A simple procedure for preparing biotinylated immunoglobulin M from hybridoma culture medium.

We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N',N'-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.

A liquid static culture using a gas-permeable film bag contributes to microbiology.

We propose a simple tool for liquid static culture using a copolymer film with high gas permeability. The film bags were successfully used to culture microorganisms Escherichia coli, Komagataella phaffii (methylotrophic) and Bacillus sp. (biofilm-forming), with cells cultured under physical stress-free conditions with sufficient oxygen supply. Similar growth curves and plasmid productivity were observed for liquid shake and film bag E. coli cultures. The early growth response of the film bag culture following colony inoculation of liquid media differed from conventional shake cultures. Our results indicate that a gas-permeable film bag is a promising liquid culture tool and provides novel microbiology materials.

Adding-value to Ganoderma lingzhi by producing enzymes and antioxidant compounds under submerged fermentation using different culture media.

Ganoderma lingzhi is widely reported for its medicinal properties, presenting several bioactive substances with potential pharmaceutical and industrial application. This study aimed to evaluate the production of mycelial biomass, extracellular enzymes and antioxidant compounds by G. lingzhi under submerged fermentation. G. lingzhi was cultured in Polysaccharide (POL) and Melin-Norkrans (MNM) media for 7 days. The cellulases, xylanases, pectinases, laccases, and proteases activities were quantified in the culture broth, while the antioxidant potential was evaluated for the mycelial biomass. G. lingzhi showed higher biomass production in MNM. However, it exhibited similar microstructural characteristics in both culture media. In the POL there was greater activity of CMCase (0.229 U/mL), xylanase (0.780 U/mL), pectinase (0.447 U/mL) and proteases (16.13 U/mL). FPase did not differ (0.01 U/mL), and laccase was detected only in MNM (0.122 U/mL). The biomass water extract from MNM showed high levels of phenolic compounds (951.97 mg AGE/100 g). DPPH• inhibition (90.55%) and reducing power (0.456) were higher in MNM medium, while ABTS•+ inhibition (99.95%) and chelating ability (54.86%) were higher in POL. Thus, the MNM medium was more favorable to the production of mycelial biomass and phenolic compounds, while the POL medium favored the synthesis and excretion of hydrolytic enzymes.

The yeast Dothiora sorbi IOJ-3 naturally produced various filamentous sectors with distinct abilities by undergoing DNA demethylation.

Some fungi have demonstrated the ability to adapt rapidly to changing environments by exhibiting morphological plasticity, a trait influenced by species and environmental factors. Here, an anamorphic yeast strain IOJ-3 exhibited unique sectorization characteristics, naturally producing diverse filamentous sectors when cultivated on potato dextrose agar (PDA) medium or natural culture medium for durations exceeding 13 days. The strain IOJ-3 and its filamentous sectors were identified as Dothiora sorbi. The morphology of the sectors was consistent and heritable. The life cycle of strain IOJ-3 was investigated through microscopic observation, emphasizing the development of conidiogenous cells as a crucial stage, from which filamentous sectors originate. Some physiological characteristics of IOJ-3 and filamentous sectors are compared, and strain IOJ-3 has a higher antibiotic tolerance than two filamentous sectors, IOJ-3a expands faster on the culture medium, and IOJ-3b can penetrate cellophane. A transcriptomic analysis was conducted to investigate the differentially expressed genes between the yeast form IOJ-3 and its two filamentous sectors, revealing a total of 594 genes that exhibited consistent differential expression relative to IOJ-3, including 44 silencing genes in IOJ-3 that were activated. Gene Ontology analysis indicated that these differentially expressed genes were primarily associated with the cellular component category. Furthermore, adding 5-Azacytidine accelerated filamentous sectorization and increased the proportion of filamentous cells of strain IOJ-3 in PD liquid media, suggesting that the filamentous sectorization observed in strain IOJ-3 is linked to processes of DNA demethylation. In conclusion, this study sheds light on the biological characteristics of D. sorbi regarding morphological transitions and provides substantial direction for exploring genes related to fungal filamentous development.

