In Vitro Culture Alters Cell Lineage Composition and Cellular Metabolism of Bovine Blastocyst.

Profiling bovine blastocyst transcriptome at the single-cell level has enabled us to reveal the first cell lineage segregation, during which the inner cell mass (ICM), trophectoderm (TE), and an undefined population of transitional cells were identified. By comparing the transcriptome of blastocysts derived in vivo (IVV), in vitro from a conventional culture medium (IVC), and in vitro from an optimized reduced nutrient culture medium (IVR), we found a delay of the cell fate commitment to ICM in the IVC and IVR embryos. Developmental potential differences between IVV, IVC, and IVR embryos were mainly contributed by ICM and transitional cells. Pathway analysis of these non-TE cells between groups revealed highly active metabolic and biosynthetic processes, reduced cellular signaling, and reduced transmembrane transport activities in IVC embryos that may lead to reduced developmental potential. IVR embryos had lower activities in metabolic and biosynthetic processes but increased cellular signaling and transmembrane transport, suggesting these cellular mechanisms may contribute to improved blastocyst development compared to IVC embryos. However, the IVR embryos had compromised development compared to IVV embryos with notably over-active transmembrane transport activities that impaired ion homeostasis.

Study of the polysaccharide production by the microalgae C-1509 Nannochloris sp. Naumann.

Biologically active compounds, including polysaccharides isolated from microalgae, have various properties. Although Nannochloropsis spp. have the potential to produce secondary metabolites important for biotechnology, only a small part of the research on these microalgae has focused on their ability to produce polysaccharide fractions. This study aims to evaluate the physicochemical growth factors of Nannochloropsis spp. microalgae, which ensure the maximum accumulation of polysaccharides, as well as to optimize the parameters of polysaccharide extraction. The optimal nutrient medium composition was selected to maximize biomass and polysaccharide accumulation. The significance of selecting the extraction module and extraction temperature regime, as well as the cultivation conditions (temperature and active acidity value) is emphasized. Important chemical components of polysaccharides responsible for their biological activity were identified.

Comparison of protein profiles obtained by matrix-assisted laser desorption/ionization-Time-of-flight mass spectrometry in representatives of the family Mycobacteriaceae grown on various nutrient media.

Background: The nutrient medium effects on the quality of the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-ToF) mass spectra. The standard library includes spectra of microorganisms of the family Mycobacteriaceae grown on the Lowenstein-Jensen and Middlebrook Media. There are new methods for culturing microorganisms from this group, including inoculation on chromogenic media. Methods: The study included 240 strains of NTM isolated from patients during tuberculosis examination. The inoculation of the biological material was carried out on solid culture media of Lowenstein-Jensen and universal chromogenic media. Identification of bacteria from both types of media was performed by MALDI-ToF mass spectrometry (Bruker Daltonik GmbH, Germany). Analysis of protein spectra was performed. Results: For all strains, the spectra revealed both coinciding peaks (regardless of the cultivation medium) and significant differences, including the complete absence of some peaks depending on the medium. The results of a greater divergence of peaks in mass and intensity were obtained for slow-growing species than for fast-growing species. For all analyzed cultures, the number of peaks in the mass spectra was significantly higher when cultivating on a universal chromogenic medium than on a Lowenstein-Jensen medium. Conclusions: The use for NTM cultivation of a universal chromogenic medium makes it possible to obtain acceptable identification results by MALDI-ToF mass spectrometry using a standard library.

In Vitro Culture Alters Cell Lineage Composition and Cellular Metabolism of Bovine Blastocyst.

Profiling transcriptome at single cell level of bovine blastocysts derived in vivo (IVV), in vitro from conventional culture medium (IVC), and reduced nutrient culture medium (IVR) has enabled us to reveal cell lineage segregation, during which forming inner cell mass (ICM), trophectoderm (TE), and an undefined population of transitional cells. Only IVV embryos had well-defined ICM, indicating in vitro culture may delay the first cell fate commitment to ICM. Differences between IVV, IVC and IVR embryos were mainly contributed by ICM and transitional cells. Pathway analysis by using the differentially expressed genes of these non-TE cells between groups pointed to highly active metabolic and biosynthetic processes, with reduced cellular signaling and membrane transport in IVC embryos, which may lead to reduced developmental potential. IVR embryos had lower activities in metabolic and biosynthetic processes, but increased cellular signaling and membrane transport, suggesting these cellular mechanisms may contribute to the improved blastocyst development compared to IVC embryos. However, the IVR embryos had compromised development when compared to IVV embryos with notably over-active membrane transport activities that led to impaired ion homeostasis.

