Viable Cell Concentration Research Articles

Agronomic Efficiency of Signum Inoculant in Pre-inoculation of Soybean at 35 and 20 Days Before Sowing in Treated Seeds

Pre-inoculating soybean seeds can make sowing faster and provide additional benefits to farmers. However, it needs to guarantee the nitrogen supply to maintain the viability and sustainability of the technique. In this study, we evaluated the agronomic efficiency of SIGNUM® inoculant in the pre-inoculation at 20 and 35 days before sowing chemically treated soybean seeds. Experiments were conducted in four field experiments located at Paraná, Brazil, with a history of soybean cultivation managed under no-tillage systems, with crop rotation according to regional edaphoclimatic conditions. Agronomic efficiency in fields were compared with standard inoculation with a registered product used by farmers. Chemical treatment of soybean seeds with Standak Top® or Maxin XL® + Cruiser® associated with pre-inoculation of the inoculant SIGNUM® for 25 and 30 days reduced the concentration of viable Bradyrhizobium cells recovered from seeds. However, no significant difference was observed regarding nodulation, biological nitrogen fixation, and yield between the soybean inoculated with standard inoculation on farm or pre-inoculation with SIGNUM® in most studied areas.

Aerosolization triggers immediate antibiotic resistance in bacteria

Antibiotic resistance poses a threat in many institutions including hospitals, meat processing facilities and other agricultural and occupational entities. The goal of this research is to uncover how bacteria react to environmental conditions including aerosolization and delineate factors that trigger expression of antibiotic resistance genes (ARGs) using the laboratory strain Escherichia coli (E. coli) MG 1655 as surrogate bacterium. Fresh mid-log phase bacterial suspensions were aerosolized at different environmental conditions for 5 different time durations into a sterile chamber and collected using a bioaerosol collector. Each sample was analyzed based on its response to each environmental factor by antibiotic susceptibility testing and polymerase chain reaction (PCR) using specific primers to target ARGs. Strong resistance was detected to cell wall and protein synthesis inhibitors Cephalothin, and Gentamicin after 5 min compared to 30 min and 45 min of aerosolizations in reference to viable cell concentrations. Resistance to the highest number of antibiotics was detected in the shortest aerosolization samples while exposure for longer aerosolization durations caused an increased amount of stress which bacteria could not alleviate, indicating the involvement of different mechanisms to respond to short-term and long-term exposures. A small amount of resistance was detected after intermediate durations of aerosolization, maybe due to a transient response of the cells to compensate for mechanical, osmotic and other stresses during aerosolization. This study is the first to demonstrate that aerosolization triggers antibiotic resistance within minutes in E. coli.

Limosilactobacillus fermentum from buffalo milk is suitable for potential biotechnological process development and inhibits Helicobacter pylori in a gastric epithelial cell model

Probiotics are living microorganisms that give beneficial health effects while consumed, and each strain possesses diverse and unique properties and also different technological characteristics that affect its ability to be produced at large scale. Limosilactobacillus fermentum is a widely studied member of probiotics, however, few data are available on the development of fermentation and downstream processes for the production of viable biomasses for potential industrial applications.In the present study a novel L. fermentum strain was isolated from buffalo milk and used as test example for biotechnological process development. The strain was able to produce up to 109 CFU/mL on a (glucose based) semi-defined medium deprived of animal-derived raw materials up to the pilot scale (150 L), demonstrating improved results compared to commonly used, although industrially not suitable, media rich of casein and beef extract. The study of strain behavior in batch experiments indicated that the highest concentration of viable cells was reached after only 8 h of growth, greatly shortening the process. Moreover, initial concentrations of glucose in the medium above 30 g/L, if not supported by higher nitrogen concentrations, reduced the yield of biomass and increased production of heterolactic fermentation by-products. Biomass concentration via microfiltration on hollow fibers, and subsequent spray-drying allowed to recover about 5.7 × 1010CFU/gpowder of viable cells, indicating strain resistance to harsh processing conditions.Overall, these data demonstrate the possibility to obtain and maintain adequate levels of viable L. fermentum cells by using a simple approach that is potentially suitable for industrial development. Moreover, since often exopolysaccharides produced by lactobacilli contribute to the strain's functionality, a partial characterization of the EPS produced by the newly identified L. fermentum strain was carried out.Finally, the effect of L. fermentum versus H. pylori in a gastric epithelial cell model was evaluated demonstrating its ability to stimulate the response of the immune system and displace the infective agent.

