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.

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.

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.

Enhancement of paclitaxel production by Neopestalotiopsis vitis via optimization of growth conditions.

Accessibility of paclitaxel and other taxoids from natural resources is restricted. Endophytic fungi are novel, rapidly growing resources for producing these compounds. Neopestalotiopsis vitis (N. vitis) has been recently isolated from Corylus avellana, and its ability to produce a variety of taxoids has been detected and confirmed by analytical methods. Simultaneous growth and high production of taxoids by application of different sorts and concentrations of carbon and nitrogen were targeted in the present research. These criteria were assessed in different acidities (pH 4.0-7.0), carbon sources (sucrose, fructose, glucose, mannitol, sorbitol, and malt extract), and nitrogen forms (urea, ammonium nitrate, potassium nitrate, ammonium phosphate, and ammonium sulfate) by testing one parameter at a time approach. The first analysis introduced pH 7.0 as the best acidity of the medium for N. vitis, where the highest paclitaxel yield was generated. Further analysis introduced 3% Malt extract as the best carbon-providing medium. In the next step, the effects of nitrogen forms on the growth rate, paclitaxel yield, alkaloids, and amino acid contents were evaluated. Based on the results of this experiment, 5 mM ammonium sulfate was selected as the best nitrogen source to obtain the maximum biomass and paclitaxel yield. Overall, the results introduce a medium containing 3% (w/v) malt extract and 5 mM ammonium sulfate at pH 7.0 as the best medium in which N. vitis produces the highest paclitaxel yield coincident with rapid and sustainable growth. The findings pave the way for industrial manufacturing of taxoids.

Optimizing Exopolysaccharide Production by Bacillus subtilis Using Spoiled Fig and Grape.

The study aimed to enhance exopolysaccharides (EPSs) production by the bacterial strain Bacillus subtilis ES (OR501464) isolated from sugar cane juice. Spoiled grape and fig extract were utilized as cost-effective substrates for EPS synthesis by B. subtilis ES (OR501464), and the impact of nutritional factors on EPS synthesis was assessed. Among nineteen bacterial isolates evaluated for EPS production, the isolate with the highest EPS yield was identified through a combination of phenotypic and genotypic analyses. The optimization process revealed that the highest EPS yield of 4.7 g/L was achieved in a production medium containing 4% sucrose, 0.1% NaNO3, 0.002% Na2SO4, and 2% NaCl at 30 °C and pH 9. Additionally, the study explored EPS generation by B. subtilis ES (OR501464) using spoiled grape and fig extract as substrates. The addition of 2% NaCl to spoiled grape extract increased EPS production to 4.357 mg/mL compared to 3.977 mg/mL with grape alone. However, 2% NaCl did not enhance EPSs production in fig waste. Supplementing spoiled fig or grape extract with 0.2 g/L Na2SO4 and 1 g/L NaNO3 increased EPS production by B. subtilis ES (OR501464). The EPS was analyzed using GC-MS and FTIR spectroscopy for partial characterization. The study found that spoiled figs and grapes can be used as effective substrates for EPS production. The highest yield was achieved by adding 0.2 g/L Na2SO4 and 1 g/L NaNO3. This study highlights the use of spoiled figs and grapes to produce valuable biopolymers, promoting sustainable and eco-friendly bioprocessing technologies.

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.

Optimization of culture conditions for HBV-specific T cell expansion in vitro from chronically infected patients.

BACKGROUND: Hepatitis B virus (HBV) clearance depends on an effective adaptive immune response, especially HBV-specific T cell-mediated cellular immunity; however, it is difficult to produce enough HBV-specific T cells effectively. RESULTS: In this work, we investigated the proportions of stimulated cells, serum, and culture media as the three primary factors to determine the most effective procedure and applied it to HLA-A2 (+) people. In parallel, we also examined the correlation between clinical parameters and HBV-specific immunity. Concerning amplification efficiency, 4 × 105 cells stimulation was superior to 2 × 106 cells stimulation, AIM-V medium outperformed 1640 medium, and fetal bovine serum (FBS) exceeded human AB serum under comparable conditions. As expected, this procedure is also suitable for developing HBV-specific CD8 + T cells in HLA-A2(+) individuals. Expanded HBV-specific T cell responses decreased with treatment time and were negatively correlated with HBV DNA and HBsAg. Furthermore, the number of HBV-specific IFN-γ + SFCs was strongly correlated with the ALT level and negatively correlated with the absolute lymphocyte count and the ALB concentration. CONCLUSIONS: We confirm that stimulating 4 × 105 PBMCs in AIM-V medium supplemented with 10% FBS is the best approach and that HBeAg, HBsAg, and ALB are independent predictors of HBV-specific T-cell responses.

