Data on the determining influence of Shewanella oneidensis MR-1 (gram−) and Bacillus subtilis 168 (gram+) bacterial strains used for the biosynthesis of CdS nanoparticles (NPs) on the quantitative and qualitative composition of the protein coating and functional characteristics of the nanomaterial such as the biocidal and photocatalytic activity are presented. The novelty of the study is quantitative assessment of the proteins adsorbed on the surface of CdS NPs/Shewanella and CdS NPs/Bacillus. It is shown that the protein content on the surface of CdS NPs/Shewanella and CdS NPs/Bacillus substantially differs and is 34 and 5% of the total weight of the nanoparticles, respectively. The fundamental difference in the qualitative composition of the “protein corona” of CdS NPs/Shewanella and CdS NPs/Bacillus is confirmed. The presence of nitrogen-containing organic compounds of protein nature on the surface of the nanoparticles is confirmed by Fourier-transform infrared (IR) spectroscopy. The possibility of using biogenic CdS NPs as biocidal agents of a new generation against a wide range of microorganisms as well as photonanocatalysts for the decoloration of synthetic dyes is proven. A difference in the level of functional activity of CdS NPs depending on the strain used for their biosynthesis is found and higher biocidal and photocatalytic activity of CdS NPs/Bacillus in comparison with CdS NPs/Shewanella is shown. The need for selecting bacterial strains to produce nanoparticles with high functionality is demonstrated.

-In this work, we report on the effect of Shewanella oneidensis MR-1 and Bacillus subtilis 168 bacteria used for the biosynthesis of CdS nanoparticles (CdS NPs) on the level of antimicrobial activity of CdS NPs/Shewanella and CdS NPs/Bacillus against a wide range of microorganisms: gram-positive, gram-negative bacteria, yeast and mold fungi. It is shown that S. oneidensis MR-1 and B. subtilis determine the quantitative characteristics of the nanomaterial, the content of protein and the CdS fraction in CdS NP samples. The use of the atomic emission spectroscopy method to determine the elemental content of cadmium and sulfur, as well as the densitometric analysis of electropherograms of protein adsorbed on the surface of biogenic CdS NPs demonstrate a significant difference in the ratio of these components in CdS NPs/Shewanella and CdS NPs/Bacillus. It is found that the proportion of CdS in CdS NPs/Bacillus exceeds by 1.4 times this parameter for CdS NPs/Shewanella. At the same time, the proportion of protein in CdS NPs/Bacillus is reduced compared to CdS NPs/Shewanella. It is shown that the level of antimicrobial activity of CdS NPs/Bacillus in relation to all test cultures studied exceeds the same parameter for CdS NPs/Shewanella. It is assumed that an increase in the proportion of CdS in CdS NPs/Bacillus may be the cause of a higher level of biocidal activity of CdS NPs due to an increased amount of released Cd2+ ions, which lead to the generation of reactive oxygen species and the destruction of microbial cells. Thus, the importance and necessity of selecting bacterial strains for the creation of functionally active nanoparticles with optimal characteristics is proved. The results of this research confirm the prospects of utilizing biogenic CdS NPs as targeted antimicrobial agents.

The survival time of the E. coli K-12 strain and the genetically modified E. coli K-12 SGM2.0pyc-int strain developed on its basis, a producer of succinic acid, has been determined in various natural and anthropogenically contaminated water and soil samples. The dynamics of the abundance of both strains in water samples from a well, a pond and a sewer collector was evaluated. It was found that within 10 days of the experiment, the cells of both strains died in all water samples. The highest dynamics of cell death, within 4 days, was observed in the wastewater from the collector. The survival rate of both strains in the same environment was similar; however, there was a tendency towards lower viability of the genetically modified producer. In the sod-podzolic and urbanozem soil samples, the growth of the studied strains was suppressed regardless of the type of soil. The absence of viable E. coli K-12 and E. coli K-12 SGM2.0pyc-int cells was observed after 10 and 7 days of incubation, respectively. The results indicate a high level of safety of the genetically modified E. coli K-12 SGM2.0pyc-int strain and the lack of its competition with microflora of water and soil samples from natural and anthropogenic sources.Escherichia coli K-12, genetically modified microorganisms, survival, natural ecosystems, environmental monitoringThe authors are grateful to the Russian State Collection of Industrial Microorganisms National Bio-Resource Center (BRC VKPM), State Research Institute for Genetics and Selection of Industrial Microorganisms, for providing bacterial strains. We thank A. D. Novikov, a researcher of the Kurchatov Genomic Center - GosNIIgenetica, for participation in the planning of experiments and discussion of the results and Zakharova M.V., Associate Professor of the Department of Theory of State and Law of the O.E. Kutafin University (MGUA) for legal advice in the preparation of regulatory and legal documentation on genetic safety.The work was carried out with the financial support of the Russian Foundation for Basic Research (project no. 18-29-14005).

