustainable strategy for improving crop productivity in diverse environments under different conditions. Our earlier study demonstrated that Pseudomonas sivasensis 2RO45 significantly stimulated canola (Brassica napus L. var. napus) growth. The aim of the present study was to investigate the structural and functional dynamics in the canola rhizosphere microbiome after inoculation with PGPR P. sivasensis 2RO45. The results based on alpha diversity metrics showed that P. sivasensis 2RO45 did not significantly alter the diversity of the native soil microbiota. However, the introduced strain modified the taxonomic structure of microbial communities, increasing the abundance of plant beneficial microorganisms, e.g., bacteria affiliated with families Comamonadaceae, Vicinamibacteraceae, genus Streptomyces, and fungi assigned to Nectriaceae, Didymellaceae, Exophiala, Cyphellophora vermispora, and Mortierella minutissima. The analysis of community level physiological profiling (CLPP) revealed that microbial communities in the P. sivasensis 2RO45 treated canola rhizospheres were more metabolically active than those in the non-treated canola rhizosphere. Four carbon sources (phenols, polymers, carboxylic acids, and amino acids) were better metabolized by the microbial communities from the rhizosphere of plants inoculated with the P. sivasensis 2RO45 than non-inoculated canola rhizospheres. Based on the community-level physiological profiles, the functional diversity of the rhizosphere microbiome was altered by the P. sivasensis 2RO45 inoculation. Substrate utilization Shannon diversity (H) index and evenness (E) index were significantly increased in the treated canola plants. The study provides new insight into PGPR-canola interactions for sustainable agriculture development.

Polycaprolactone-Based Films Incorporated with Birch Tar-Thermal, Physicochemical, Antibacterial, and Biodegradable Properties.

We present new polymer materials consisting of polycaprolactone (PCL), polyethylene glycol (PEG), and birch tar (D). PEG was introduced into the polymer matrix in order to obtain a plasticizing effect, while tar was added to obtain antibacterial properties and to change the physicochemical properties of the film. The materials were obtained by the solvent method and characterized using a variety of methods to test their performance and susceptibility to biodegradation. The obtained data indicate that the introduction of the bioactive substance (D) into PCL improved the thermal stability and significantly lowered the Young's modulus values of the tested polymers. Moreover, the addition of birch tar improved the barrier and bacteriostatic properties, resulting in a reduction in the growth of pathogenic bacteria on the surface of the film. The films are not mutagenic but are susceptible to biodegradation in various environments. Due to their properties, they have potential for application in agriculture and horticulture and for packaging food, mainly vegetables grown in the field.

Engineering Antioxidant Surfaces for Titanium-Based Metallic Biomaterials.

Prolonged inflammation induced by orthopedic metallic implants can critically affect the success rates, which can even lead to aseptic loosening and consequent implant failure. In the case of adverse clinical conditions involving osteoporosis, orthopedic trauma and implant corrosion-wear in peri-implant region, the reactive oxygen species (ROS) activity is enhanced which leads to increased oxidative stress. Metallic implant materials (such as titanium and its alloys) can induce increased amount of ROS, thereby critically influencing the healing process. This will consequently affect the bone remodeling process and increase healing time. The current review explores the ROS generation aspects associated with Ti-based metallic biomaterials and the various surface modification strategies developed specifically to improve antioxidant aspects of Ti surfaces. The initial part of this review explores the ROS generation associated with Ti implant materials and the associated ROS metabolism resulting in the formation of superoxide anion, hydroxyl radical and hydrogen peroxide radicals. This is followed by a comprehensive overview of various organic and inorganic coatings/materials for effective antioxidant surfaces and outlook in this research direction. Overall, this review highlights the critical need to consider the aspects of ROS generation as well as oxidative stress while designing an implant material and its effective surface engineering.

Application Potential of Trichoderma in the Degradation of Phenolic Acid-Modified Chitosan.

The aim of the study was to determine the potential use of fungi of the genus Trichoderma for the degradation of phenolic acid-modified chitosan in compost. At the same time, the enzymatic activity in the compost was checked after the application of a preparation containing a suspension of the fungi Trichoderma (spores concentration 105/mL). The Trichoderma strains were characterized by high lipase and aminopeptidase activity, chitinase, and ß-1,3-glucanases. T. atroviride TN1 and T. citrinoviride TN3 metabolized the modified chitosan films best. Biodegradation of modified chitosan films by native microorganisms in the compost was significantly less effective than after the application of a formulation composed of Trichoderma TN1 and TN3. Bioaugmentation with a Trichoderma preparation had a significant effect on the activity of all enzymes in the compost. The highest oxygen consumption in the presence of chitosan with tannic acid film was found after the application of the consortium of these strains (861 mg O2/kg after 21 days of incubation). Similarly, chitosan with gallic acid and chitosan with ferulic acid were found after the application of the consortium of these strains (849 mgO2/kg and 725 mg O2/kg after 21 days of incubation). The use of the Trichoderma consortium significantly increased the chitinase activity. The application of Trichoderma also offers many possibilities in sustainable agriculture. Trichoderma can not only degrade chitosan films, but also protect plants against fungal pathogens by synthesizing chitinases and ß-1,3 glucanases with antifungal properties.

Purification, characterization and cloning of a chitinase from Stenotrophomonas rhizophila G22.