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.

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.

PRP Influences Maturation and Fertilisation of Immature Mouse Oocytes.

In vitro maturation (IVM) of immature oocytes is a valuable method to enhance the rate of mature oocytes available for fertilisation. In the current study, platelet-rich plasma (PRP) was employed in IVM medium of immature oocytes. Harvested germinal vesicle stage oocytes with cumulus cells from female mature BALB/c mice divided into two groups of control and experiment. In the experimental group, GV oocytes matured in the IVM medium supplemented with 5% PRP, while in the control group, GV oocytes matured in the IVM medium without PRP. The percentage of GV, MI, MII and degenerated oocytes, zona pellucida thickness, perivitelline space size, diameter of mature oocytes, gene expression of apoptosis-related factors and subsequent development of matured oocytes were assessed. The PRP group displayed significantly improved outcomes in various parameters, including a higher proportion of MII and fertilised oocytes, cleavage and blastocyst embryos, compared to the control group. Moreover, the thickness of the zona pellucida was significantly lower in the PRP group than in the control group (p < 0.05). Furthermore, the PRP group demonstrated a significant decrease in the expression of transcripts associated with apoptosis (Bax and caspase-3); however, in the PRP group, a substantial increase in the expression of Bcl2l1, an apoptosis inhibitor, was observed when compared to the control group (p < 0.05). In conclusion, addition of PRP to the IVM culture media significantly increased oocyte maturation rate, leading to improved fertilisation and subsequent embryonic development. This enhancement highlights the positive influence of PRP on overall in vitro maturation efficiency and early embryonic stages.

Monocultures vs. polyculture of microalgae: unveiling physiological changes to facilitate growth in ammonium rich-medium.

Due to the increasing production of wastewater from human activities, the use of algal consortia for phytoremediation has become well-established over the past decade. Understanding how interspecific interactions and cultivation modes (monocultures vs. polyculture) influence algal growth and behaviour is a cutting-edge topic in both fundamental and applied science. Ammonium-rich growth media were used to challenge the monocultures of Auxenochlorella protothecoides, Chlamydomonas reinhardtii and Tetradesmus obliquus, as well as their polyculture; NO3 - was also used as the sole nitrogen chemical form in control cultures. The study primarily compared the growth, carbon and nitrogen metabolisms, and protein content of the green microalgae monocultures to those of their consortium. Overall, the cultivation mode significantly affected all the measured parameters. Notably, at 50 mM NH4 +, the assimilation rates of carbon and nitrogen were at least twice as high as those in the monoculture counterparts, and the protein content was three times more abundant.Additionally, the consortium's response to NH4 + toxicity was investigated by observing a linear relationship between the indicator of tolerance to NH4 + nutrition and the N isotopic signature. The study highlighted a high degree of acclimation through metabolic flexibility and diversity, as well as species abundance plasticity in the consortium, resulting in a functional resilience that would otherwise have been unattainable by the respective monocultures.

Effect of type of anticoagulant, transportation time, and glucose in the culture media on neutrophil viability and function test results in dairy cattle.