Regulatory assessment and analysis were carried out of the compliance of the characteristics of a genetically modified microorganism (GMM) Escherichia coli K-12 VKPM B-13285, a producer of succinic acid (SA) containing a heterologous pyruvate carboxylase gene ( pyc A from the Bacillus subtilis strain) in the chromosome, with the requirements of Russian and international legislation applied to industrial GMM producers of biologically active substances. Comparative analysis was performed of a number of characteristics of the initial E. coli K-12 MG1655 and GMM under the influence of environmental factors. Methods for studying the growth characteristics of the strains, methods for estimating the effect of such parameters as temperature, UV radiation, incubation in water, soil, wastewater on the viability of bacteria are presented. A method of the identification of GMM among other representatives of enterobacteria by PCR analysis using pairs of locus-specific primers in the regions that distinguish a GMM from the initial E. coli K-12 strain, as well as a method of identification of enterobacteria on diagnostic media, are presented. The regulatory documents of Russian and international legislation with regard to the requirements for microorganisms obtained using the methods of genetic engineering were analyzed. The compliance of characteristics of E. coli K-12 VKPM B-13285 (a plasmid-free SA producer containing no antibiotic resistance genes) with the requirements of legislative documents was established. It was demonstrated that GMM differs from E. coli K-12 MG1655 in terms of growth parameters and the level of biomass accumulation and is characterized by reduced viability under UV irradiation, an increase in temperature, and cultivation in soil and wastewater. The possibility of identifying GMM under different conditions was established and the sensitivity of the methods using PCR analysis and diagnostic media was determined. Based on a comparative analysis of regulatory documents and of the characteristics and behavior of E. coli K-12 MG1655 and GMM under the influence of environmental factors, it is possible to state that the application of precision genomic editing using chromosomal recombination engineering is valid for the construction of microorganisms that meet international safety requirements in industrial biotechnology.

The ability of biogenic cadmium sulfide nanoparticles obtained by microbial synthesis to the photocatalytic decolorization of nine synthetic organic dyes (thiazine, dyes of triphenylmethane types, and azo dyes) under the effect of ultraviolet (UV) radiation (365 nm) is studied. Microbial synthesis is carried out using the strain Bacillus subtilis 168 (NPsCdS/Bacillus) under aerobic conditions at a temperature optimum for the bacterial strain. Characteristics of NPsCdS were determined by the following methods: transmission electron microscopy with analysis of the elemental composition, spectrophotometry, dynamic light scattering, and mass spectroscopy. The dyes of the triphenylmethane types: brilliant green, malachite green, and bromocresol purple, are shown to be most effectively decolorized. The dynamics of dye decolorization depending on the time and power of UV irradiation, as well as the concentration of NPsCdS/Bacillus in the solution, are studied. The possibility of the three-fold efficient use of NPsCdS/Bacillus for the decolorization of methylene blue and brilliant green is shown. This indicates the photostability of the nanomaterial obtained by microbial synthesis and makes it promising for ecological applications.