Modelling of Lemna minor L. growth as influenced by nutrient supply, supplemental light, CO2 and harvest intervals for a continuous indoor cultivation.

Given the proper conditions, Lemna spp. rapidly produce a high amount of valuable biomass which is considered as an alternative source for feed and food. For a continuous and long-term indoor production under controlled conditions, environmental and harvest parameters have to be optimized to suppress algal growth and constantly yield a high-quality product. Experimentally assessing the effect of a larger number of parameters on the growth rate ri is impossible due to the theoretically high number of parameter combinations. Thus, a SIMILE® - based model has been developed. This enables production parameters to be assessed individually for its effect on the growth rate r i by a differential equation. Start values for numerical integration were taken from measured data and analytical solutions of the differential growth equation. At 400 ppm CO2, the regrowth rate ri in an optimized laboratory set-up amounted to 216 g FM·m-2d-1, harvesting one third of the biomass at intervals of 5 days. In up-scaled set-ups, lower regrowth rates ri of about 173 g FM·m-2d-1 (Kalkar) and 190 g FM·m-2d-1 (Berlin) were obtained, because temperature and light conditions were below optimum. At 3,500 ppm CO2, the regrowth rate ri in laboratory set-up increased to 323 g FM·m-2d-1 by shortening the harvest interval to three days. Maximum growth rates ri were obtained with an NH4 +/NO3 - ratio of 1/9 at 1.14 mM total N concentration. The results indicate how to optimize culture conditions and harvest intervals. Model runs closely match the experimental data taken from the three different approaches and thus confirm the validity of the model.

Study of the Polysaccharide Production by the Microalga Vischeria punctata in Relation to Cultivation Conditions.

Vischeria punctata is a unicellular microalga that has industrial potential, as it can produce substances with beneficial properties. Among them, endopolysaccharides (accumulated in cells) and exopolysaccharides (released by cells into the culture medium) are of particular interest. This study aimed to investigate the effect of nutrient medium composition on the growth of V. punctata biomass and the synthesis of polysaccharides by microalgae. The effect of modifying a standard nutrient medium and varying cultivation parameters (temperature, time, and extractant type) on the yield of exopolysaccharides produced by the microalgae V. punctate was investigated. The methods of spectrophotometry, ultrasonic extraction, and alcohol precipitation were used in the study. It was found that after 61 days of cultivation, the concentration of polysaccharides in the culture medium was statistically significantly higher (p <0.05) when using a Prat nutrient medium (984.9 mg/g d.w.) than BBM 3N (63.0 mg/g d.w.). It was found that the increase in the V. punctata biomass when cultivated on different nutrient media did not differ significantly. The maximum biomass values on Prat and BBM 3N media were 1.101 mg/g d.w. and 1.120 mg/g d.w., respectively. Neutral sugars and uronic acids were found in the culture media. It follows on from the obtained data that the modified PratM medium was more efficient for extracting polysaccharides from V. punctata. The potential of microalgae as new sources of valuable chemicals (polysaccharides), which can be widely used in technologies for developing novel functional foods, biologically active food supplements, and pharmaceutical substances, was studied.

Development and study of nutrient media for determining the biological properties of Listeria.

The commercial nutrient media for investigation of the biologic properties of L. monocytogenes is developed. The efficiency of the use of developed media for determination of motility and lecithinase activity of Listeria in the establishment of the isolated culture to the pathogenic species is shown. These nutrient media are developed for visually accurate registration of the motility and lecithinase activity of Listeria when identifying the isolated cultures improving the diagnostics of listeriosis.

Features of the Formation of Communities of Microorganisms Adapting to the Existing Conditions in Different Types of Agrophytocenoses.

The article is devoted to studying the features of the formation of communities of microorganisms, adapting to the conditions of existence in various types of agrophytocenoses. The study was carried out on model weed plant species, the most common in agrophytocenoses of the Belgorod region. Two nutrient media were prepared for microbiological examination: nutrient agar (BPA - the number of mesophilic aerobic and facultative anaerobic microorganisms) and ENDO - an elective medium for CB (Escherichia coli bacteria). It was found that up to 80% of the total numbers of epiphytes are Erwinia herbicola (Pseudomonas herbicola) cells. The most common among the epiphytic bacteria of terrestrial higher weeds are traditional representatives of cosmopolitan bacteria such as Pseudomonas fluorescens, Ps. furbicolaaurum, Ps. putida, Pantoea agglomerans, Arthrobacter flavescens, A. album, Lactobacillus Plantarum, Васillus subtilis, В. megaterium, Klebsiella rosea, Agrobacterium.