In dairy cattle research, in vitro assessment of innate immune function is commonly evaluated by flow cytometry via the quantitative analysis of circulating polymorphonuclear leukocytes (PMN) functionalities specifically focusing on the capacities for phagocytosis (PC) and oxidative burst (OB). Variations in these PMN functions, however, may not only be influenced by the health status of the animals but also by technical, non-animal related factors. Our objectives were to assess the PMN viability, PC and OB capacities from blood samples collected in tubes coated with different anticoagulants (acid citrate dextrose (ACD) and ethylenediaminetetraacetic acid (EDTA)) and stored for 0, 3, 6, 9, and 12 h at 4°C (to mimic transportation timeframe). Furthermore, we evaluated the PMN functionalities (PC and OB) in samples incubated in culture medium with glucose (7.2 mM) versus no glucose. Over five replicates, coccygeal blood samples were collected from three nulliparous Holstein heifers (5 ACD and 5 EDTA per heifer) and allocated in a refrigerated container (4°C) for 0, 3, 6, 9, and 12 h. At each time point, PMN were isolated using gradient centrifugation. Immunolabeled PMN (CH138A) were subjected to a tricolor fluorescent staining to evaluate their viability (viable, apoptotic, and necrotic PMN). Phagocytosis and OB were assessed by incubating PMN with fluorescent beads and by phorbol 12-myristate 13-acetate stimulation, respectively. The effects of anticoagulant type, storage time, and presence of glucose in the culture medium on PMN viability and function parameters were fitted in mixed linear regression models. The proportion of viable PMN at 0 h was similar for ACD and EDTA (92 ± 4.6% and 93 ± 4.6%, respectively) but it decreased to 78 ± 4.6% for ACD and 79 ± 4.6% for EDTA after 6 h of storage. The proportion of viable PMN was not different between ACD and EDTA at any time point. The proportion of PMN that engulfed beads (PC percentage) and the PC median fluorescence intensity (MFI) reached their highest value after 3 h of storage compared with the other time points. However, the anticoagulant type (ACD versus EDTA) and the presence of glucose in the culture medium did not influence these PC parameters. Oxidative burst MFI was higher in PMN incubated in glucose-supplemented culture medium versus no glucose. We demonstrated that technical factors interfere with the evaluation of PMN viability and functionality, which can potentially lead to bias in the findings of a research hypothesis. To conclude, the present study showed that the optimal timeframe for performing PMN function analyses is within 3 hours after blood sampling. Furthermore, the presence of 7.2 mM glucose in the culture medium, a common concentration in formulation of cell culture medium, increases the in vitro OB capacity, potentially masking any impairments in in vivo PMN dysfunctionality.

Efficient Production of Some Bioactive Depsides and Simple Phenolic Acids by Microshoots of Aronia × Prunifolia (Purple Aronia) Agitated Cultures as the Result of Feeding Strategy with Four Different Biogenetic Precursors.

A precursor feeding strategy was used for the first time in agitated microshoot cultures of Aronia × prunifolia. This strategy involved the addition of biogenetic precursors of simple phenolic acids (phenylalanine, cinnamic acid, and benzoic acid) and depsides (caffeic acid) into the culture media, with an assessment of its effect on the production of these bioactive compounds. The in vitro cultures were maintained in Murashige-Skoog medium (1 mg/L BAP and 1 mg/L NAA). Precursors at five concentrations (0.1, 0.5, 1.0, 5.0, and 10.0 mmol/L) were fed into the medium at the time of culture initiation (point "0") and independently on the 10th day of growth cycles. The contents of 23 compounds were determined in methanolic extracts of biomass collected after 20 days of growth cycles using an HPLC method. All extracts contained the same four depsides (chlorogenic, neochlorogenic, rosmarinic, and cryptochlorogenic acids) and the same four simple phenolic acids (protocatechuic, vanillic, caffeic, and syringic acids). Chlorogenic and neochlorogenic acids were the predominant compounds in all extracts (max. 388.39 and 263.54 mg/100 g d.w.). The maximal total contents of all compounds were confirmed after feeding with cinnamic acid (5 mmol/L, point "0") and caffeic acid (10 mmol/L, point "0"), which caused a 2.68-fold and 2.49-fold increase in the contents of the estimated compounds vs. control cultures (603.03 and 558.48 mg/100 g d.w., respectively). The obtained results documented the efficacy of the precursor feeding strategy in enhancing the production of bioactive compounds in agitated cultures of A. × prunifolia and suggest a potential practical application value.

Prolonged incubation of frozen-thawed equine spermatozoa for in vitro fertilization: A preliminary study using low temperature and INRA96 medium.

