A forced aeration system for microbial culture of multiple shaken vessels suppresses volatilization.

Shake-flask culture, an aerobic submerged culture, has been used in various applications involving cell cultivation. However, it is not designed for forced aeration. Hence, this study aimed to develop a small-scale submerged shaking culture system enabling forced aeration into the medium. A forced aeration control system for multiple vessels allows shaking, suppresses volatilization, and is attachable externally to existing shaking tables. Using a specially developed plug, medium volatilization was reduced to less than 10%, even after 45 h of continuous aeration (~ 60 mL/min of dry air) in a 50 mL working volume. Escherichia coli IFO3301 cultivation with aeration was completed within a shorter period than that without aeration, with a 35% reduction in the time-to-reach maximum bacterial concentration (26.5 g-dry cell/L) and a 1.25-fold increase in maximum concentration. The maximum bacterial concentration achieved with aeration was identical to that obtained using the Erlenmeyer flask, with a 65% reduction in the time required to reach it.

Development of an insertion device selectively operational as a helical/figure-8 undulator.

An insertion device capable of switching the operation mode between helical and figure-8 undulators, and thus referred to as a helical-8 undulator, has been developed. It has the advantage that the on-axis heat load can be kept low regardless of the polarization state, even when a high K value is required to lower the fundamental photon energy. This is in contrast to conventional undulators in which the on-axis heat load tends to be significantly high to generate linearly polarized radiation with a high K value, and optical elements can be seriously damaged. The principle of operation, specification and light source performance of the developed helical-8 undulator are presented together with further options to enhance its capability.

Understudied Hyperphosphatemia (Chronic Kidney Disease) Treatment Targets and New Biological Approaches.

Hyperphosphatemia is a secondary disorder of chronic kidney disease that causes vascular calcifications and bone-mineral disorders. As per the US Centers for Disease Control and Prevention, renal damage requires first-priority medical attention for patients with COVID-19; according to a Johns Hopkins Medicine report, SARS-CoV-2 can cause renal damage. Therefore, addressing the research inputs required to manage hyperphosphatemia is currently in great demand. This review highlights research inputs, such as defects in the diagnosis of hyperphosphatemia, flaws in understanding the mechanisms associated with understudied tertiary toxicities, less cited adverse effects of phosphate binders that question their use in the market, socioeconomic challenges of renal treatment and public awareness regarding the management of a phosphate-controlled diet, novel biological approaches (synbiotics) to prevent hyperphosphatemia as safer strategies with potential additional health benefits, and future functional food formulations to enhance the quality of life. We have not only introduced our contributions to emphasise the hidden aspects and research gaps in comprehending hyperphosphatemia but also suggested new research areas to strengthen approaches to prevent hyperphosphatemia in the near future.

Effects of organic carbon sources on growth and oil accumulation by Desmodesmus subspicatus LC172266 under mixotrophic condition.

Energy crisis and environmental sustainability have attracted global attention to microalgal biofuels. The present study investigated the impact of organic carbon sources on growth and bio-oil accumulation by an oleaginous microalga Desmodesmus subspicatus LC172266 under mixotrophic culture condition. Glucose and glycerol supported higher growth rates and lipid productivities than sucrose, fructose, mannitol and acetate. Each of the organic carbon source tested supported significantly (P < 0.05) higher growth rates and lipid productivities than the photoautotrophic culture (without organic carbon source). The lipid productivity obtained with a mixture of optima concentrations of glucose and glycerol (5.0 gL-1 glycerol + 10.0 gL-1glucose) (0.14875 ± 0.002 g/L/day) was about 25% and 66% higher than the values obtained with only 10.0 gL-1glucose and 5.0 gL-1glycerol, respectively. When a batch culture with 5gL-1glycerol was fed with 0.5 gL-1glucose daily the cell growth and lipid productivity were lower than the values obtained in a batch culture with a mixture of glucose and glycerol. The lipid productivity obtained in a 4-L photobioreactor was 94% (0.217 gL-1 day-1), higher than the value obtained in a flask culture with 10.0 g/Lglucose (0.112 gL-1 day-1) and 46% higher than the value obtained in a flask culture with 5.0 gL-1glycerol (0.086 gL-1 day-1).