Influence of the Nitrate-N to Ammonium-N Ratio on Relative Growth Rate and Crude Protein Content in the Duckweeds Lemna minor and Wolffiella hyalina.

In order to produce protein-rich duckweed for human and animal consumption, a stable cultivation process, including an optimal nutrient supply for each species, must be implemented. Modified nutrient media, based on the N-medium for duckweed cultivation, were tested on the relative growth rate (RGR) and crude protein content (CPC) of Lemna minor and Wolffiella hyalina, as well as the decrease of nitrate-N and ammonium-N in the media. Five different nitrate-N to ammonium-N molar ratios were diluted to 10% and 50% of the original N-medium concentration. The media mainly consisted of agricultural fertilizers. A ratio of 75% nitrate-N and 25% ammonium-N, with a dilution of 50%, yielded the best results for both species. Based on the dry weight (DW), L. minor achieved a RGR of 0.23 ± 0.009 d-1 and a CPC of 37.8 ± 0.42%, while W. hyalina's maximum RGR was 0.22 ± 0.017 d-1, with a CPC of 43.9 ± 0.34%. The relative protein yield per week and m2 was highest at this ratio and dilution, as well as the ammonium-N decrease in the corresponding medium. These results could be implemented in duckweed research and applications if a high protein content or protein yield is the aim.

Effects of Conventional and Bokashi Hydroponics on Vegetative Growth, Yield and Quality Attributes of Bell Peppers.

Due to consumers' awareness and concern about nutrition and health in different parts of the world, the adoption of organic hydroponics is increasing. This has led to a search for organic nutrient media. One of the viable nutrient sources for organic hydroponics is bokashi compost. The principal objective of this study was to compare the performance of 10% bokashi hydroponics with convention hydroponics for bell pepper production. The different hydroponics influenced vegetative growth parameters largely due to considerable differences in the mineral elements in both hydroponic systems. Stems of conventionally grown plants were significantly (p ≤ 0.05) thicker (10.2 mm) compared to those of the bokashi grown plants (7.3 mm). Conventionally grown plants had significantly (p ≤ 0.05) higher photosynthetic performance than bokashi grown plants; normalized difference vegetation index (NDVI) (78.80 versus 67.49), soil plant analysis development (SPAD; 73.89 versus 38.43), and quantum yield (QY; 0.64 versus 0.49). Leaf superoxide dismutase (SOD) activity in the leaves of bokashi grown plants (0.32 units/mg protein) was significantly (p ≤ 0.05) lower than in the leaves of conventionally grown plants (0.37 units/mg protein). This also corresponded to significantly (p ≤ 0.05) higher leaf sap content in the conventionally grown plant than bokashi grown plants. Furthermore, conventional hydroponics yielded three-fold greater pepper fruit per plant compared to bokashi. After 14 days of storage at 7 °C and 95% relative humidity, the firmness of both groups declined, especially for the bokashi grown fruit (27.73 shore unit), which was significantly lower compared to conventionally grown fruit (35.65 shore unit). However, there was an increase in carotenoid content in fruit grown in both hydroponic systems after storage. In conclusion, although bell pepper plant was successfully cultivated in bokashi hydroponics, the plant performance, fruit yield and postharvest quality were lower than conventional hydroponics. We believe that this study and its approach will provide future research with baseline information on optimizing media of bokashi hydroponics to produce bell pepper.

Plant Tissue Cultures.

Plant tissue cultures are an efficient system to study cell wall biosynthesis in living cells in vivo. Tissue cultures also provide cells and culture medium from which enzymes and cell wall polymers can easily be separated for further studies. Tissue cultures with tracheary element differentiation or extracellular lignin formation have provided useful information related to several aspects of xylem and lignin formation. In this chapter, methods for nutrient medium preparation and callus culture initiation and its maintenance as well as those for protoplast isolation and viability observation are described. As a case study, we describe the establishment of a xylogenic culture of Zinnia elegans mesophyll cells.

[Trials of the domestically produced nutrient medium «Agar Muller-Hinton II - Obolensk¼.]