Introduction. The productivity of microbial synthesis of stable nanoparticles is determined by the growth stage of the populations of bacterial cultures used to obtain nanostructures. The study of the biocidal activity of biogenic nanoparticles of cadmium sulfide (NPsCdS), comparable in properties with nanomaterials obtained by physicochemical methods, is promising.The aim of this work was to evaluate the effect of the cell growth phase of the bacterial strains Bacillus subtilis 168 and Shewanella oneidensis MR-1 on the efficiency of biosynthesis of NPsCdS and to study their bactericidal properties against a number of gram-positive and gram-negative strains of microorganisms.Material and methods. Nanoparticles were obtained by introducing Na2S and CdCl2 salts to a final concentration of 2 mM : 2 mM in liquid bacterial cultures with cells in different phases of growth. The efficiency of NPsCdS biosynthesis was evaluated by the optical density of aqueous nanoparticles solutions. The bactericidal properties of NPsCdS were determined by the diameter of zone of inhibition growth of gram-positive bacteria B. subtilis 168, B. amyloliquefaciens, Streptococcus salivarius, Rhodococcus rhodochrous and gram-negative S. oneidensis MR-1, Escherichia coli K-12, Pseudomonas putida.Results. It was found that the use of cells in the stationary phase of growth (18–24 hours) contributes to obtaining the maximum amount of NPsCdS corresponding to concentrations of 1.0–1.2 mg/ml. The high antimicrobial activity of NPsCdS was shown against gram-positive microorganisms, among gram-negative bacteria, P. putida strain showed insignificant sensitivity.Discussion. The experimental results expand scientific data about the effect of the phase of bacterial growth cycle on biosynthesis of nanoparticles. The stationary phase of growth of B. subtilis 168, S. oneidensis MR-1 is optimal for obtaining of NPsCdS. For the first time, the cytotoxicity of NPsCdS/Shewanella against bacteria of various taxonomic groups was demonstrated.Conclusion. An effective method for obtaining extracellular NPsCdS using bacteria B. subtilis 168, S. oneidensis MR-1 in the stationary phase of growth has been developed. The biocidal activity of biogenic NPsCdS was shown, which allows to consider them as a new class of antimicrobial agents.

Cadmium sulfide (CdS) and zinc sulfide (ZnS) biogenic nanoparticles (NPs) were produced by microbial synthesis using bacteria of different taxonomic groups: Gram-negative (Shewanella oneidensis MR-1) and Gram-positive (Bacillus subtilis 168) bacteria in a liquid medium under aerobic conditions in the presence of salts of the respective metals and sulfur. It was shown that the stabilization of nanoparticles in aqueous suspensions is due to the presence of certain protein molecules of the outer membrane of cells, that is, proteins of the families of various receptors, porins, and flagellin, on the nanoparticle (NP) surface. The effect of the protein coating on stability, luminescence, zeta-potential, hydrodynamics diameter and other physiochemical characteristics of nanoparticles was studied. Decolorization of methylene blue dye under the exposure to UV irradiation was used as a model to demonstrate the photocatalytic properties of NPsCdS. This opens the possibility of using biogenic nanoparticles in photocatalysis for industrial wastewater treatment.

NpCdS nanocrystals are prepared by a microbial synthesis technique at the NRC “Kurchatov Institute”—GOSNIIGENETIKA. The stabilizing layer of nanocrystals consists of proteins, and the composition of the protein layer depends on the strain used in nanoparticle (NP) biosynthesis. The morphology and size, hydrodynamic diameter, zeta-potential, and luminescence properties of the biogenic NPs are investigated using electron microscopy, dynamic light scattering, and spectrofluorimetry, and the NPs are identified as quantum dots. The effects that temperature, pressure, and solvents have on the stability and luminescence intensity of biogenic NPs are studied in collaboration with the National Research Center Kurchatov Institute—IREA. For the aqueous NpCdS suspension, the dependence of luminescence intensity on the NP concentration range is established. The feasibility of incorporation and identification of NpCdS in an epoxy resin, polyimide, and polyvinyl alcohol is evaluated. Polymer nanocomposites find use in optoelectronics, biomedicine, and agriculture.

At the NRC “Kurchatov Institute” – GOSNIIGENETIKA, NpCdS nanocrystals were obtained by microbial synthesis. They were stabilized with proteins, which composition is determined by the strain used for biosynthesis of nanoparticles. Biogenic nanoparticles were studied and described by size, shape, hydrodynamic diameter, ζ potential, luminescence level, and defined as quantum dots applying methods of electron microscopy, dynamic light scattering, and spectrofluorimetry. The influence of temperature, pressure and solvents on the stability of biogenic nanoparticles and the luminescence intensity was evaluated in collaboration with IREA (NRC “Kurchatov Institute”). The luminescence intensity of the aqueous suspension of NpCdS was determined depending on the range of nanoparticle concentrations. The possibility of introducing and identifying NpCdS in epoxy resin, polyimide, and polyvinyl alcohol was assessed. Polymer nanocomposites are used for optoelectronic, biomedical and agricultural applications.