Non-invasive and time-resolved measurement of the respiration activity of Chinese hamster ovary cells enables prediction of key culture parameters in shake flasks.

Shake flasks are frequently used for mammalian cell suspension cultures. For process development and routine culture monitoring, information on culture behavior is needed early on. Here, cell-specific oxygen uptake rates (qO2 ) of two CHO cell lines were determined from shake flask experiments by simultaneous measurement of oxygen transfer rates (OTR) and viable cell concentrations (VCC). For cell line one, qO2 decreased from 2.38·10-10 to 1.02·10-10 mmol cell-1 h-1 during batch growth. For cell line two, qO2 was constant (1.90·10-10 mmol h-1 ). Determined qO2 values were used to calculate the VCC from OTR data. Cumulated oxygen consumption and glucose consumption were correlated for both cell lines and enabled calculation of glucose concentrations from OTR data. IgG producing cell line one had an oxygen demand of ∼15 mmoloxygen gglucose -1 , cell line two consumed ∼5 mmoloxygen gglucose -1 . The established correlations for determination of VCC and glucose were successfully transferred to subsequent cultivations for both cell lines. Combined measurement of the OTR and the carbon dioxide transfer rate enabled quantitative determination of the lactate concentration (production and consumption) without sampling. Taken together, non-invasive measurement of the respiration activity enabled time-resolved determination of key culture parameters for increased process understanding in shake flasks.

Preliminary study on centrifugation time of liquid plasmatrix for soft tissue regeneration

Objective: To provide reference for clinical application of liquid plasmatrix, and to investigate the optimal centrifugation time of liquid plasmatrix prepared by horizontal centrifugation for soft tissue regeneration from the aspects of mechanical properties, biological properties, and the effect of promoting soft tissue regeneration. Methods: Venous blood was collected from 6 healthy volunteers [3 males and 3 females, aged (26±2) years, with informed consent] who volunteered to donate blood at School of Stomatology, Wuhan University from September to November 2021. The collected venous blood was centrifuged at 500 ×g for 3, 5, 8 and 12 min to obtain liquid plasmatrix. The volume, weight, solidification time, and mechanical properties of liquid plasmatrix prepared at different centrifugation time were measured and recorded (the sample size at each time point was 3). The microstructure of different groups of liquid plasmatrix clot was observed by scanning electron microscope (SEM). The rheological properties of each group of liquid plasmatrix clot were measured by rheological test. The number and concentration of cells in the whole blood group and in each liquid plasmatrix group were measured using complete blood count test. The distribution of cells in the liquid plasmatrix clots was observed by hematoxylin-eosin staining. The effect of control group (Dulbecco's modified Eagle's medium containing 20% fetal bovine serum) and liquid plasmatrix clot exudates in 3, 5, 8, 12 min group (the sample size at each time point was 3) on gingival fibroblast migration was detected by cell migration method. Finally, the effects of control group and liquid plasmatrix clot exudates on the morphology of gingival fibroblasts were observed by fluorescence microscope. Results: The volume of liquid plasmatrix in 3, 5, 8 and 12 min group were approximately (2.47±0.12), (2.67±0.12), (3.53±0.12) and (3.73±0.12) ml, respectively. The weight of liquid plasmatrix in 3, 5, 8 and 12 min group were approximately (0.35±0.01), (0.46±0.02), (0.88±0.06) and (1.03±0.01) g, respectively. The maximum tensile force of liquid plasmatrix clots in 3, 5, 8 and 12 min group were (0.55±0.03), (0.56±0.03), (1.31±0.05) and (1.38±0.02) N, respectively. SEM results showed that the fibers inside the liquid plasmatrix clot became denser with increased centrifugation time. Compared with other groups, the concentrations of leukocytes, neutrophils, monocytes and lymphocytes in 8 min group were the highest, and the distribution of cell was more even. Compared with other groups, the efficiency of stimulating gingival fibroblast migration in 8 min group was the best (1.60±0.01). Fluorescence staining test showed that the liquid plasmatrix clot exudates could make gingival fibroblasts more stretched compared with control group. Conclusions: The present study shows that liquid plasmatrix prepared by centrifugation with 500 ×g centrifugal force for 8 min has higher concentration of viable cells and the ability to promote the migration of gingival fibroblasts.