Cadmium and calcium ions' effects on the growth of Pleurotus ostreatus mycelia are related to phosphatidylethanolamine content.

Heavy metal Cd2+ can easily be accumulated by fungi, causing significant stress, with the fungal cell membrane being one of the primary targets. However, the understanding of the mechanisms behind this stress remains limited. This study investigated the changes in membrane lipid molecules of Pleurotus ostreatus mycelia under Cd2+ stress and the antagonistic effect of Ca2+ on this stress. Cd2+ in the growth media significantly inhibited mycelial growth, with increasing intensity at higher concentrations. The addition of Ca2+ mitigated this Cd2+-induced growth inhibition. Lipidomic analysis showed that Cd2+ reduced membrane lipid content and altered lipid composition, while Ca2+ counteracted these changes. The effects of both Cd2+ and Ca2+ on lipids are dose dependent and phosphatidylethanolamine appeared most affected. Cd2+ also caused a phosphatidylcholine/phosphatidylethanolamine ratio increase at high concentrations, but Ca2+ helped maintain normal levels. The acyl chain length and unsaturation of lipids remained unaffected, suggesting Cd2+ doesn't alter acyl chain structure of lipids. These findings suggest that Cd2+ may affect the growth of mycelia by inhibiting the synthesis of membrane lipids, particular the synthesis of phosphatidylethanolamine, providing novel insights into the mechanisms of Cd2+ stress in fungi and the role of Ca2+ in mitigating the stress.

In vitro culture and morphological observation of human eye demodex mites.

The prevalence of human Demodex mites has surged in recent years, prompting significant concern among both patients and the medical community. This study aimed to investigate the survival duration and morphological alterations of Demodex folliculorum under diverse temperature conditions and in various culture media. We employed the eyelash sampling technique to procure the mites. The collected specimens were then subjected to culture at two distinct temperature ranges (16-22 °C and 4 °C) across a spectrum of media, including 30% tea tree oil (TTO), phosphate-buffered saline (PBS), pure water, 0.9% physiological saline, 5 µg/ml propidium iodide (PI), liquid paraffin, glycerol, and a blank culture medium. Post-administration, the mites' activity and morphological changes were meticulously documented. Our findings indicate that the survival span of Demodex mites within the same medium was notably extended at 4 °C compared to room temperature. Specifically, under 4 °C, the use of liquid paraffin as a culture medium yielded the longest survival time of 12 days, surpassing all other conditions. Remarkably, the morphological integrity of the mites in this group remained largely unaltered. These results suggest that 4 °C is the optimal temperature for the in vitro cultivation of Demodex mites, offering insights into the environmental preferences of these organisms and potentially informing future therapeutic strategies.

Conditional lethality profiling reveals anticancer mechanisms of action and drug-nutrient interactions.

Chemical screens across hundreds of cell lines have shown that the drug sensitivities of human cancers can vary by genotype or lineage. However, most drug discovery studies have relied on culture media that poorly reflect metabolite levels in human blood. Here, we perform drug screens in traditional and Human Plasma-Like Medium (HPLM). Sets of compounds that show conditional anticancer activity span different phases of global development and include non-oncology drugs. Comparisons of the synthetic and serum-derived components that comprise typical media trace sets of conditional phenotypes to nucleotide synthesis substrates. We also characterize a unique dual mechanism for brivudine, a compound approved for antiviral use. Brivudine selectively impairs cell growth in low folate conditions by targeting two enzymes involved in one-carbon metabolism. Cataloged gene essentiality data further suggest that conditional phenotypes for other compounds are linked to off-target effects. Our findings establish general strategies for identifying drug-nutrient interactions and mechanisms of action by exploiting conditional lethality in cancer cells.

Improvement strategies for transient gene expression in mammalian cells.