Production and stability of pigments by Talaromyces purpurogenus LC128689 in an alternating air phase-liquid phase cultivation system.

Effects of carbon source, nitrogen source, and alternatingly submerging the cells and exposing to gaseous oxygen on pigment production by Talaromyces purpurogenus LC128689, as well as pH, temperature, and UV stability of the pigments were investigated. Although fructose supported higher cell growth, a mixture of glucose and glycerol resulted in higher pigment production. Out of the organic and inorganic nitrogen sources investigated, peptone gave the highest cell concentration (7.2 ± 1.1 g/L) and pigment production (p ≤ 0 .05). The cells were then immobilized in loofa sponge and cultivated under alternating liquid phase-air phase (ALAP) system whereby the cells were alternatingly submerged and exposed to gaseous oxygen. After 20 days of cultivation, the concentrations of the red, orange, and yellow pigments were 30.15 AU500 nm , 15 AU460 nm , and 6.25 AU400 nm , respectively. In comparison with submerged culture in flasks, the red and orange pigments were 100% and 50% higher (p ≤ 0.05) in ALAP system. On the other hand, the yellow pigment was 100% higher in flask cultures than in ALAP. The three pigments were stable within a pH range of 2-12, retained more than 80% of their color intensity after autoclaving at (121°C and 1.0 atm) for 15 min and exposure to UV (3 uW/cm2 ) for 24 h.

Analysis of porous breathable stopper and development of PID control for gas phase during shake-flask culture with microorganisms.

We evaluated the ventilation ability of two types (plug-type and cap-type) of culture-stoppers having standard air permeability. The culture-stoppers were evaluated using the circulation direct monitoring and sampling system with CO2 concentration in the gas phase of a shake-flask culture as an index. The half-lives of CO2 in the headspace of the shake flask with the plug-type and cap-type stoppers were about 51.5 min and about 30.3 min, respectively. Based on these half-lives, we formulated a model equation to simulate the behaviour of CO2 with different culture-stoppers. After validating the model equation by shake-flask culture with Saccharomyces cerevisiae, we investigated the effect of different ventilation abilities of the culture-stoppers on the growth of Pelomonas saccharophila and Escherichia coli: the sensitivity of the culture-stopper to the ventilation ability was dependent on the microorganism species. In the case of P. saccharophila, when the plug-type culture-stopper was combined with controlled CO2 concentration (6%) in the flask, the maximum yield increased by twofold compared to that of the control. This study shows the importance of ventilation in headspace and conventional culture-stoppers during the shake-flask culture of microorganisms. The problems that may occur between the conventional shake-flask culture approach using a breathable culture-stopper and the next-generation shake-flask culture without a conventional culture-stopper were clarified from the evaluation of gas-permeable culture-stoppers. The importance of controlled gaseous phase in the headspace during shake-flask culture of the microorganisms was also elucidated. KEY POINTS: • Ventilation capacity of culture-stoppers was evaluated using the CO2 half-life concentration. • Behaviour of microorganisms varies with the type of culture-stopper. • Developed a PID system for control of CO2 in flask gas phase to enhance the shake-flask culture.

Monitoring of CO2 and O2 concentrations in the headspace of Sakaguchi flasks during liquid culture of microorganism.