Impact of Influenza A Virus Infection on Growth and Metabolism of Suspension MDCK Cells Using a Dynamic Model.

Cell cultured-based influenza virus production is a viable option for vaccine manufacturing. In order to achieve a high concentration of viable cells, is requirement to have not only optimal process conditions, but also an active metabolism capable of intracellular synthesis of viral components. Experimental metabolic data collected in such processes are complex and difficult to interpret, for which mathematical models are an appropriate way to simulate and analyze the complex and dynamic interaction between the virus and its host cell. A dynamic model with 35 states was developed in this study to describe growth, metabolism, and influenza A virus production in shake flask cultivations of suspension Madin-Darby Canine Kidney (MDCK) cells. It considers cell growth (concentration of viable cells, mean cell diameters, volume of viable cells), concentrations of key metabolites both at the intracellular and extracellular level and virus titers. Using one set of parameters, the model accurately simulates the dynamics of mock-infected cells and correctly predicts the overall dynamics of virus-infected cells for up to 60 h post infection (hpi). The model clearly suggests that most changes observed after infection are related to cessation of cell growth and the subsequent transition to apoptosis and cell death. However, predictions do not cover late phases of infection, particularly for the extracellular concentrations of glutamate and ammonium after about 12 hpi. Results obtained from additional in silico studies performed indicated that amino acid degradation by extracellular enzymes resulting from cell lysis during late infection stages may contribute to this observed discrepancy.

Mathematical Modeling of Fed-Batch Ethanol Fermentation Under Very High Gravity and High Cell Density at Different Temperatures.

The use of more appropriate kinetic models can assist in improving ethanol fermentation under conditions of very high gravity (VHG) and high cell density (HCD), in order to obtain higher amounts of ethanol in the broth combined with high productivity. The aim of this study was to model fed-batch ethanol fermentation under VHG/HCD conditions, at different temperatures, considering three types of inhibition (substrate, ethanol, and cells). Fermentations were carried out using different temperatures (28 ≤ [Formula: see text] (°C) ≤ 34), inoculum sizes (50 ≤ [Formula: see text] (g L-1) ≤ 125), and substrate concentrations in the must (258 ≤ [Formula: see text] (g L-1) ≤ 436). In the proposed model, the cell inhibition power parameter varied with the temperature and inoculum size, while the cell yield coefficient varied with inoculum size and substrate concentration in the must. Hence, it was possible to propose correlations for the cell inhibition power parameter ([Formula: see text]) and for the cell yield coefficient ([Formula: see text]), as functions of the fermentation conditions. Simulations of fed-batch ethanol fermentations at different temperatures, under VHG/HCD conditions, were performed using the proposed correlations. Experimental validation showed that the model was able to accurately predict the dynamic behavior of the fermentations in terms of the concentrations of viable cells, total cells, ethanol, and substrate.

Stress-induced increase of monoclonal antibody production in CHO cells.