Mammalian cells are suitable hosts for producing recombinant therapeutic proteins, with Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells being the most commonly used cell lines. Mammalian cell expression system includes stable and transient gene expression (TGE) system, with the TGE system having the advantages of short cycles and simple operation. By optimizing the TGE system, the expression of recombinant proteins has been significantly improved. Here, the TGE system and the detailed and up-to-date improvement strategies of mammalian cells, including cell line, expression vector, culture media, culture processes, transfection conditions, and co-expression of helper genes, are reviewed. KEY POINTS: • Detailed improvement strategies of transient gene expression system of mammalian cells are reviewed • The composition of transient expression system of mammalian cell are summarized • Proposed optimization prospects for transient gene expression systems.

Using Mycobacterium smegmatis as a Bioindicator for Zinc-limited Growth Conditions in Mycobacteria.

Many bacteria build alternative ribosomes in Zn2+-limiting growth conditions by replacing Zn2+-binding ribosomal proteins with Zn2+-independent paralogs. Defining a system to study these alternative ribosomes has proven difficult because Zn2+ contamination in the laboratory is common. To address this issue, chelating agents are sometimes added to growth media, but this approach convolutes the biological response to gradual Zn2+ limitation and is associated with ribosome hibernation. Here, detailed instructions are outlined for preparing media and seeding cultures for Zn2+-limited growth without adding chelators. Following this method, the model bacterium, Mycobacterium smegmatis, undergoes morphogenesis, which depends on alternative ribosomes. Because morphogenesis is tractable and only occurs in Zn2+-limiting conditions, M. smegmatis can be used as a bioindicator to verify biologically relevant growth conditions. Three bioindicator phenotypes (cell density, cell length, and coenzyme F420 fluorescence) that indicate Zn2+ limitation in the wild-type are described, and changes in these bioindicators for a deletion mutant that cannot build alternative ribosomes are outlined. Since trace Zn2+ contamination is difficult to control for each batch of media, and precise quantification of Zn2+ in each media preparation is overly burdensome, following this bioindicator phenotype is an accessible way to validate the preparation of Zn2+-limited growth media. To help identify proper conditions for Zn2+-limiting growth and alternative ribosome production, changes in the bioindicator phenotypes were profiled for Zn2+-contaminated or severely Zn2+-depleted preparations of Zn2+-limited media as well. Further details to achieve Zn2+-limiting growth and alternative ribosome production in M. tuberculosis are presented, along with the associated bioindicator phenotype. Overall, the detailed instructions and bioindicator phenotypes described here will help standardize the production of translationally active alternative ribosomes in mycobacteria.

Novel and effective screening system for recombinant protein production in CHO cells.

At present, biopharmaceuticals have received extensive attention from the society, among which recombinant proteins have a good growth trend and a large market share. Chinese hamster ovary (CHO) cells are the preferred mammalian system to produce glycosylated recombinant protein drugs. A highly efficient and stable cell screening method needs to be developed to obtain more and useful recombinant proteins. Limited dilution method, cell sorting, and semi-solid medium screening are currently the commonly used cell cloning methods. These methods are time-consuming and labor-intensive, and they have the disadvantage of low clone survival rate. Here, a method based on semi-solid medium was developed to screen out high-yielding and stable cell line within 3 weeks to improve the screening efficiency. The semi-solid medium was combined with an expression vector containing red fluorescent protein (RFP) for early cell line development. In accordance with the fluorescence intensity of RFP, the expression of upstream target gene could be indicated, and the fluorescence intensity was in direct proportion to the expression of upstream target gene. In conclusion, semi-solid medium combined with bicistronic expression vector provides an efficient method for screening stable and highly expressed cell lines.

Analysis of secondary metabolites and morphology in Streptomyces rimosus microparticle-enhanced cultivation (MPEC) at various initial organic nitrogen concentrations.