CO2 and O2 in the Sakaguchi flask headspace during culture was monitored via circulation direct monitoring and sampling system (CDMSS), a device with circulation bypass system. In static culture with Saccharomyces cerevisiae (circulation rate, 50 mL/min), a vertical CO2 concentration gradient (maximum gap ~ 2% (v/v) [height from the bottom of flask 45 mm, 7%; 155 mm, 5%]) in the Sakaguchi flask headspace was observed; no concentration O2 gradient was observed. However, shake flask culture showed vertical gradient concentrations for both CO2 and O2 (maximum gap of CO2 and O2 concentrations: 2 and 4% [heights from the bottom of flask 115 mm, 6.0 and 9.5%; 175 mm, 4.0 and 13.5%], respectively). When the CDMSS circulation rate in the Sakaguchi flask headspace was 300 or 400 mL/min, the gaseous environment was uniformly distributed so that no vertical gradient concentration was observed. In shaking culture with Escherichia coli under these conditions, CO2 was accumulated at high concentrations in the headspace and culture broth (maximum values 8%, in the headspace; 120 mg/L, in the culture broth). Most of the accumulated CO2 in the headspace could be removed by inserting a column packed with CO2 adsorbent at the bypass port of the CDMSS gaseous circulation. Thus, dissolved CO2 was maintained at a lower concentration, and the final UOD (unit optical density) value of culture was increased compared with that of the control. This study is the first to demonstrate that vertical gradients of CO2 and O2 concentrations exist in the headspace of Sakaguchi flask during culture.

Adsorption preference for divalent metal ions by Lactobacillus casei JCM1134.

The removal of harmful metals from the intestinal environment can be inhibited by various ions which can interfere with the adsorption of target metal ions. Therefore, it is important to understand the ion selectivity and adsorption mechanism of the adsorbent. In this study, we estimated the adsorption properties of Lactobacillus casei JCM1134 by analyzing the correlation between its maximum adsorption level (qmax) for seven metals and their ion characteristics. Some metal ions showed altered adsorption levels by L. casei JCM1134 as culture growth time increased. Although it was impossible to identify specific adsorption components, adsorption of Sr and Ba may depend on capsular polysaccharide levels. The maximum adsorption of L. casei JCM1134 (9 h of growth in culture) for divalent metal ions was in the following order: Cu2+ > Ba2+ > Sr2+ > Cd2+ > Co2+ > Mg2+ > Ni2+. The qmax showed a high positive correlation with the ionic radius. Because this tendency is similar to adsorption occurring through an ion exchange mechanism, it was inferred that an ion exchange mechanism contributed greatly to adsorption by L. casei JCM1134. Because the decrease in the amount of adsorption due to prolonged culture time was remarkable for metals with a large ion radius, it is likely that the adsorption components involved in the ion exchange mechanism decomposed over time. These results and analytical concept may be helpful for designing means to remove harmful metals from the intestinal tract.

Practices of shake-flask culture and advances in monitoring CO2 and O2.

About 85 years have passed since the shaking culture was devised. Since then, various monitoring devices have been developed to measure culture parameters. O2 consumed and CO2 produced by the respiration of cells in shaking cultures are of paramount importance due to their presence in both the culture broth and headspace of shake flask. Monitoring in situ conditions during shake-flask culture is useful for analysing the behaviour of O2 and CO2, which interact according to Henry's law, and is more convenient than conventional sampling that requires interruption of shaking. In situ monitoring devices for shake-flask cultures are classified as direct or the recently developed bypass type. It is important to understand the characteristics of each type along with their unintended effect on shake-flask cultures, in order to improve the existing devices and culture conditions. Technical developments in the bypass monitoring devices are strongly desired in the future. It is also necessary to understand the mechanism underlying conventional shake-flask culture. The existing shaking culture methodology can be expanded into next-generation shake-flask cultures constituting a novel culture environment through a judicious selection of monitoring devices depending on the intended purpose of shake-flask culture. Construction and sharing the databases compatible with the various types of the monitoring devices and measurement instruments adapted for shaking culture can provide a valuable resource for broadening the application of cells with shake-flask culture.

Novel method for screening probiotic candidates tolerant to human gastrointestinal stress.