Monoclonal antibodies (mAbs) are of great interest to the biopharmaceutical industry due to their widely used application as human therapeutic and diagnostic agents. As such, mAb require to exhibit human‐like glycolization patterns. Therefore, recombinant Chinese hamster ovary (CHO) cells are the favored production organisms; many relevant biopharmaceuticals are already produced by this cell type. To optimize the mAb yield in CHO DG44 cells a corelation between stress‐induced cell size expansion and increased specific productivity was investigated. CO2 and macronutrient supply of the cells during a 12‐day fed‐batch cultivation process were tested as stress factors. Shake flasks (500 mL) and a small‐scale bioreactor system (15 mL) were used for the cultivation experiments and compared in terms of their effect on cell diameter, integral viable cell concentration (IVCC), and cell‐specific productivity. The achieved stress‐induced increase in cell‐specific productivity of up to 94.94.9%–134.4% correlates to a cell diameter shift of up to 7.34 μm. The highest final product titer of 4 g/L was reached by glucose oversupply during the batch phase of the process.

The efficient development of a novel recombinant adenovirus zoster vaccine perfusion production process

The adenovirus vector vaccines induce humoral and cellular immune responses and have been used to develop vaccines for effective prevention of life-threating viruses, such as Ebola and Coronaviruses. High demand of vaccines worldwide requires optimization of the production process. Perfusion process increases cell concentration and volumetric productivity, so that it becomes the commonly used strategy in vaccine production In this study, we optimized and developed a perfusion process for the adenovirus-based zoster vaccine production efficiently. We first tested different perfusion strategies in shake flasks, showing semi-continuous strategies for optimal HEK 293 cell growth. We then evaluated three empirical key process parameters (cell concentration at the time of infection (VCC), multiplicity of infection (MOI), virus production pH) by the design of experiment (DoE) method, from which the robust setpoint (VCC 1.04 × 107 cells/mL, MOI 9, and virus production pH 7.17) was confirmed in both shake flask and 2 L benchtop bioreactor. In the bioreactor, we compared the performances of two perfusion systems, the commercially-available XCell ATF® system and a novel peristaltic pump-driven alternating tangential flow perfusion system (PATFP system) that we developed. During cell cultivation stage, both perfusion systems have comparable performances regarding viable cell concentration and cell viability. At 2 dpi, the PATFP system resulted in an adenovirus titer of 2.1 × 1010 IFU/mL and cell-specific virus yield of 2,062 IFU/cell, reaching 75% and 77% of values for XCell ATF® system. This study demonstrates the perfusion process to be superior strategy for adenovirus-based vaccine production compared to the batch-mode strategy (1,467 IFU/cell). Furthermore, our PATFP system shows potential to be comparable to the XCell ATF® system, and it would become an alternative perfusion strategy for the vaccine production.

Plant Growth Promoting Activities of Spore-Forming and Vegetative Cells of Salt-Tolerant Rhizobacteria under Salinity Condition

Plant growth promoting rhizobacteria are able to enhance plant growth. This study isolated spore-forming rhizobacteria from vegetable rhizosphere soil samples. These isolates were tested for their abilities to promote plant growth and to be used in bio-fertilizers. Thirty-nine isolates with different characteristics were obtained. Three isolates, TYS1.1, TYS3.3, and TYS3.5, showed multifunctional activities on nitrogen fixation and potassium solubilization. They were tested for IAA production in liquid medium supplemented with tryptophan and NaCl, with the vegetative cells of isolate TYS3.5 showing the highest IAA production. The colonization of the three isolates on okra roots was checked by spread plate technique and scanning electron microscope. It was found that rhizobacteria could colonize plant roots with a concentration of 8.19 log CFU/g in the presence of 50 mM NaCl solution. Bio-fertilizer was produced by immobilizing the mixture of three isolates on carriers. The viable cells were enumerated during the storage at room temperature for 60 days. The results showed that the highest number of survival cells in the form of vegetative and spore-forming cells were obtained when using rice husk ash and vermiculite as a carrier. The concentration of viable cells was in a range of 8.14-8.44 log CFU/g. These isolates were Bacillus sp. according to the 16S rDNA sequencing analysis.