In the presented research the extracellular chitinase of Stenotrophomonas rhizophila G22 was biochemically and molecularly characterized. The studied enzyme was purified from a 72-h bacterial culture about 14 times, with a recovery of 63%. The molecular weight of the purified protein was estimated at 50 kDa by SDS-PAGE. The enzyme showed high activity against colloidal chitin. Significantly lower activities were observed with native chitin powder and chitosan. Adsorption of the enzyme to colloidal chitin and to powdered chitin at the level of 75% and 37%, respectively, was observed after 30 min of reaction. Optimum temperature and pH were 37 °C and 5.9, respectively. The enzyme demonstrated higher activity against nitrophenyl-ß d N, N', N″-triacetylchitotriose and approx. 5 times lower activity for 4-nitrophenyl-N, N'-diacetyl-ß-d-chitobiose. The enzyme is an endochitinase, which is confirmed by the K m and V max values determined in the studies. S. rhizophila G22 endochitinase was inhibited in the presence of cysteine-specific inhibitors, which indicates the role of cysteine moieties in the mechanism of catalysis or in stabilisation of the enzyme molecule. Also Ca2+ and Mn2+ ions may stabilise the protein's spatial structure. SDS and ions: Fe2+, Cu2+, Co2+, Zn2+ inhibited the activity of enzyme. A full-length (2109 bp) gene coding chitinase from S. rhizophila G22 was obtained. Four domains typical for glycoside hydrolase family 18 (GH 18) chitinases were identified: catalytic Gly_18, chitin-binding-ChtBD3, type-III fibronectin-FN3 and polycystic kidney disease domain-PKD domain.

Biofilm formation during biodegradation of polylactide, poly (3,4 hydroxybutyrate) and poly(ε-caprolactone) in activated sludge.

Biodegradable materials, namely pure polylactide (PLA), poly (3,4-hydroxybutyrate) (PHB), poly(ε-caprolactone) (PCL) were investigated to assess their degradability by activated sludge. The study aimed at the isolation of biofilm-forming bacteria and the determination of their hydrolytic activity toward the PLA, PHB, and PCL with embedded PHMG derivatives. The biological oxygen demand and physical properties (tensile strength, water vapor permeability, surface structure) of materials indicated that PCL was the best biodegradable film. Aeromonas and Rhodococcus isolated from the polymers' surface during the process of decomposition showed the ability to form biofilms. The introduction of PHMG derivatives into PLA, PCL, and PHB films did not affect biofilm formation and hydrolase activity for most of the isolates. PHMG derivatives at the concentration of 1% disturbed the degradation process.

Production, characterization, gene cloning, and nematocidal activity of the extracellular protease from Stenotrophomonas maltophilia N4.

A rhizosphere strain of the bacterium Stenotrophomonas maltophilia N4 secretes the serine protease PN4, whose molecular mass is approximately 42 kDa. The optimal temperature for the enzyme activity of the 11-fold purified protein was 50°C and the optimal pH was 10.5. The activity of the enzyme was strongly inhibited by specific serine protease inhibitors, which allowed for its classification as an alkaline serine protease family. Ca(2+) ions stimulated the activity of the protease PN4, while Mg(2+) ions stabilized its activity, and Zn(2+) and Cd(2+) ions strongly inhibited its activity. The enzyme has broad substrate specificity. For example, it is able to hydrolyse casein, keratin, albumin, haemoglobin, and gelatin, as well as the insoluble modified substrates azure keratin and azocoll. The gene that encodes the 1740 bp precursor form of the enzyme (accession number: LC031815) was cloned. We then deduced that its amino acid sequence includes the region of the conserved domain of the S8 family of peptidases as well as the catalytic triad Asp/His/Ser. The bacterial culture fluid as well as the purified protease PN4 demonstrated biocidal activity with regard to the nematodes Caenorhabditis elegans and Panagrellus spp.

Biofilm formation on the surface of polylactide during its biodegradation in different environments.

The research was aimed at determining the abundance and viability of biofilm formed on the surface of polylactide (PLA) during its biodegradation in different environments. It was also aimed at isolating biofilm forming bacteria, determining their hydrolytic activity and taxonomic status. The first step was to evaluate PLA biodegradability in lake water, compost and soil, using OxiTop Control. The next step was to assess the ability of isolated bacteria to form biofilm in the investigated environments and to evaluate the biofilm structure. The results indicate that PLA is sensitive to biodegradation in any environment, particularly in compost. During this process biofilm of high viability was observed on the surface of PLA. Based on the 16S rRNA gene sequence, the biofilm-forming bacteria were classified as the following species: Acidovorax sp. LW9, Chryseobacterium sp. LW2, Aeromonas veronii LW8, Arthrobacter aurescens LG2, Arthrobacter sp. LG12, A. aurescens LG9, Elizabethkingia meningoseptica LK3, A. aurescens LK9, A. aurescens and LK7. The results show that different bacterial species formed biofilm of different abundance and hydrolytic activitiy levels.

Microbiological contamination of water in fountains located in the city of Torun, Poland.

OBJECTIVE: The aim of this study was determination of the level of water contamination in fountains in Torun, Poland. METHODS: The studies were conducted at monthly intervals from May - September 2009, and consisted in the determination of the number of heterotrophic bacteria capable of growing at a temperature of 22 and 37°C (PN-ISO 6222), as well as the number of coliform bacteria (PN-75/C-04615/05), E. coli (ISO 9308-1) and faecal streptococci (PN-EN ISO 7899-2). RESULTS: The 'Rafter' fountain in the Old Town Market Square, continuously supplied with municipal water, had the cleanest water. The highest number of heterotrophic bacteria capable of development at 22 and 37°C was found in the fountain located in the City Park of Bydgoskie suburb. Throughout the entire research period, water from the fountain in the Old Town Moat had the worst sanitary (health) indices. CONCLUSION: The recorded results indicate that water from the 3 out of 4 examined fountains, with inappropriate exploitation--bathing, water drinking, hands washing--may constitute a danger to human health. The fountains should be carefully monitored for the presence of microorganisms, or a ban on bathing in fountains should be more rigorously enforced, in order to prevent possible infections.