Tolerance to human gastrointestinal stressors is crucial for probiotics to exhibit their health benefits; however, there is no standardised method for screening their stress tolerance. In this study, we proposed a novel method for screening probiotic candidates tolerant to human gastrointestinal stress-gastrointestinal tolerance assay and culture (GTA-C) method-using black polyethylene terephthalate (PET) non-woven fabric as a scaffold to modify the specialized cellulose film (SCF) method. The modified SCF method showed excellent pH-based diffusion of medium components, had minimal effect on the growth of Escherichia coli K12, and improved the visibility of the colonies. Analysis of kimchi samples cultured using the SCF and modified SCF methods revealed that the modified method diversified the cultured bacteria. GTA in a simulated human fasting state using the modified SCF method showed that acid stress significantly affected the growth of four bacteria used as probiotics and that tolerance to acid stress may be species-dependent. Screening of probiotics in kimchi samples resulted in the identification of lactic acid bacteria tolerant to human gastrointestinal stress during fasting. Our results indicate that the modified SCF method (GTA-C method) is useful for screening probiotics resistant to the gastrointestinal environment during fasting.

Simultaneous removal of caesium and strontium using different removal mechanisms of probiotic bacteria.

When radioactive materials are released into the environment due to nuclear power plant accidents, they may enter into the body, and exposing it to internal radiation for long periods of time. Although several agents have been developed that help excrete radioactive elements from the digestive tract, only one type of radioactive element can be removed using a single agent. Therefore, we considered the simultaneous removal of caesium (Cs) and strontium (Sr) by utilising the multiple metal removal mechanisms of probiotic bacteria. In this study, the Cs and Sr removal capacities of lactobacilli and bifidobacteria were investigated. Observation using an electron probe micro analyser suggested that Cs was accumulated within the bacterial cells. Since Sr was removed non metabolically, it is likely that it was removed by a mechanism different from that of Cs. The amount of Cs and Sr that the cells could simultaneously retain decreased when compared to that for each element alone, but some strains showed only a slight reduction in removal. For example, Bifidobacterium adolescentis JCM1275 could simultaneously retain 55.7 mg-Cs/g-dry cell and 8.1 mg-Sr/g-dry cell. These results demonstrated the potentials of utilizing complex biological system in simultaneous removal of multiple metal species.

Development of a simple cultured cell-anaerobic microbial co-culture system using liquid paraffin.

Conventional co-culture systems are complicated, lack versatility, and do not adequately replicate the intestinal lumen environment. This study aimed to devise a system that allows for (i) arbitrary sampling of the culture medium, (ii) monitoring the growth of co-cultured cells, (iii) aerobic-anaerobic co-culture, (iv) simple operation, and (v) evaluation of multiple samples. We built a simple cell-anaerobic microorganism co-culture system using liquid paraffin to separate growth spaces for aerobic cells and anaerobic bacteria. Mineral oil was added to the top of the anaerobic bacterial cultivation space to seal the space and reduce gas exchange. Co-culture of anaerobic, Bifidobacterium bifidum and aerobic, epithelial Madin-Darby canine kidney (MDCK) cells demonstrated that the barrier function and viability of co-cultured MDCK cells were comparable to those of a pure MDCK culture after 24 h, and the growth curve of co-cultured B. bifidum was similar to that of pure B. bifidum. Furthermore, the growth of B. bifidum pure culture under sealed conditions was approximately 1.5 times greater than that under non-sealed conditions at 24 h. Glucose consumption at 24 h of co-culture under sealed conditions was 10%-15% higher than that under non-sealed conditions. This highly versatile culture method enabled the quantitative characterisation of B. bifidum and MDCK cells upon co-culture. The newly established co-culture system could be applied to various aerobic cell-anaerobic bacteria co-culture which will provide a strategy for basic and applied research on host-microbe interactions.

Secretory production of cell wall components by Saccharomyces cerevisiae protoplasts in static liquid culture.