Influence of Culture Medium on Cementation of Coarse Grains Based on Microbially Induced Carbonate Precipitation

A main challenge in the large-scale application of the microbially induced carbonate precipitation (MICP) technique includes the low efficiency of the cementation of coarse grains. Actually, in the MICP treatment process, the cementation effect of the bonding points was more important than pore filling due to the large porosity for coarse grains. To achieve a better cementation effect at bonding points between coarse particles, the quick formation and growth of a biofilm is necessary. In this study, an optimized medium was proposed to improve the cementation effects for coarse materials. The optimized medium and other different media were used for bio-cementation tests with MICP. The viable cell concentrations, strengths, microscopic characteristics, biofilm contents, and calcium carbonate (CaCO3) contents were used to evaluate the bio-cementation and its effects. In bio-cementation tests, the optimized medium led to increased CaCO3 precipitation at the bonding points and better cementation effects compared to other media. Indeed, the strength of the sample treated with the optimized medium was more than 1.2–4 times higher that of the values for other media. The advantages of the optimized medium were demonstrated via bio-cementation tests.

Process intensification for the continuous production of an antimicrobial peptide in stably-transformed Sf-9 insect cells

The antibiotic resistance crisis has prompted research into alternative candidates such as antimicrobial peptides (AMPs). However, the demand for such molecules can only be met by continuous production processes, which achieve high product yields and offer compatibility with the Quality-by-Design initiative by implementing process analytical technologies such as turbidimetry and dielectric spectroscopy. We developed batch and perfusion processes at the 2-L scale for the production of BR033, a cecropin-like AMP from Lucilia sericata, in stably-transformed polyclonal Sf-9 cells. This is the first time that BR033 has been expressed as a recombinant peptide. Process analytical technology facilitated the online monitoring and control of cell growth, viability and concentration. The perfusion process increased productivity by ~ 180% compared to the batch process and achieved a viable cell concentration of 1.1 × 107 cells/mL. Acoustic separation enabled the consistent retention of 98.5–100% of the cells, viability was > 90.5%. The recombinant AMP was recovered from the culture broth by immobilized metal affinity chromatography and gel filtration and was able to inhibit the growth of Escherichia coli K12. These results demonstrate a successful, integrated approach for the development and intensification of a process from cloning to activity testing for the production of new biopharmaceutical candidates.

Cole-Cole modeling of real-time capacitance data for estimation of cell physiological properties in recombinant Escherichia coli cultivation.

Real-time estimation of physiological properties of the cell during recombinant protein production would ensure enhanced process monitoring. In this study, we explored the application of dielectric spectroscopy to track the fed-batch phase of recombinant Escherichia coli cultivation for estimating the physiological properties, namely, cell diameter and viable cell concentration (VCC). The scanning capacitance data from the dielectric spectroscopy were pre-processed using moving average. Later, it was modeled through a nonlinear theoretical Cole-Cole model and further solved using a global evolutionary genetic algorithm (GA). The parameters obtained from the GA were further applied for the estimation of the aforementioned physiological properties. The offline cell diameter and cell viability data were obtained from particle size analyzer and flow cytometry measurements to validate the Cole-Cole model. The offline VCC was calculated from the cell viability % from flow cytometry data and dry cell weight concentration. The Cole-Cole model predicted the cell diameter and VCC with an error of 1.03% and 7.72%, respectively. The proposed approach can enable the operator to take real-time process decisions to achieve desired productivity and product quality.

Model Transferability and Reduced Experimental Burden in Cell Culture Process Development Facilitated by Hybrid Modeling and Intensified Design of Experiments.