A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE at 30 or 38.2°C for 22 h. Following incubation, total motility (TM), progressive motility (PM), viability and acrosome integrity were evaluated. The results showed that TM was significantly higher (p < .001) at 30°C in both media, while PM was higher for INRA96 at 30°C compared to 38°C (p < .05). Moreover, INRA96 at 30°C exhibited higher sperm viability and acrosome integrity (p < .001) compared to the other experimental groups. These preliminary results suggest that incubating thawed equine spermatozoa at 30°C with PHE in INRA96 successfully maintains motility, viability and acrosome integrity in equine spermatozoa, indicating its potential use for conventional equine IVF.

Enhancing felid conservation: Exploring the impact of in vitro culture media on domestic cat blastocyst production.

This study investigated the optimization of assisted reproductive techniques for wild felid conservation, focusing on in vitro procedures using the domestic cat as a model species. The research evaluated the impact of three different in vitro culture media on blastocyst formation. Oocytes and spermatozoa were collected and processed, followed by in vitro fertilization and culture. Results returned a similar blastocyst rate (ANOVA, p > .05), over 16% across all groups. While demonstrating the potential of these techniques, further investigations are warranted to evaluate embryo quality to refine optimal protocols and their applicability in felid conservation efforts.

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.

Biological characteristics of marine Streptomyces SK3 and optimization of cultivation conditions for production of compounds against Vibiriosis pathogen isolated from cultured white shrimp (Litopenaeus vannamei).

Antibiotic resistance in shrimp farms has emerged as an extremely serious situation worldwide. The main aim of this study was to optimize the cultural conditions for producing new antibiotic agents from marine Streptomyces species. Streptomyces SK3 was isolated from marine sediment and was identified by its 16S rDNA as well as biochemical characteristics. This microbe produced the highest concentration of bioactive secondary metabolites (BSMs) when cultured in YM medium (YM/2). It produced the maximum total protein (41.8 ± 6.36 mg/ml) during the late lag phase period. The optimum incubation temperature was recorded at 30 °C; BSMs were not produced at ≤10 °C within an incubation period of 3-4 days. The suitable agitation speed was found to be 200 rpm with pH 7.00. The proper carbon, nitrogen, and trace elements supplementation consisted of starch, malt extract, calcium carbonate (CaCO3), and magnesium sulfate (MgSO4). The ethyl acetate extract was found to act strongly against three vibriosis pathogens, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vunificus, as indicated by the inhibition zones at 34.5, 35.4, and 34.3 mm, respectively. The extract showed the strongest anti-V. harveyi activity, as indicated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 0.101 ± 0.02 and 0.610 ± 0.04 mg/ml, respectively. Basic chemical investigation of the crude extract using thin layer chromatography (TLC), bioautography, liquid chromatography tandem mass spectrometry (LC‒MS/MS), Fourier transform infrared spectroscopy (FTIR), and proton nuclear magnetic resonance (1H-NMR) revealed that the active components were the terpenoid and steroid groups of compounds. They showed carboxylic acid and ester functions in their molecules.

2PP-Hydrogel Covered Electrodes to Compensate for Media Effects in the Determination of Biomass in a Capillary Wave Micro Bioreactor.

Microbioreactors increase information output in biopharmaceutical screening applications because they can be operated in parallel without consuming large quantities of the pharmaceutical formulations being tested. A capillary wave microbioreactor (cwMBR) has recently been reported, allowing cost-efficient parallelization in an array that can be activated for mixing as a whole. Although impedance spectroscopy can directly distinguish between dead and viable cells, the monitoring of cells in suspension within bioreactors is challenging because the signal is influenced by the potentially varying properties of the culture medium. In order to address this challenge, an impedance sensor consisting of two sets of microelectrodes in a cwMBR is presented. Only one set of electrodes was covered by a two-photon cross-linked hydrogel to become insensitive to the influence of cells while remaining sensitive to the culture medium. With this impedance sensor, the biomass of Saccharomyces cerevisiae could be measured in a range from 1 to 20 g L-1. In addition, the sensor can compensate for a change in the conductivity of the suspension of 5 to 15 mS cm-1. Moreover, the two-photon cross-linking of hydroxyethyl starch methacrylate hydrogel, which has been studied in detail, recommends itself for even much broader sensing applications in miniaturized bioreactors and biosensors.