Increase In Cell Concentration Research Articles

Highly efficient inoculum propagation in perfusion culture using WAVE Bioreactor™ systems

A perfusion-based process was developed to increase the split ratio during the scale-up of CHO-S™ cell cultures. Fedbatch cultures were inoculated with cells propagated in either batch or perfusion cultures. All cultures were grown in disposable Cellbag™ bioreactors using the WAVE Bioreactor system. Cell concentrations of 4.8 × 107 cells/mL were achieved in the perfusion culture, whereas the final cell concentration in the batch culture was 5.1 × 106 cells/mL. The higher cell concentration of the perfusion culture allowed for a more than six-fold increase of the split ratio to about 1:30. The method described here, can reduce the number of required expansion steps and eliminate the need for one or two bioreactors in the seed train. Single-use bioreactors at benchtop scale can be used for direct inoculation of production bioreactors. Alternatively, high biomass concentrations accumulated in perfusion culture can be used to seed production vessels at increased cell concentrations. Thus, the process time in these bioreactors, which often is the bottleneck in plant throughput, can be shortened.

Continuing Multiplication of Salmonella enteritidis Strains in Egg Yolk During Refrigeration at 7.2oC

The continuing attribution of human illness caused by Salmonella enteritidis to the consumption of contaminated eggs has led to widespread implementation of risk reduction programs for commercial egg production, often emphasizing prompt refrigeration of eggs to prevent bacterial multiplication to dangerously high levels. However, microbial growth may not cease immediately inside warm eggs after transfer to refrigerated storage. The present study compared the abilities of 8 S. enteritidis strains (of 4 phage types) to continue multiplying in experimentally contaminated egg yolk during the first 24 h after transition from warm to refrigeration temperatures. After 15 mL samples of egg yolk were inoculated with 10 CFU/ml of S. enteritidis, they were incubated at 37°C for 16 h and then transferred into refrigeration at 7.2°C for 24 h. Bacterial cell concentrations were determined following 37°C incubation and again after both 8 and 24 h at 7.2°C. All 8 S. enteritidis isolates multiplied significantly during 16 h of incubation, reaching an overall mean of log 8.790 CFU/ml. After refrigeration, the observed mean values for cell concentrations in yolk samples 10 were log10 8.780 CFU/mL at 8 h and log10 8.849 CFU/mL at 24 h. For 3 of 8 strains, a significant (p<0.05) increase in cell concentrations in egg yolk occurred during 24 h of refrigeration. These results support the importance of prompt egg refrigeration for minimizing the numbers of S. enteritidis in marketed table eggs, although refrigeration at 7.2 C may not immediately or completely arrest multiplication by all strains. o

Immobilization of New Isolated Iron Oxidizing Bacteria on Natural Carriers

The bioleaching of sulfide minerals by iron oxidizing bacteria are implemented by direct and indirect mechanisms. The direct dissolution of minerals is caused by the attack of sulfide ions by enzymatic system of bacteria. In the indirect mechanism the ferric ion from oxidation of ferrous iron serves as a leaching agent that reacts chemically with the minerals. The increase of ferrous ion oxidation contributes to the intensification of bioleaching process. On this purpose the immobilization of iron oxidizing bacteria on different organic and inorganic carriers (calcium alginate, carragiran, ceramic support, activated carbon, porous glass, etc.) has been implemented which allows to increase cell concentration. In the present work for the first time the native shungit, zeolit and their chemically modified forms have been used for immobilization of new isolated iron oxidizing bacteria of the genera Leptospirillum and Sulfobacillus. Efficient physico-chemical conditions for immobilization on the mentioned carriers have been developed. The ferrous ion oxidation by immobilized cells has been studied both in shake-flasks experiments and in the glass column using air-lift process. It has been shown that in both cases the rate of iron oxidation considerably enhances in comparison with free cells.

