Comparison of the Wild-Type Obligate Methylotrophic Bacterium Methylophilus quaylei and its Isogenic Streptomycin-Resistant Mutant via Metal Nanoparticle Generation.

Metal nanoparticles synthesized by green methods with the use of microorganisms are currently one of the most closely studied types of nanomaterials. It has accurately been shown that the characteristics of metal nanoparticles generated in the presence of different bacteria vary. For the two isogenic strains of obligate methylotrophic bacteria of the wild type (M. quaylei MTT) and its streptomycin-resistant mutant (M. quaylei SMR), the pleiotropic character of streptomycin resistance mutation in the SMR cells has been revealed. It has been shown that both cultures can generate silver nanoparticles. There is a dramatic difference in the formation of palladium nanoparticles, which are formed only in the presence of cells of the streptomycin-resistant mutant M. quaylei SMR. This study shows that closely related isogenic strains of obligate methylotrophic bacteria can be distinguished by the spectra of biogenic nanoparticles of two noble metals. While palladium nanoparticles are only generated by the cells of the streptomycin-resistant mutant M. quaylei SMR, biogenic silver nanoparticles can be generated from both cultures. Thus, the assessment of the ability of microorganisms to form biogenic nanoparticles of different metals allows the revelation of subtle metabolic differences of even close cultures.

Antimicrobial activity of silver salt and silver nanoparticles in different forms against microorganisms of different taxonomic groups.

The development of antiseptics and medical products (bandaging materials, sponges, etc.) based on silver nanoparticles is an essential task due to the growing resistance of pathogenic microorganisms to medicines long used in clinical practice. Using silver nanoparticles for the same purpose is promising, but the potential hazards and cumulative effects in the application of nanoparticles requires a thorough study of those materials. To evaluate the efficiency of antiseptics and medical products based on silver nanoparticles, it is necessary to conduct an in-depth study of the activity of silver nanoparticles in different forms and immobilized in carriers. The study examines the resistance of bacterial and fungal cultures to silver nanoparticles produced by chemical reduction and microbiological synthesis. The study of resistance was carried out in different growth phases of pathogenic microorganisms and in both liquid and solid media. Chemically and microbiologically synthesized nanoparticles were added in the form of a suspension, as well as encapsulated in chitosan-PVA matrices. It was experimentally discovered that, depending on the medium and form of the silver, the antibacterial effect would significantly differ due to changes in the mechanisms regarding the release of nanoparticles and their activity against the cells of pathogenic and potentially pathogenic microorganisms.

Halobacterium salinarum storage and rehydration after spray drying and optimization of the processes for preservation of carotenoids.

Spray drying is appropriate for the preservation of halophilic microorganisms due to the nature of these microorganisms, as they survive in adverse environmental conditions by being encapsulated in salt crystals. Artificial neural networks were in this study used to optimize practically significant spray-drying regimes of the C50-carotenoids producer Halobacterium salinarum. Immediately after drying, the samples contained up to 54% halobacterial biomass and less than 5% moisture, and the level of preservation of carotenoids was 95-97%. The storage of biomass at 4 °C resulted in the gradual degradation of the carotenoids, which reached 58-64% in the best samples after 1 year. A comprehensive study of changes in halobacteria biomass after spray drying and the nature of the damage provided new data on the survival and preservation of cells and biologically active substances in the various spray-drying regimes and at different storage times.

Efficient non-cytotoxic fluorescent staining of halophiles.

Research on halophilic microorganisms is important due to their relation to fundamental questions of survival of living organisms in a hostile environment. Here we introduce a novel method to stain halophiles with MitoTracker fluorescent dyes in their growth medium. The method is based on membrane-potential sensitive dyes, which were originally used to label mitochondria in eukaryotic cells. We demonstrate that these fluorescent dyes provide high staining efficiency and are beneficial for multi-staining purposes due to the spectral range covered (from orange to deep red). In contrast with other fluorescent dyes used so far, MitoTracker does not affect growth rate, and remains in cells after several washing steps and several generations in cell culture. The suggested dyes were tested on three archaeal (Hbt. salinarum, Haloferax sp., Halorubrum sp.) and two bacterial (Salicola sp., Halomonas sp.) strains of halophilic microorganisms. The new staining approach provides new insights into biology of Hbt. salinarum. We demonstrated the interconversion of rod-shaped cells of Hbt. salinarium to spheroplasts and submicron-sized spheres, as well as the cytoplasmic integrity of giant rod Hbt. salinarum species. By expanding the variety of tools available for halophile detection, MitoTracker dyes overcome long-standing limitations in fluorescence microscopy studies of halophiles.