Reliable process development is accompanied by intense experimental effort. The utilization of an intensified design of experiments (iDoE) (intra-experimental critical process parameter (CPP) shifts combined) with hybrid modeling potentially reduces process development burden. The iDoE can provide more process response information in less overall process time, whereas hybrid modeling serves as a commodity to describe this behavior the best way. Therefore, a combination of both approaches appears beneficial for faster design screening and is especially of interest at larger scales where the costs per experiment rise significantly. Ideally, profound process knowledge is gathered at a small scale and only complemented with few validation experiments on a larger scale, saving valuable resources. In this work, the transferability of hybrid modeling for Chinese hamster ovary cell bioprocess development along process scales was investigated. A two-dimensional DoE was fully characterized in shake flask duplicates (300 ml), containing three different levels for the cultivation temperature and the glucose concentration in the feed. Based on these data, a hybrid model was developed, and its performance was assessed by estimating the viable cell concentration and product titer in 15 L bioprocesses with the same DoE settings. To challenge the modeling approach, 15 L bioprocesses also comprised iDoE runs with intra-experimental CPP shifts, impacting specific cell rates such as growth, consumption, and formation. Subsequently, the applicability of the iDoE cultivations to estimate static cultivations was also investigated. The shaker-scale hybrid model proved suitable for application to a 15 L scale (1:50), estimating the viable cell concentration and the product titer with an NRMSE of 10.92% and 17.79%, respectively. Additionally, the iDoE hybrid model performed comparably, displaying NRMSE values of 13.75% and 21.13%. The low errors when transferring the models from shaker to reactor and between the DoE and the iDoE approach highlight the suitability of hybrid modeling for mammalian cell culture bioprocess development and the potential of iDoE to accelerate process characterization and to improve process understanding.

Silk Fibroin and Pomegranate By-Products to Develop Sustainable Active Pad for Food Packaging Applications.

In this study, a bio-based polymeric system loaded with fruit by-products was developed. It was based on silk fibroin produced by the silkworm Bombyx mori and pomegranate peel powder, selected as active agent. The weight ratio between fibroin and pomegranate powder was 30:70. Pads also contained 20% w/w of glycerol vs. fibroin to induce water insolubility. Control systems, consisting of only fibroin and glycerol, were produced as reference. Both control and active systems were characterized for structural and morphological characterization (Fourier-transform infrared spectroscopy and optical microscope), antioxidant properties and antimicrobial activity against two foodborne spoilage microorganisms. Results demonstrate that under investigated conditions, an active system was obtained. The pad showed a good water stability, with weight loss of about 28% due to the release of the active agent and not to the fibroin loss. In addition, this edible system has interesting antioxidant and antimicrobial properties. In particular, the pad based on fibroin with pomegranate peel recorded an antioxidant activity of the same order of magnitude of that of vitamin C, which is one of the most well-known antioxidant compounds. As regards the antimicrobial properties, results underlined that pomegranate peel in the pad allowed maintaining microbial concentration around the same initial level (104 CFU/mL) for more than 70 h of monitoring, compared to the control system where viable cell concentration increased very rapidly up to 108 CFU/mL.