Differential Modulation of Human Intestinal Bifidobacterium Populations after Consumption of a Wild Blueberry (Vaccinium angustifolium) Drink

Bifidobacteria are gaining increasing interest as health-promoting bacteria. Nonetheless, the genus comprises several species, which can exert different effects on human host. Previous studies showed that wild blueberry drink consumption could selectively increase intestinal bifidobacteria, suggesting an important role for the polyphenols and fiber present in wild blueberries. This study evaluated the modulation of the most common and abundant bifidobacterial taxonomic groups inhabiting the human gut in the same fecal samples. The analyses carried out showed that B. adolescentis, B. breve, B. catenulatum/pseudocatelulatum, and B. longum subsp. longum were always present in the group of subjects enrolled, whereas B. bifidum and B. longum subsp. infantis were not. Furthermore, it was found that the most predominant bifidobacterial species were B. longum subsp. longum and B. adolescentis. The results obtained revealed a high interindividual variability; however, a significant increase of B. longum subsp. infantis cell concentration was observed in the feces of volunteers after the wild blueberry drink treatment. This bifidobacterial group was shown to possess immunomodulatory abilities and to relieve symptoms and promote the regression of several gastrointestinal disorders. Thus, an increased cell concentration of B. longum subsp. infantis in the human gut could be considered of potential health benefit. In conclusion, wild blueberry consumption resulted in a specific bifidogenic effect that could positively affect certain populations of bifidobacteria with demonstrated health-promoting properties.

아가로스 겔에 포함된 세포의 농도가 확산 계수에 미치는 영향 측정

In this study, diffusion coefficients of 20 kDa FITC-dextran in 2% agarose gel with different cell concentrations were measured using fiberoptic-based fluorescence recovery after photobleaching technique. As increasing cell concentration suspended in agarose gel, the diffusion coefficients were decreased. The diffusion coefficient of agarose gel which contains cells/ml was decreased to 11% that of in agarose gel without cells. The distribution of fluorescence dye in 3D scaffold was also simulated. The simulation result shows that the diffusion coefficient is more significant factor than the scaffold structure.

Scale‐up and intensification of (S)‐1‐(2‐chlorophenyl)ethanol bioproduction: Economic evaluation of whole cell‐catalyzed reduction of o‐Chloroacetophenone

Escherichia coli cells co-expressing genes coding for Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase were used for the bioreduction of o-chloroacetophenone with in situ coenzyme recycling. The product, (S)-1-(2-chlorophenyl)ethanol, is a key chiral intermediate in the synthesis of polo-like kinase 1 inhibitors, a new class of chemotherapeutic drugs. Production of the alcohol in multi-gram scale requires intensification and scale-up of the biocatalyst production, biotransformation, and downstream processing. Cell cultivation in a 6.9-L bioreactor led to a more than tenfold increase in cell concentration compared to shaken flask cultivation. The resultant cells were used in conversions of 300 mM substrate to (S)-1-(2-chlorophenyl)ethanol (e.e. >99.9%) in high yield (96%). Results obtained in a reaction volume of 500 mL were identical to biotransformations carried out in 1 mL (analytical) and 15 mL (preparative) scale. Optimization of product isolation based on hexane extraction yielded 86% isolated product. Biotransformation and extraction were accomplished in a stirred tank reactor equipped with pH and temperature control. The developed process lowered production costs by 80% and enabled (S)-1-(2-chlorophenyl)ethanol production within previously defined economic boundaries. A simple and efficient way to synthesize (S)-1-(2-chlorophenyl)ethanol in an isolated amount of 20 g product per reaction batch was demonstrated.

An experimental model approach of biologically-assisted silicate dissolution with olivine and Escherichia coli – Impact on chemical weathering of mafic rocks and atmospheric CO2 drawdown

Chemical weathering of Mg, Ca-silicates and alumino-silicates contributes significantly to the drawdown of atmospheric CO2 over long time scales. The present work focuses on how this mode of weathering may change in the presence of free-living bacteria in oligotrophic waters, which compose most of the surface freshwaters of the Earth. Forsterite (Fo90) was reacted for 1week with a stable Escherichia coli population in water maintained at 37°C and neutral pH in a batch reactor. Control samples with suspensions of pure olivine powders and E. coli cells in pure water were also used for reference. Olivine controls reproduce the Mg, Si and Fe release in solutions predicted from rates published in the literature with pH shifts of less than 0.5 unit. After 1week, under abiotic conditions, weathered surfaces are enriched in Fe and Fe3+ relative to the initial composition of the mineral. Bacterial controls (without minerals) show decreasing Eh with increasing cell concentrations (−50mV with 7×107cells/mL and −160mV with 8×108cells/mL). Magnesium concentrations in bacterial control solutions are in the μg/L range and can be accounted for by the release of Mg from dead cells. More than 80% of the cells were still alive after 1week. The solutions obtained in the experiments in which olivine reacts in the presence of cells show Mg and Si concentrations a few tens of percent lower than in the mineral control samples, with a prominent depletion of Fe(III) content of the mineral surfaces. Magnesium mass balance discounts both significant bacterial uptake and inhibition of the Mg dissolution rates as a consequence of changing pH and Eh. Coating by bacterial cell layers is also negligible. E. coli reduces the chemical weathering of olivine. This study infers that the presence of free-living Proteobacteria, a prevalent group of subsurface bacteria, should decrease the amount of riverine Mg released by chemical weathering of mafic rocks.