The results of the comparative tests of the «Agar Muller-Hinton II - Obolensk¼ nutrient medium developed in SRCAMB, Obolensk, and the control nutrient medium imported «Mueller Hinton II Agar¼ are presented in the study. The susceptibility of bacterial clinical strains to antimicrobial agents (AMP) was determined by the disc diffusion method and the method of gradient diffusion (E-test). The carbapenemase activity of the strains carrying the carbapenemase genes was determined by CIM-test. Total 173 characterized bacterial strains of species Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Serratia marcescens, Enterobacter aerogenes, Escherichia coli; Photorhabdus spp., Staphylococcus aureus, Enterococcus spp. were used in the study, including producers of OXA- and NDM-types carbapenemases for gram negative bacteria. A high degree of coincidence of the results obtained on both nutrient media was shown. The consistency index of the strain sensitivity categories to AMPs (S, I, and R) was 98.2% for the disc diffusion method, and 94.4-100% - for E-test and CIM-test methods. Thus, within the framework of the Import Substitution Program, the domestic nutrient medium «MHA II-Obolensk¼ has been successfully developed. The nutrient medium meets the requirements of GOST R ISO 20776-2-2010 «Clinical laboratory testing and in vitro diagnostic test systems - Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices¼.

Effect of Nutrient Sources on the Alginate Accumulation in the Culture Liquid of Azotobacter vinelandii D-05 and Obtaining Biocomposite Materials

ABSTRACT Using the classic biotechnological methods, the dependence of A. vinelandii D-05 culture alginate production from the media carbon and nitrogen content was investigated. The maximal alginate production was observed during cultivation bacterium in the medium with 2 to 4% of sucrose, but the maximal growth was found in the medium with 4% glucose. It was found that for the alginate production the optimal nitrogen contents could take from 0.05% yeast extract (carbon: nitrogen ratio 168:1). For the first time we demonstrated possibility the A. vinelandii growth during the cultivation in a medium with molasses (a by-product of sugar production) and the significant polysaccharide production (16.6 g/l) was obtained. It was established, that A. vinelandii culture broth could be used as a biological binder for obtaining the biocomposite materials.

High frequency plant regeneration with histological analysis of organogenic callus from internode explants of Asteracantha longifolia Nees.

Asteracantha longifolia Nees is an ayurvedic medicinal herb. The internode explants of this plant were used for high frequency plant regeneration on Murashige and Skoog (MS) medium supplemented with various plant growth regulators (PGRs) in different concentrations. Apical meristem and leaf primordium formations were confirmed through microscopic analysis of histological sections of the organogenic callus tissues. The synergistic effect of α-naphthaleneacetic acid (NAA) 0.5 mg/l with N 6 benzyladenine (BA) 0.25 mg/l increased the percentage of explants response for callus induction while comparing other treatments. Various concentrations of NAA were also found to be best for explants response to callus induction than 2,4-dichlorophenoxyacetic acid (2,4-D). The callus morphology (color and texture) was different according to the growth regulators and their concentrations. The highest percentage of response per culture for shoot bud regeneration was noted for the concentration of NAA 0.5 mg/l with BA 2.0 mg/l, the same concentration effectively increased the number of shoots per culture. Different concentrations of indol-3-butyric acid (IBA) and NAA were used in half strength MS medium for in vitro rooting of regenerated shoots. The maximum percentage of shoot response for rooting and the highest number of root formations per shoot were observed on the medium containing 0.5 mg/l of IBA. The survival rate (86.7%) of the regenerated plants was noted after 20 days of transplantation.

Extractability of polyhydroxyalkanoate synthesized by Bacillus flexus cultivated in organic and inorganic nutrient media.

Bacillus flexus was isolated from local soil sample and identified by molecular methods. In inorganic nutrient medium (IM) containing sucrose as carbon source, yield of biomass and polyhydroxyalkanoate (PHA) were 2 g/l and 1 g/l (50% of biomass), respectively. Substitution of inorganic nitrogen by peptone, yeast extract or beef extract resulted in biomass yields of 4.1, 3.9 and 1.6 g/l, respectively. Corresponding yields of PHA in biomass was 30%, 40% and 44%. Cells subjected to change in nutrient condition from organic to inorganic, lacked diaminopimelic acid in the cell wall and the concentration of amino acids also decreased. Under these conditions the extractability of the polymer from the cells by hot chloroform or mild alkali hydrolysis was 86-100% compared to those grown in yeast extract or peptone (32-56%). The results demonstrated that growth, PHA production and the composition of cell wall of B. flexus are influenced by the organic or inorganic nutrients present in the growth medium. Cells grown in inorganic medium lysed easily and this can be further exploited for easier recovery of the intracellular PHA.