Efficient continuous biosynthesis of silver nanoparticles by activated sludge micromycetes with enhanced tolerance to metal ion toxicity.

The method for producing AgNPs by granules of activated sludge micromycetes with enhanced tolerance to metal ion toxicity - Penicillium glabrum, Fusarium nivale and Fusarium oxysporum has been developed; the optimum conditions for AgNP biosynthesis being found: the Ag+ ion concentration, duration of the contact of microbial cells with silver ions, a growth phase of microorganisms, medium composition, a рН value, mixing conditions, and also lighting intensity. The effect of Cl-, SO42- and HPO42- ions binding Ag+ ions was eliminated, that brought to significant increase of the yield of NPs. Under batch conditions, silver particles of 60-110 nanometers in size were formed with a 65% yield. It was established that the nanoparticles were covered with microbial cell membrane proteins composed up to 70% by weight of the NPs that prevented their aggregation. In addition, it was the first time stable AgNPs had been formed by continuous AgNP biosynthesis by living cells of F. oxysporum with an 80% yield for a long time.

High-effective cultivation of Halobacterium salinarum providing with bacteriorhodopsin production under controlled stress.

Submerged growth of Halobacterium salinarum and therefore synthesis of bacteriorhodopsin (BR) and carotenoids depend greatly on products of both chemical and/or photochemical oxidation of medium components and cellular metabolism which act as inhibitors. Some cultivation variants which allowed eliminating an adverse effect of inhibitors on biomass accumulation and BR synthesis are reviewed. The application of activated charcoal or ion exchange resin as adsorbents at preparing inoculums and the main cultivation stages was shown to allow controlling, namely lowering overstress of the halobacterial cells by metabolites. The halobacterial biomass containing BR up to 1,750mgL(-1) and the minimum amount of carotinoids that would BR greatly facilitate isolation was accumulated up to 45gL(-1) during eight-day cultivation with cell recycling through adsorbent suspension in a fed-batch mode. To control BR biosynthesis the express method of BR quantification based on colour shades of cell suspension was developed.

Toxic influence of silver and uranium salts on activated sludge of wastewater treatment plants and synthetic activated sludge associates modeled on its pure cultures.

Toxic impact of silver and uranium salts on activated sludge of wastewater treatment facilities has been studied. Some dominating cultures (an active nitrogen fixer Agrobacterium tumifaciens (A.t) and micromyces such as Fusarium nivale, Fusarium oxysporum, and Penicillium glabrum) have been isolated and identified as a result of selection of the activated sludge microorganisms being steadiest under stressful conditions. For these cultures, the lethal doses of silver amounted 1, 600, 50, and 300 µg/l and the lethal doses of uranium were 120, 1,500, 1,000, and 1,000 mg/l, respectively. A.tumifaciens is shown to be more sensitive to heavy metals than micromyces. Synthetic granular activated sludge was formed on the basis of three cultures of the isolated micromyces steadiest against stress. Its granules were much more resistant to silver than the whole native activated sludge was. The concentration of silver causing 50 % inhibition of synthetic granular activated sludge growth reached 160-170 µg/l as far as for the native activated sludge it came only to 100-110 µg/l.

The role of stress agents as operating factors in formation and functioning of granular aerobic activated sludge at model domestic wastewater treatment.

Maintenance of the wastewater treatment plants and increasing the efficiency of existing aerobic biological reactors depend on the stability of activated sludge characteristics under varying wastewater parameters within significant limits and/or influence of some environmental factors. The steady microbial communities observed in biofilms and anaerobic granules of activated sludge can serve as successful samples of formation of the similar aerobic systems. The granular aerobic sludge obtained in the course of our researches is an ideal "plant" on treatment of biogenic pollution at both low and high concentrations. It demonstrates high ability for treatment and stability to adverse factors. To improve aerobic wastewater treatment characteristics, a possibility of using impact of stress conditions upon activated sludge has been studied. Under conditions of fractional hydrogen peroxide addition at diffused lighting, the granular aerobic activated sludge adapted to hydrogen peroxide has been obtained. This sludge has got good sedimentary properties and it differs from the control sample in the species diversity, improved treatment characteristics and also resistance to the stressor. It also endures an impact of one-time hydrogen peroxide addition up to 1.2-1.5 g H2O2/l. The conditions under which the steady aerobic granules of the diameter from 2 to 5 mm were formed with high treatment ability have been chosen. The granules were being stabilized at passages with hydrogen peroxide treatment and they endured up to 2.4-3.0 g/l of one-time H2O2 addition.