Microbial Decolorization and Degradation of Orange 16 Dye by a Newly Isolated Aeromonas Spp. Etl-1949

A bacterial strain ETL-1949, identified as Aeromonas spp. was isolated from sediment from a textile wastewater treatment plant in Ankleshwar, Gujarat, India. The strain could decolorize an azo dye, Orange 16 under the static condition after the shaking culture. In order to increase cell concentration by the shaking culture, the optimum culture condition was investigated for following the decolorization incubation. The dye was decolorized completely up to concentration of 750 mg/l for 50 hr under static condition by using 12 hr-cultured cells at optimal shaking culture condition. Effect of initial cell mass showed that higher cell mass concentration can accelerate decolorization process with maximum of 92% decolorization observed at 2.5 g/l cell mass within 6.5 h. Effect of various metal ions showed Mn & Mg has positive inducing effect whereas Zn strongly inhibited the decolorization process at 5 mM concentration. Analysis of biodegradation products carried out with UV–vis spectroscopy, HPTLC and FTIR confirmed the decolorization and degradation of Orange 16.

Continuous butanol fermentation from xylose with high cell density by cell recycling system

A continuous butanol production system with high-density Clostridium saccharoperbutylacetonicum N1-4 generated by cell recycling was established to examine the characteristics of butanol fermentation from xylose. In continuous culture without cell recycling, cell washout was avoided by maintaining pH>5.6 at a dilution rate of 0.26h−1, indicating pH control was critical to this experiment. Subsequently, continuous culture with cell recycling increased cell concentration to 17.4gL−1, which increased butanol productivity to 1.20gL−1h−1 at a dilution rate of 0.26h−1 from 0.529gL−1h−1 without cell recycling. The effect of dilution rates on butanol production was also investigated in continuous culture with cell recycling. Maximum butanol productivity (3.32gL−1h−1) was observed at a dilution rate of 0.78h−1, approximately 6-fold higher than observed in continuous culture without cell recycling (0.529gL−1h−1).

Decolorization of azo dyes by Geobacter metallireducens

Geobacter metallireducens was found to be capable of decolorizing several azo dyes with different structures to various extents. Pyruvate, ethanol, acetate, propionate, and benzoate could support 66.3 ± 2.6-93.7 ± 2.1% decolorization of 0.1mM acid red 27 (AR27) in 40h. The dependence of the specific decolorization rate on AR27 concentration (25 to 800μM) followed Michaelis-Menten kinetics (K m = 186.9 ± 1.4μΜ, V max = 0.65 ± 0.02μmol mg protein(-1)h(-1)). Enhanced AR27 decolorization was observed with the increase of cell concentrations ranging from 7.5 to 45mgL(-1). AR27 decolorization by G. metallireducens was retarded by the presence of goethite, which competed electrons with AR27 and was reduced to Fe(II). The addition of low concentrations of humic acid (1-100mgL(-1)) or 2-hydroxy-1,4-naphthoquinone (0.5-50μM) could improve the decolorization performance of G. metallireducens. High-performance liquid chromatography analysis suggested reductive pathway to be responsible for decolorization. This was the first study on azo dye decolorization by Geobacter strain and might improve our understanding of natural attenuation and bioremediation of environments polluted by azo dyes.