PRODUCTION OF LACTIC ACID WORT-BASED BEVERAGES WITH MINT ESSENTIAL OIL ADDITION

Lactic acid wort-based beverages are functional, non-alcoholic, with low pH value and produced by the fermentation of wort by lactic acid bacteria. They are not well accepted by consumers because of their poor sensory characteristics. Therefore, 0.025 and 0.05 % (v/v) mint (Mentha piperita) essential oil was used as a tool for improvement of lactic acid wort-based beverages organoleptic profile. Wort was produced by 60% Pilsen malt, 20% Vienna malt, and 20% Caramel Munich ІІ malt. It was inoculated with probiotic lactic acid bacteria Lactobacillus casei ssp. rhamnosus LBRC11 at a concentration of 107 cells/ml and fermentation was carried out at constant temperature of 25°C. The dynamics of pH, concentration of viable cells, phenolic compounds and antioxidant activity were monitored and the beverages obtained were evaluated by a tasting panel. The results showed that addition of mint essential oil in concentration of 0.025 and 0.05 % (v/v) inhibited lactic acid fermentation but improved the sensory profile of the beverage obtained only when 0.025% mint essential oil was added. Mint essential oil addition led to an increase in the total phenolic compounds concentration, phenolic acids and flavonoid phenolic compounds, measured by Folin–Ciocalteu and modified Glories method but resulted in a decrease in the antioxidant activity, measured by the DPPH radical scavenging assay, cupric reducing antioxidant power (CUPRAC) and ferric reducing antioxidant power (FRAP). The antioxidant activity measured by the ABTS radical scavenging assay was almost equal for the beverages with and without mint essential oil addition. The results obtained will be used for modeling of lactic acids fermentation with addition of mint essential oil for the production of functional wort-based beverages. Keywords: lactic acid fermentation, wort, mint essential oil, phenolic compounds, antioxidant activity

PRODUCTION OF LACTIC ACID WORT-BASED BEVERAGES WITH MINT ESSENTIAL OIL ADDITION

Lactic acid wort-based beverages are functional, non-alcoholic, with low pH value and produced by the fermentation of wort by lactic acid bacteria. They are not well accepted by consumers because of their poor sensory characteristics. Therefore, 0.025 and 0.05 % (v/v) mint (Mentha piperita) essential oil was used as a tool for improvement of lactic acid wort-based beverages organoleptic profile. Wort was produced by 60% Pilsen malt, 20% Vienna malt, and 20% Caramel Munich ІІ malt. It was inoculated with probiotic lactic acid bacteria Lactobacillus casei ssp. rhamnosus LBRC11 at a concentration of 107 cells/ml and fermentation was carried out at constant temperature of 25°C. The dynamics of pH, concentration of viable cells, phenolic compounds and antioxidant activity were monitored and the beverages obtained were evaluated by a tasting panel. The results showed that addition of mint essential oil in concentration of 0.025 and 0.05 % (v/v) inhibited lactic acid fermentation but improved the sensory profile of the beverage obtained only when 0.025% mint essential oil was added. Mint essential oil addition led to an increase in the total phenolic compounds concentration, phenolic acids and flavonoid phenolic compounds, measured by Folin–Ciocalteu and modified Glories method but resulted in a decrease in the antioxidant activity, measured by the DPPH radical scavenging assay, cupric reducing antioxidant power (CUPRAC) and ferric reducing antioxidant power (FRAP). The antioxidant activity measured by the ABTS radical scavenging assay was almost equal for the beverages with and without mint essential oil addition. The results obtained will be used for modeling of lactic acids fermentation with addition of mint essential oil for the production of functional wort-based beverages.

Thermally induced oxygen related defects in eco-friendly ZnFe2O4 nanoparticles for enhanced wastewater treatment efficiencies