When protoplasts of Saccharomyces cerevisiae T7 and IFO 0309 are cultured in a static liquid culture at 2.5 × 10(6) protoplasts/ml, cell wall regeneration does not occur and cell wall components (CWC) are released into the culture broth. By using a specialized fluorometer, the concentrations of CWC could be measured on the basis of the fluorescence intensity of the CWC after staining with Fluostain I. The inoculum concentration, pH, and osmotic pressure of the medium were important factors for the production of CWC in culture. Under optimal culture conditions, S. cerevisiae T7 protoplasts produced 0.91 mg/ml CWC after 24 h. The CWC induced the tumor necrosis factor-α production about 1.3 times higher than that of the commercially available ß-1,3/1,6-glucan from baker's yeast cells.

Enhancement of microbubble generation in a pressurized dissolution process by packing the nozzle with porous ceramics.

The pressurized dissolution method is often used for microbubble generation. However, the main disadvantage of this method is that a large amount of energy (more than 0.3 MPa) is required to generate many microbubbles, each of which have a diameter of several dozen µm. To overcome this problem, we investigated the effectiveness of porous ceramic when used as the packing material in the pressurized dissolution method. The results showed that when compared with the control (no porous ceramics), use of porous ceramics resulted in a 39% increase in the number of microbubbles. Furthermore, when this system was used for the flotation separation of artificial suspended solids and activated sludge, the level of separation achieved with porous ceramics at 0.15 MPa was the same as that achieved using no porous ceramics at 0.25 MPa. It was estimated that the use of porous ceramics led to a 40% reduction in the energy required for the dissolved air flotation, with subsequent decreases in the operating cost.

Stabilization of a raw starch digesting amylase from Aspergillus carbonarius via immobilization on activated and non-activated agarose gel.

Applications of raw starch digesting amylases (RSDAs) are limited due to instability, product inhibition of enzyme and contamination. RSDA from Aspergillus carbonarius was stabilized through immobilization on agarose gel by adsorption, spontaneous crosslinking and conjugation using glycidol, glutaraldehyde or polyglutaraldehyde. Effects of immobilization on kinetics, catalytic, storage and operational stability of immobilized enzyme were evaluated. Polyglutaraldehyde activated agarose RSDA (PGAg-RSDA) gave the highest immobilization yield (100%) with expressed activity of 86.7% while that of glycidol activated RSDA (GlyAg-RSDA) was 80.4%. A shift in pH from optimum of 5 for the soluble enzyme to 6 for RSDA adsorbed on agarose followed by crosslinking with glutaraldehyde (AgRSDA-CROSS) and simultaneous adsorption and crosslinking (AgRSDA-RET), and pH 7 for PGAg-RSDA was seen. PGAg-RSDA and AgRSDA-CROSS were most pH stable and retained over 82% of their activities between pH 3.5 and 9 compared to 59% for the soluble enzyme. Thermoinactivation studies showed that immobilized RSDAs with the exception of GAg-RSDA retained over 90% of their activities at 60°C for 120 min while soluble enzyme retained only 76% activity under the same condition. AgRSDA-CROSS, PGAg-RSDA, Gly-RSDA and GAg-RSDA retained approximately 100% of their activities after 30 days storage at 4°C. GlyAg-RSDA retained 99.6%, PGAg-RSDA 94%, AgRSDA-CROSS 90%, GAg-RSDA 86.5% and Ag-RSDA-RET 80% activity after 10 batch reactions. Immobilization stabilized RSDA and permits processing at higher temperatures to reduce contamination.

Control of carbon dioxide concentration in headspace of multiple flasks using both non-electric bellows pump and shaking incubator.

Current methods of controlling gas in the headspace involve constant speed aeration and proportional-integral-differential (PID) controlled aeration using improved monitoring devices or gas cylinders. However, these approaches are restricted and inconvenient to use. In this study, we propose a method to control the CO2 concentration in the headspace while maintaining the convenience of shake-flask culture. A combination of a non-electric bellows pump for shake-flask (NeBP-sf) and a CO2 incubator was used to control the flask gas phase by shaking without additional external power. The CO2 half-life, as an indicator of the ventilation ability of the system, was measured using a circulation direct monitoring and sampling system, and the NeBP-sf was optimised. The ventilation capacity varied depending on the shaking speed, and under optimal conditions, was 10 min compared with 45 min when only a breathable culture plug was used. In conventional microbial shaking culture, the CO2 concentration in the flask gas phase remained higher than the 5% set-value with a maximum of 9%, resulting in a large concentration difference with the set point. Therefore, the ventilation capacity of the conventional shake-flask culture was insufficient for aerobic culture. Cultivation of Escherichia coli and Lactiplantibacillus plantarum using the system showed no significant difference between the set point and real point values. Thus, the system combined an NeBP-sf and a gas incubator built-in shaking table to achieve the reproducibility of gas control while maintaining a high level of convenience.