Erratum to: Luciferase-transfected colon adenocarcinoma cell line (DLD-1) for use in Orthotopic Xenotransplantation studies

Renilla Luciferase reporter gene (rLuc) GL4.82 and GL4.13 promoter are key player in transfection, but precise knowledge of its targets in colon cancer remains limited. The aim of this study was to characterize the best transfection technique to produce a stable transfected colon DLD1 (colorectal adenocarcinoma cell line), therefore imaging based approaches were employed.DLD1 cells were transfected with a Plasmid (SV40-RLuc) carrying Renilla luciferase under the control of the SV-40 promoter, by using two different transfection techniques. Cells expressing the required DNA were isolated after antibiotic (Puramycin) selection. Clones of DLD-1/SV40-RLuc were produced using two different techniques (96 well plates and Petri dish) and their florescence intensity was recorded using IVIS machine (Calliper Life Sciences, Hopkinton, USA). Both techniques were characterized with the help of serial dilution technique. Results from this study substantiated that electroporation is the best. As expected, clones varied in their specific luciferase activity along with the dilutions. With the increase in cell concentration increase in intensity of florescence was recorded.Based on the results we are confident that this transfected cell line DLD-1/SV40-RLuc (colorectal adenocarcinoma cell line) is the best for further Orthotopic Xenotransplantation Studies and in-vivo experiments as well. Investigation shows that DLD1/SV-rLuc cells have gained little bit resistance against both drugs therefore further study is suggested to know the reasons.

Aggregation by depletion attraction in cultures of bacteria producing exopolysaccharide

In bacteria, the production of exopolysaccharides--polysaccharides secreted by the cells into their growth medium--is integral to the formation of aggregates and biofilms. These exopolysaccharides often form part of a matrix that holds the cells together. Investigating the bacterium Sinorhizobium meliloti, we found that a mutant that overproduces the exopolysaccharide succinoglycan showed enhanced aggregation, resulting in phase separation of the cultures. However, the aggregates did not appear to be covered in polysaccharides. Succinoglycan purified from cultures was applied to different concentrations of cells, and observation of the phase behaviour showed that the limiting polymer concentration for aggregation and phase separation to occur decreased with increasing cell concentration, suggesting a 'crowding mechanism' was occurring. We suggest that, as found in colloidal dispersions, the presence of a non-adsorbing polymer in the form of the exopolysaccharide succinoglycan drives aggregation of S. meliloti by depletion attraction. This force leads to self-organization of the bacteria into small clusters of laterally aligned cells, and, furthermore, leads to aggregates clustering into biofilm-like structures on a surface.

Magnetite nanoparticles doped photoresist derived carbon as a suitable substratum for nerve cell culture

A method which alters the substrate's physical and electrochemical properties by doping photoresist derived carbon with magnetite nanoparticles has been developed to enhance the existing substrate's ability to foster cell growth. Cyclic voltammetry, scanning electron microscopy and atomic force microscopy are used to evaluate the characters of the prepared film. And then, the magnetite nanoparticles doped carbon film is used as substrate for the growth of nerve cell. Here, rat pheochromocytoma cells are used for culture to test substrate–cell interactions. The results showed an increase in cell concentration and average neurite length with the increase of nanoparticle concentration on the surface. Importantly, the nerve cells can be grown on the magnetite nanoparticles doped carbon even in the absence of nerve growth factor. This finding will potentially provide a new material for nerve regeneration.

Multifractal dynamics of turbulent flows in swimming bacterial suspensions

We experimentally investigate the self-propelled two-dimensional turbulent flows of Escherichia coli suspensions in thin liquid films at two different cell concentrations. It is found that the flow has fluctuating vortices with a broad range of scales and intensities through the nonlinear interaction of the swimming bacteria. Increasing cell concentration increases the total propelling power and the nonlinear interaction. It causes the generation of vortices with larger scale, lower frequency, and higher intensity. It also widens the histograms of the flow velocity and the velocity increment between two spatially separated points with more stretched non-Gaussian tails. From the scaling analysis of the structure function S(q)(r) of the qth moment of the velocity increment between two points with spatial separation r, nonlinear relations between the scaling exponent ζ(q) of S(q)(r) and q are found for both cell concentrations, which manifests the multifractal dynamics. The multifractality can be enhanced by increasing cell concentration.