The influence of talc microparticles on metabolism and morphology of S. rimosus at various initial organic nitrogen concentrations was investigated. The shake flask cultivations were conducted in the media with yeast extract (nitrogen source) concentration equal to 1 g YE L- 1 and 20 g YE L- 1. Two talc microparticle concentrations of 5 g TALC L- 1 and 10 g TALC L- 1 were tested in microparticle-enhanced cultivation (MPEC) runs. A high nitrogen concentration of 20 g YE L- 1 promoted the development of small agglomerates (pellets) of projected area lower than 105 µm2 and dispersed pseudohyphae. A low nitrogen concentration of 1 g YE L- 1 led to the limitation of S. rimosus growth and, in consequence, the development of the smaller number of large pseudohyphal agglomerates (pellets) of projected area higher than 105 µm2 compared to the culture containing a high amount of nitrogen source. In both cases talc microparticles were embedded into pellets and caused the decrease in their sizes. The lower amount of talc (5 g TALC L- 1) usually caused the weaker effect on S. rimosus morphology and metabolite production than the higher one. This correlation between the microparticles effect on morphology and metabolism of S. rimosus was especially noticeable in the biosynthesis of oxytetracycline, 2-acetyl-2-dicarboxamide oxytetracycline (ADOTC) and spinoxazine A. Compared to the control run, in MPEC their levels increased 4-fold, 5-fold and 1.6-fold respectively. The addition of talc also improved the production of 2-methylthio-cis-zeatin, lorneic acid J and milbemycin A3.

Cultured fecal microbial community and its impact as fecal microbiota transplantation treatment in mice gut inflammation.

The fecal microbiome is identical to the gut microbial communities and provides an easy access to the gut microbiome. Therefore, fecal microbial transplantation (FMT) strategies have been used to alter dysbiotic gut microbiomes with healthy fecal microbiota, successfully alleviating various metabolic disorders, such as obesity, type 2 diabetes, and inflammatory bowel disease (IBD). However, the success of FMT treatment is donor-dependent and variations in gut microbes cannot be avoided. This problem may be overcome by using a cultured fecal microbiome. In this study, a human fecal microbiome was cultured using five different media; growth in brain heart infusion (BHI) media resulted in the highest microbial community cell count. The microbiome (16S rRNA) data demonstrated that the cultured microbial communities were similar to that of the original fecal sample. Therefore, the BHI-cultured fecal microbiome was selected for cultured FMT (cFMT). Furthermore, a dextran sodium sulfate (DSS)-induced mice-IBD model was used to confirm the impact of cFMT. Results showed that cFMT effectively alleviated IBD-associated symptoms, including improved gut permeability, restoration of the inflamed gut epithelium, decreased expression of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1, IL-6, IL-12, and IL-17), and increased expression of anti-inflammatory cytokines (IL-4 and IL-10). Thus, study's findings suggest that cFMT can be a potential alternative to nFMT. KEY POINTS: • In vitro fecal microbial communities were grown in a batch culture using five different media. • Fecal microbial transplantation was performed on DSS-treated mice using cultured and normal fecal microbes. • Cultured fecal microbes effectively alleviated IBD-associated symptoms.

Effect of histidine and L-Tyrosine supplementation in maturation medium on in-vitro developmental outcomes of buffalo oocytes.