Herein, a simple but highly effective strategy of thermal annealing to modulate oxygen vacancies related defects in ZnFe2O4 (ZFO) nanoparticles for obtaining enhanced wastewater treatment efficiencies is reported. The as-prepared nanoparticles were thermally annealed at three different temperatures (500 °C, 600 °C and 700 °C) and their phase purity was confirmed by X-ray diffraction (XRD). All samples were found to exhibit pure phases of ZFO with different crystallite sizes ranging from 10 nm to 25 nm. The transmission electron microscope (TEM) images showed well dispersed nanoparticles and a strong correlation of grain size growth with annealing temperature was established. The optical absorption and emission characteristics were estimated through UV–visible and Photoluminescence (PL) spectroscopy. Raman spectroscopy and X-ray Photoelectron Spectroscopy (XPS) confirmed the variation of oxygen vacancies in the synthesized samples’ lattice. The photocatalytic activities of all samples were investigated and the highest efficiencies were recorded for the ZFO samples annealed at 500 °C. Under high salinity condition, the organic dye degradation efficiency of the same sample remained the highest among all. The excellent dye degradation abilities in ZFO samples can be attributed to the abundance of oxygen vacancies in the crystal lattice that slow down the recombination rate during the photocatalysis process. Moreover, cytotoxicity tests revealed that all prepared ZFO samples showed insignificant cell structure effects on Picochlorum sp microalgae, as verified by Fourier-transform infrared (FTIR) spectroscopy. On the other hand, no significant changes were detected on the viable cell concentration and Chlorophyll a content. This work presents a systematic way to finely tune the crystal sizes and to modulate oxygen related defects in ZFO through a highly effective annealing approach to signify their potential in industrial wastewater and seawater treatment processes.

Equal mixing time enables scale‐down and optimization of a CHO cell culture process using a shaken microbioreactor system

The advancement of microbioreactor technology in recent years has transformed early- and mid-stage process development. The monitoring and control capabilities of microbioreactors not only promote the quick accumulation of process knowledge but has also led to an increased scalability when compared to traditionally used systems such as shake flasks and microtitre plates. This study seeks to establish a framework for the micro-Matrix microbioreactor (Applikon-Biotechnology BV) as process development tool. Using the Dual Indicator System for Mixing Time, the system was initially characterized for mixing properties at varying operating conditions, which was found to yield mixing times between 0.9 and 41.8 s. A matched mixing time was proposed as scale-down criterion for an IgG4 producing GS-CHO fed-batch process between a 5 L stirred tank reactor (STR) and the micro-Matrix microbioreactor. Growth trends, maximum viable cell concentrations, final titre, and glycoprofiles were nearly identical at both scales. The scale-down model was then employed to optimize a bolus feeding regime using response surface methodology, which led to a 25.4% increase of the space-time yield and a 25% increase of the final titre. The optimized feeding strategy was validated at the small-scale and successfully scaled up to the 5 L STR. This work for the first time provides a framework of how the micro-Matrix microbioreactor can be implemented in a bioprocess development workflow and demonstrates scalability of growth and production kinetics as well as IgG4 glycosylation between the micro-Matrix and a benchtop-scale STR system.

Cell culture\u2013based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

Respiratory diseases including influenza A virus (IAV) infections represent a major threat to human health. While the development of a vaccine requires a lot of time, a fast countermeasure could be the use of defective interfering particles (DIPs) for antiviral therapy. IAV DIPs are usually characterized by a large internal deletion in one viral RNA segment. Consequentially, DIPs can only propagate in presence of infectious standard viruses (STVs), compensating the missing gene function. Here, they interfere with and suppress the STV replication and might act “universally” against many IAV subtypes. We recently reported a production system for purely clonal DIPs utilizing genetically modified cells. In the present study, we established an automated perfusion process for production of a DIP, called DI244, using an alternating tangential flow filtration (ATF) system for cell retention. Viable cell concentrations and DIP titers more than 10 times higher than for a previously reported batch cultivation were observed. Furthermore, we investigated a novel tubular cell retention device for its potential for continuous virus harvesting into the permeate. Very comparable performances to typically used hollow fiber membranes were found during the cell growth phase. During the virus replication phase, the tubular membrane, in contrast to the hollow fiber membrane, allowed 100% of the produced virus particles to pass through. To our knowledge, this is the first time a continuous virus harvest was shown for a membrane-based perfusion process. Overall, the process established offers interesting possibilities for advanced process integration strategies for next-generation virus particle and virus vector manufacturing.Key points• An automated perfusion process for production of IAV DIPs was established.• DIP titers of 7.40E + 9 plaque forming units per mL were reached.• A novel tubular cell retention device enabled continuous virus harvesting.