Retracted: Luciferase-transfected colon adenocarcinoma cell line (DLD-1) for use in Orthotopic Xenotransplantation studies.

BackgroundRenilla Luciferase reporter gene (rLuc) GL4.82 and GL4.13 promoter are key player in transfection, but precise knowledge of its targets in colon cancer remains limited. The aim of this study was to characterize the best transfection technique to produce a stable transfected colon DLD1 (colorectal adenocarcinoma cell line), therefore imaging based approaches were employed.ResultsDLD1 cells were transfected with a Plasmid (SV40-RLuc) carrying Renilla luciferase under the control of the SV-40 promoter, by using two different transfection techniques. Cells expressing the required DNA were isolated after antibiotic (Puramycin) selection. Clones of DLD-1/SV40-RLuc were produced using two different techniques (96 well plates and Petri dish) and their florescence intensity was recorded using IVIS machine (Calliper Life Sciences, Hopkinton, USA). Both techniques were characterized with the help of serial dilution technique. Results from this study substantiated that electroporation is the best. As expected, clones varied in their specific luciferase activity along with the dilutions. With the increase in cell concentration increase in intensity of florescence was recorded.ConclusionsBased on the results we are confident that this transfected cell line DLD-1/SV40-RLuc (colorectal adenocarcinoma cell line) is the best for further Orthotopic Xenotransplantation Studies and in-vivo experiments as well. Investigation shows that DLD1/SV-rLuc cells have gained little bit resistance against both drugs therefore further study is suggested to know the reasons.

Effect of organic load and nutrient ratio on the operation stability of the moving bed bioreactor for kraft mill wastewater treatment and the incidence of polyhydroxyalkanoate biosynthesis

This paper studies the effect of organic load rate (OLR) and nutrient ratio on operation stability of the moving bed bioreactor (MBBR) for kraft mill wastewater treatment, analyzing the incidence of polyhydroxyalkanoate (PHA) production. The MBBR operating strategy was to increase OLR from 0.25 ± 0.05 to 2.41 ± 0.19 kg COD m(-3) d(-1) between phases I and IV. The BOD(5):N:P ratio (100:5:1 and 100:1:0.2) was evaluated as an operation strategy for phases IV to V. A stable MBBR operation was found when the OLR was increased during 225 days in five phases. The maximum absolute fluorescence against the proportion of cells accumulating PHA was obtained for an OLR of 2.41 ± 0.19 kg COD m(-3)d(-1) and a BOD(5):N:P relationship of 100:1:0.2. The increase of PHA biosynthesis is due to the increased OLR and is not attributable to the increased cell concentration, which is maintained constant in stationary status during bioreactor biosynthesis.

Cells adhered and cultured on microcantilevers

This paper shows a novel method to cultivate cells on a π-shape microcantilever inside a polydimethylsiloxane microfluidic system. Only one lithography step was needed to precisely align and pattern a poly(2-hydroxyethyl methacrylate) hydrogel microstructure, of size 200 × 200 μm, onto a silicon nitride microcantilever inside the PDMS microfluidic device. Gelatin was used as a sacrificial layer to resolve the issue of the microfluidic and hydrogel microstructure sticking together, successfully releasing the microcantilevers. BHK-21 cells were successfully laden and cultivated on the hydrogel microstructures of microcantilevers for 24 h. The optical system consisted of a He–Ne laser, a charge-coupled device camera, and a position-sensitive detector, which was used to measure the deflections of the microcantilevers due to the laden cells. The deflection increased continually during the cell-laden period. Meanwhile, the deflection increased with increasing cell concentration. By repeating the cell-laden and culture experiment three times, the magnitude and trend of deflection of microcantilevers were almost the same. It demonstrates that the microcantilever-based biochip has adequate stability and provides reliable measurement results for drug screening applications in the future.