The present study was designed to investigate the effects of amino acid (histidine and L-Tyrosine) on in vitro maturation (IVM), in vitro fertilization (IVF), cleavage (CR) rates, and in vitro embryonic cultivation (IVC; Morula and Blastocyst stage) in buffaloes. Within two hours of buffalo slaughter, the ovaries were collected and transported to the laboratory. Follicles with a diameter of 2 to 8 mm were aspirated to recover the cumulus oocyte complexes (COCs). Histidine (0.5, 1, and 3 mg/ml) or L-Tyrosine (1, 5, and 10 mg/ml) were added to the synthetic oviductal fluid (SOF) and Ferticult media. The IVM, IVF, CR, and IVC (Morula and Blastocyst) rates were evaluated. The results showed that SOF maturation media containing histidine at 0.5 mg/ml significantly (P ≤ 0.01) improved the oocyte maturation when compared to control and other concentrations. The addition of histidine to FertiCult media at 0.5, 1, and 3 mg/ml did not improve the IVM, IVF, CR, or IVC percentages. However, the embryos in the control group were unable to grow into a morula or blastocyst in the SOF or Ferticult, while addition of L-Tyrosine to the SOF or Ferticult at various concentrations improved IVC (morula and blastocyst rates). There was a significant (P ≤ 0.01) increase in IVM when histidine was added to SOF medium at a concentration of 0.5 mg/ml compared with L-Tyrosine. Also, there were significant (P ≤ 0.01) increases in IVC when L-Tyrosine was added to SOF medium at concentrations of 1 and 10 mg/ml compared with histidine. In conclusion, the supplementation of the SOF and FertiCult with the amino acids histidine and L-Tyrosine improve the maturation rate of oocytes and development of in vitro-produced buffalo embryos.

Kinetics of Riboflavin Production by Hyphopichia wangnamkhiaoensis under Varying Nutritional Conditions.

Riboflavin, an essential vitamin for humans, is extensively used in various industries, with its global demand being met through fermentative processes. Hyphopichia wangnamkhiaoensis is a novel dimorphic yeast species capable of producing riboflavin. However, the nutritional factors affecting riboflavin production in this yeast species remain unknown. Therefore, we conducted a kinetic study on the effects of various nutritional factors-carbon and energy sources, nitrogen sources, vitamins, and amino acids-on batch riboflavin production by H. wangnamkhiaoensis. Batch experiments were performed in a bubble column bioreactor to evaluate cell growth, substrate consumption, and riboflavin production. The highest riboflavin production was obtained when the yeast growth medium was supplemented with glucose, ammonium sulfate, biotin, and glycine. Using these chemical components, along with the mineral salts from Castañeda-Agullo's culture medium, we formulated a novel, low-cost, and effective culture medium (the RGE medium) for riboflavin production by H. wangnamkhiaoensis. This medium resulted in the highest levels of riboflavin production and volumetric productivity, reaching 16.68 mg/L and 0.713 mg/L·h, respectively, within 21 h of incubation. These findings suggest that H. wangnamkhiaoensis, with its shorter incubation time, could improve the efficiency and cost-effectiveness of industrial riboflavin production, paving the way for more sustainable production methods.

The antagonistic activity of Streptomyces spiroverticillatus (No. HS1) against of poplar canker pathogen Botryosphaeria dothidea.

BACKGROUND: Poplar canker caused by Botryosphaeria dothidea is one of the most severe plant disease of poplars worldwide. In our study, we aimed to investigate the modes of antagonism by fermentation broth supernatant (FBS) of Streptomyces spiroverticillatus HS1 against B. dothidea. RESULTS: In vitro, the strain and FBS of S. spiroverticillatus HS1 significantly inhibited mycelial growth and biomass accumulation, and also disrupted the mycelium morphology of B. dothidea. On the 3rd day after treatment, the inhibition rates of colony growth and dry weight were 80.72% and 52.53%, respectively. In addition, FBS treatment damaged the plasma membrane of B. dothidea based on increased electrical conductivity in the culture medium, and malondialdehyde content of B. dothidea mycelia. Notably, the analysis of key enzymes in glycolysis pathway showed that the activity of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK), Ca2+Mg2+-ATPase were significantly increased after FBS treatment. But the glucose contents were significantly reduced, and pyruvate contents were significantly increased in B. dothidea after treatment with FBS. CONCLUSIONS: The inhibitory mechanism of S. spiroverticillatus HS1 against B. dothidea was a complex process, which was associated with multiple levels of mycelial growth, cell membrane structure, material and energy metabolism. The FBS of S. spiroverticillatus HS1 could provide an alternative approach to biological control strategies against B. dothidea.

Characterization of Bacillus pacificus KUMBNGBT-39-Derived Polyhydroxybutyrate and Impact of Physicochemical Factors on its Production.