Screening and isolation of acid-tolerant bacteria using a novel pH shift culture method.

Acid-tolerant bacteria, which multiply under neutral pH and can survive under acidic pH conditions, have a potential role in various applications under acidic conditions. Despite higher biomass productivity, their isolation and utilisation are not sufficiently developed compared to those of acidophiles. It takes considerable effort to distinguish the acid-tolerant bacteria from the rest of the bacterial community using conventional screening methods. Thus, we developed a novel screening method for acid-tolerant bacteria, which involves shifting the pH between acidic and neutral conditions. With this method, the bacterium Enterobacter sp. AC06 was isolated. Based on comparisons with the results reported in previous studies, the strain can be classified as acid-tolerant bacteria. The decreases in the live cell concentrations were 3.87 and 6.16 log cycles after 3 h acid treatment under pH 3.0 and 2.5, respectively. These results suggest that it is possible to isolate acid-tolerant bacteria using the pH shift culture method. In summary, this is the first study on bacterial screening based on acid tolerance. Our novel method potentially contributes to the understanding and utilisation of acid-tolerant bacteria by enhancing screening efficiency. Furthermore, our novel concept shift culture is potentially valuable for screening previously uncultured bacteria tolerant to various selective stress conditions.

Use of Catharanthus roseus Cell Cultures for the Synthesis of Metal Nanoparticles.

Metal nanoparticles have found applications in many fields owing to their unique physicochemical characteristics and ease of surface functionalization. Crucial for these applications is the development of environmentally friendly strategies for nanoparticle preparation. Nanoparticles can be prepared using several physical and chemical methods; however, the use of nontoxic and eco-friendly approaches is receiving increasing attention. Plant cell cultures are sustainable sources of bioactive compounds that can act as reducing and stabilizing agents during nanoparticle synthesis. Here, we describe the procedures used to synthesize silver and gold nanoparticles with cultured cells of Catharanthus roseus. The bioreduction of silver ions to nanoparticles with extract of seed-derived callus of C. roseus is evident from UV-Vis spectroscopy results wherein an absorption maxima is observed at 425 nm, indicating the formation of elemental silver. Similarly, reaction mixtures containing cell-free suspension culture filtrate of C. roseus and gold III ions turn wine red after 24 h incubation because of gold nanoparticle formation. These methods can be easily adapted for use in the preparation of other metal nanoparticles.

Fungal Pigment-Assisted Silver Nanoparticle Synthesis and Their Antimicrobial and Cytotoxic Potential.

The biological production of nanoparticles has taken center stage among the various generation methods available owing to its environment-friendly characteristics and reduced energy requirements. A protocol for pigment-assisted silver nanoparticle (AgNP) synthesis was established, employing an extracellular composite pigment produced by the Ascomycota Talaromyces purpurogenus (presently Talaromyces purpureogenus). The extracellular pigment can reduce the precursor silver salt into nanoparticles in the presence of sodium hydroxide and light. Transmission electron microscopy may be used to characterize the bio-generated nanoparticles, which can also be tested for their biological properties using antimicrobial and anticancer assays.

Development of a device for cultivation and isolation of microbes using a specialized cellulose film.

A device was developed for the cultivation and isolation of microbes using a specialized cellulose film (SCF). This method can solve problems associated with the agar plate method. It can also be used for other culture-related operations, which is not possible with the traditional agar plate culture method.