The role of hydrodynamic conditions and pH on algal-rich water fouling of ultrafiltration

The aim of this paper was to study the membrane fouling phenomena by eutrophic water using Microcystis aeruginosa under various operational conditions (flux and air flow rate) and solution chemistry (pH). All the experiments were performed in a lab scale employing the polyvinyl chloride ultrafiltration membrane with nominal cut-off of 10 kDa. A slight fouling appeared at the flux not more than 10 L/m2/h, and the trend of trans-membrane pressure (TMP) development varied as a function of flux from linear to exponential with the increase of cell concentration. This paper also studied an important consideration of aeration in algal fouling: shear force. Besides alleviating membrane fouling, the shear produced by the bubbling should take responsible for the breakup of cells and the release of intracellular organic matters which caused the rate of the TMP increase closed to that without aeration. The optimum aeration intensity was observed to be 2.5 m3/m2/h in this experimental condition. As another important parameter considered in the study, the pH value of the raw water changed the physical and chemical reaction between the membrane and foulants or themselves. The results showed that the final TMP reduced with the pH increase due to the electro-static repulsion strengthening between the macromolecules which developed a looser gel. The most severe fouling was obtained at pH 5.0 near to the iso-electric point of algal solution, where electrostatic repulsion between algal cells was weakest. Furthermore, low pH value had a negative impact on cell integrity which gave rise to much more dissolved algogenic organic matter in the solution. It also played a part role on the membrane fouling.

Evaluation of DNA isolation procedures for detecting and quantifying environmental Legionella by real-time quantitative PCR

Six methods, QiAamp DNA Mini Kit (Q), Q with Sepharose 4B gel column (Q/G), Q with low melting point agarose (Q/L), freeze-thaw/phenol-chloroform lysis (FT-PC), FT-PC/G, and FT-PC/L, were evaluated for their ability to isolate DNA of sufficient quality to quantify Legionella using qPCR. Samples of mixing Legionella pneumophila (ATCC33152) and humic acid (HA, 0-126.8 mg/l) were treated by the six methods. Q, Q/G, Q/L, FT-PC/G, and FT-PC/L removed HA from 1.9-126.8 to <1 mg/l determined by A260 with a spectrophotometer. Q obtained the highest DNA yield, followed by Q/G. Dilution (10- to 100-fold) of DNA arising from extraction using Q, Q/G, FT-PC, or FT-PC/G prevented qPCR inhibition. The highest recovery of cells was found in DNA extracted by Q and diluted 100-fold, and followed by Q/G. The applicability of Q and Q/G with dilution was further validated with cooling tower waters. Q or Q/G with 10-fold dilution increased L. pneumophila detection, whereas 100-fold dilution obtained the highest cell concentrations. Similar results were found for Legionella spp. except that both 10- and 100-fold dilutions increased cell concentrations. Thus, Q with 10-fold dilution is suggested to detect and quantify Legionella spp. and detect L. pneumophila. For L. pneumophila-positive samples, 100-fold diluted DNA must be re-analyzed to accurately quantify L. pneumophila.

Enzymatic Production of Ethanol from Cassava Starch Using Two Strains of Saccharomyces cerevisiae

ABSTRACT Cassava starch from TMS 30572 and Idileru were hydrolyzed with α-amylase and amylo-glucosidase before fermentation using two strains of Saccharomyces cerevisiae from palm wine and bakers’ yeast. The per cent yield of sugars and total dissolved solids were 66 % and 26% respectively while pH was 7. Spectrophotometric measurement of the cell growth revealed steady but insignificant (p ≤ 0.05) increase in cell concentrations up to 48 h fermentation time with a gradual decline by 72 h. Saccharomyces cerevisiae strain from palm wine grew best on TMS 30572 hydrolysate at 20% sugar concentration (optical density 0.663; fermentation time 48 h) while on Idileru hydrolysate it grew best at 25% (optical density 0.698; fermentation time 60 h). The pH values obtained from the fermenting hydrolysates for both yeast strains declined gradually as the fermentation progressed with the lowest pH values (3.01 for S. cerevisiae from palm wine; 3.06 for S. cerevisiae from bakers’ yeast) obtained for TMS 30572 cassava variety at 25% sugar concentration. Changes in pH were significant (p ≤ 0.05). The Saccharomyces cerevisiae strain from palm-wine had a higher conversion of available sugar into ethanol. The yield of ethanol was found to vary but the highest ethanol concentration obtained was 5.3% at 10% initial sugar concentration, which gave a sugar conversion efficiency of 37.3%. The results obtained suggest that Saccharomyces cerevisiae strains from sources other than those used conventionally can serve as good substitutes for bio-conversion processes in the industrial production of ethanol.