The extensive use of various chemicals in synthetic plastics is toxic and threatens the biosphere. To address this, the study aimed to isolate, screen, characterize, optimize, and quantify polyhydroxybutyrate (PHB)-producing bacteria using cost-effective residues. Isolated from a landfill site, the Gram-positive, rod-shaped, spore-forming, motile bacterium with intracellular PHB granules was identified as Bacillus pacificus based on phenotypic and genotypic characteristics. Optimal PHB production parameters included a nutrient broth medium, 72 h of incubation, a temperature of 37° C, a pH of 7.0, glucose as the carbon source, ammonium chloride as the nitrogen source, and a carbon-to-nitrogen ratio of 4:1, resulting in a 1.42-fold PHB production increase. B. pacificus was also cultured on various low-cost substrates. Among the oil wastes, feedstock showed the highest PHB production (1.983 ± 0.005 g/L) and among agricultural residues, the maximum PHB was obtained from rice bran (1.626 ± 0.01 g/L). UV-visible spectrophotometric, FT-IR, and HR-LCMS analysis of extracted PHB confirmed characteristics of PHB molecules (ʎ-max at 210 nm, functional groups between 1152 and 2925 cm-1). The 1H NMR analysis revealed distinct signals for protons resonating at aliphatic CH3 proton groups, bridged CH protons, and shielding CH2 proton regions that matched PHBs. Thermal gravimetric analysis (TGA) and direct scanning colorimetric (DSC) analysis revealed 89.4% degradation and melting temperature at 124.1 °C for the extracted PHB compound.

An efficient approach for overproduction of DNA polymerase from Pyrococcus furiosus using an optimized autoinduction system in Escherichia coli.

High fidelity DNA polymerase from Pyrococcus furiosus (Pfupol) is an attractive alternative to the highly popular DNA polymerase from Thermus aquaticus. Because this enzyme is in great demand for biotechnological applications, optimizing Pfupol production is essential to supplying the industry's expanding demand. T7-induced promoter expression in Escherichia coli expression systems is used to express recombinant Pfupol; however, this method is not cost-effective. Here, we have effectively developed an optimized process for the autoinduction approach of Pfupol expression in a defined medium. To better examine Pfupol's activities, its purified fraction was used. A 71 mg/L of pure Pfupol was effectively produced, resulting in a 2.6-fold increase in protein yield when glucose, glycerol, and lactose were added in a defined medium at concentrations of 0.05%, 1%, and 0.6%, respectively, and the condition for production in a 5 L bioreactor was as follow: 200 rpm, 3 vvm, and 10% inoculant. Furthermore, the protein exhibited 1445 U/mg of specific activity when synthesized in its active state. This work presents a high level of Pfupol production, which makes it an economically viable and practically useful approach.

Vitamin C enhances the in vitro development of early porcine embryos by improving mitochondrial function.

Mammalian embryos often suffer from oxidative stress in vitro, as the oxygen in the atmosphere is higher than that in the oviductal environment. Vitamin C (Vc) has been proven to enhance early embryonic development in vitro, but the underlying mechanism remains unclear. In this study, we investigated the pathways of action by which Vc promotes the in vitro development of porcine embryos. Comparative analysis of in vitro and in vivo gene expression profiles of morula found that most of the differentially expressed genes were enriched in pathways related to mitochondrial function. The addition of 12.5 µg/mL Vc to the culture medium significantly increased blastocyst production in a dose- and duration-dependent manner. Moreover, ROS levels were significantly higher in embryos cultured in the air (21% oxygen) than cultured in a hypoxic condition (5% oxygen) and were reduced by Vc supplementation. Vc also significantly increased the mitochondrial membrane potential levels and the expression levels of mitochondrial function-related genes (MFN1 and OPA1) and TCA cycle-related genes (PDHA1 and OGDH) in embryos cultured in vitro. These results suggest that the addition of Vc to the in vitro culture medium can increase the developmental potential and improve the mitochondrial function of early porcine embryos.

Comparative Analysis of the Mass Spectra of Mycobacterium abscessus Complex Strains Isolated on Various Nutrient Media.

BACKGROUND: Mycobacterium abscessus complex (MABSc) causes chronic infection in patients with concomitant structural changes in the respiratory tract, which is especially important for patients with cystic fibrosis. To isolate an MABSc culture from clinical material, a variety of nutrient media are used. For species determination of microorganisms isolated on these media, additional identification methods are used, for example, polymerase chain reaction, sequencing, or mass spectrometry. The latter method is relatively easy to implement but requires improvement, due to the identification inaccuracy of nontuberculosis mycobacterias in general. Consequently, a set of nutrient media may be important for subsequent identification by mass spectrometry. METHODS: The study was conducted on 64 strains of MABSc representatives: 56 strains were obtained from patients with cystic fibrosis and 8 strains from patients with pulmonary pathology unrelated to cystic fibrosis. The obtained MABSc strains were transplanted to the universal chromogenic medium and the selective medium for the Burkholderia cepacia complex (BCC) isolation. Species identification was carried out by mass spectrometry based on matrix-activated laser time-of-flight desorption/ionization (MALDI-ToF MS). Microbial identification is based on a comparison of the obtained mass spectra with reference spectra from the database. Microorganisms were identified based on the coincidence degree (Score value). Sample preparation for microbial identification by mass spectrometry was carried out by an extended direct application method. Fragments of the rpoB and hsp65 genes with lengths of 752 bp and 441 bp, respectively, were used as molecular markers for subspecific identification of MABSc strains. RESULTS: A comparison of the peaks obtained after mass spectrometry of MABSc strains isolated on the studied nutrient media showed significant differences between these indicators selective medium for the BCC isolation with the supplement of iron polymaltose hydroxide (III) and universal chromogenic medium (P < 0.001) and selective medium for the BCC isolation with universal chromogenic medium (P < 0.001). Twenty-five strains of MABSc representatives were sequenced: results of subspecies determination in strains isolated on the universal chromogenic medium coincided with the results sequencing in 13 (86.6%) strains out of 15. CONCLUSION: MALDI-ToF mass spectrometry allows microbial identification in a short time and with minimal cost, but it does not yet allow the proper identification of the subspecies of certain microbial groups, such as MABSc. Cultivation methods need optimization and new approaches to the extraction process of the bacterial protein fraction.

Oxygen Transfer Effect on the Growth of Limosilactobacillus reuteri ATCC 53608 and on Its Metabolic Capacity.

Limosilactobacillus reuteri is a probiotic microorganism used in the treatment of gastrointestinal disorders. The effect of oxygen transfer on cultures of L. reuteri ATCC 53608 at shake flask and stirred tank bioreactor scales was studied, using MRS and molasses-based media. At shake flask scale, in MRS medium, a maximum bacterial concentration of 2.01 ± 0.02 g L-1 was obtained; the oxygen transfer coefficient was 2.01 ± 0.04 h-1. Similarly, in a 7.5 L bioreactor, in MRS, a maximum bacterial concentration of 2.46 ± 0.16 g L-1 was achieved (kLa = 2.64 ± 0.06 h-1). In contrast, using a molasses-based medium, bacterial concentration reached 3.13 ± 0.17 g L-1 in the 7.5 L bioreactor. A progressive reduction in lactic acid concentration and yield was observed as the oxygen transfer coefficient increased, at shake flask scale. Also, the oxygen transfer coefficient strongly affected the growth of L. reuteri in shake flask and bioreactor and allowed us to successfully scale up L. reuteri culture, producing similar maximum bacterial concentrations in both scales (2.01 g L-1 and 2.46 g L-1 in MRS). This is the first study on oxygen transfer coefficients in L. reuteri, and it is a valuable contribution to the field as it provides important insights about how this organism tolerates oxygen and adapts its metabolism for larger biomass production.