Relationship between respirometric activity and community of entrapped nitrifying bacteria: Implications for partial nitrification

Activated sludge obtained from two municipal wastewater treatment facilities (WWTF) was used as seed sludge for enriched nitrifiers, which were later entrapped in polyvinyl alcohol. Seed sludge from one WWTF was acclimated to high ammonia level (1813 mg NH₃-N l^?1) through the return of sludge digester supernatant back to primary clarifier while seed sludge from the other WWTF was un-acclimated. To elucidate on how to control partial nitrification by entrapped cells, which could be different from suspended cells, kinetics of entrapped enriched nitrifiers were studied using a respirometric assay. The community of nitrifiers within the entrapment matrix, which was observed by fluorescence in situ hybridization (FISH) technique, was related to the nitritation and nitratation kinetics based on oxygen uptake rate. Maximum oxygen uptake rate, and substrate and oxygen affinities of both ammonia oxidizing bacteria (AOB) for nitritation and nitrite oxidizing bacteria (NOB) for nitratation in entrapped cells were lower than those of corresponding suspended cells. Under dissolved oxygen (DO) limiting conditions, nitratation was more suppressed than nitritation for suspended cells, while for the entrapped cells, the results were the contrary. A free ammonia (FA) inhibition affected only the un-acclimated sludge. Either FA inhibition or DO limitation might not be a sole effective control parameter to achieve partial nitrification by entrapped cells. FISH results revealed that Nitrosomonas europaea was the dominant AOB while Nitrobacter species was the dominant NOB in all cases. Heterotrophs were also present in the entrapment at 22.8 ± 18.6% and 41.5 ± 4.3% of total bacteria for acclimated and un-acclimated originated sludge. The availability of substrate and oxygen governed the distributions of AOB, NOB and heterotrophs within the entrapment and nitritation kinetics of entrapped nitrifiers.     

Effects of cell entrapment on growth rate and metabolic activity of pure cultures commonly found in biological wastewater treatment

The effects of cell entrapment on the growth rate and metabolic activity of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different cell entrapment media (alginate, carrageenan and polyvinyl alcohol) and three cell-to-matrix ratios (0.1%, 0.2% and 0.6%, w v⁻¹) were examined. Representative species of betaproteobacteria were Alcaligenes faecalis and Comamonas testosteroni whereas Pseudomonas putida was a gammaproteobacteria species studied. Free (non-entrapped) cells were included in the study for comparative purpose. Results indicated that the entrapment, type of entrapment media, and cell-to-matrix ratio had significant effects on the growth and metabolic activity of major groups of bacteria in wastewater treatment. Polyvinyl alcohol entrapped cells had the highest specific growth and specific substrate utilization rates. Increase of cell-to-matrix ratio (from 0.1% to 0.2% or 0.6%) did not improve the specific growth and specific substrate utilization rates. The relative performances provided by different entrapment media of the three species studied were quite consistent. This study showed that the suitable choices of entrapment media and cell-to-matrix ratio are important but similar for major groups of bacteria in wastewater treatment.     

Mass entrapment and lysis of Mesodinium rubrum cells in mucus threads observed in cultures with Dinophysis

The entrapment and death of the ciliate Mesodinium rubrum in the mucus threads in cultures with Dinophysis is described and quantified. Feeding experiments with different concentrations and predator–prey ratios of Dinophysis acuta, Dinophysis acuminata and M. rubrum to study the motility loss and aggregate formation of the ciliates and the feeding behaviour of Dinophysis were carried out. In cultures of either Dinophysis species, the ciliates became entrapped in the mucus, which led to the formation of immobile aggregates of M. rubrum and subsequent cell lysis. The proportion of entrapped ciliates was influenced by the concentration of Dinophysis and the ratio of predator and prey in the cultures. At high cell concentrations of prey (136 cells mL⁻¹) and predator (100 cells mL⁻¹), a maximum of 17% of M. rubrum cells became immobile and went through cell lysis. Ciliates were observed trapped in the mucus even when a single D. acuminata cell was present in a 3.4 mL growth medium. Both Dinophysis species were able to detect immobile or partly immobile ciliates at a distance and circled around the prey prior to the capture with a stretched out peduncle. Relatively high entrapment and lysis of M. rubrum cells in the mucus threads indicates that under certain conditions Dinophysis might have a considerable impact on the population of M. rubrum.     

Survival of Bifidobacterium longum Immobilized in Calcium Alginate Beads in Simulated Gastric Juices and Bile Salt Solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Ethanol-based proliposome delivery systems of paclitaxel for in vitro application against brain cancer cells

In this study the anticancer activity of paclitaxel-loaded nano-liposomes on glioma cell lines was investigated. Soya phosphatidylcholine:cholesterol (SPC:Chol), hydrogenated soya phosphatidylcholine:cholesterol (HSPC:Chol) or dipalmitoylphosphatidylcholine:cholesterol (DPPC:Chol) in 1:1?mole ratio were used to prepare ethanol-based proliposomes. Following hydration of proliposomes, the size of resulting vesicles was subsequently reduced to nanometer scale via probe-sonication. The resulting formulations were characterized in terms of size, zeta potential and morphology of the vesicles, and entrapment efficiency of paclitaxel (PX) as well as the final pH of the preparations. DPPC-liposomes entrapped 35–92% of PX compared to 27–74% and 25–60% entrapped by liposomes made from SPC and HSPC formulations respectively, depending on drug concentration. The entrapment efficiency of liposomes was dependent on the lipid bilayer properties and ability of PX to modify surface charge of the vesicles. In vitro cytotoxicity studies revealed that PX-liposome formulations were more selective at inhibiting the malignant cells. The cytotoxicity of PX-liposomes was dependent on their drug-entrapment efficiency. This study has shown PX-liposomes generated from proliposomes have selective activity against glioma cell lines, and the synthetic DPPC phospholipid was most suitable for maximized drug entrapment and highest activity against the malignant cells in vitro.     

Effects of entrapment on nucleic acid content,cell morphology,cell surface property,and stress of pure cultures commonly found in biological wastewater treatment

The effects of cell entrapment on nucleic acid content, cell morphology, cell surface property, and stress of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different entrapment media (alginate, carrageenan, and polyvinyl alcohol) were examined. Results indicated that the entrapment and type of entrapment media affected nucleic acid content, cell morphology, cell surface property, and stress of the three representative species (Alcaligenes faecalis, Comamonas testosteroni, and Pseudomonas putida) studied. The highest deoxyribonucleic acid and ribonucleic acid increases were observed with the alginate and polyvinyl alcohol (PVA) entrapment, respectively. A cell morphological change from bacilli to coccoidal was observed in the case of alginate entrapment while the PVA-entrapped cells had a slim morphology when compared to non-entrapped cells and formed putative nanowires. The entrapment increased or decreased the surface roughness of cells depending on the type of entrapment media. Expression of a nitrosative stress gene, which is linked to oxygen deprivation, was observed more in the alginate-entrapped cells. These research findings advance the fundamental understanding of the entrapped cell physiology which can lead to more efficient entrapped cell-based wastewater treatment.     

Immobilization of trypsin on polysaccharide film from Anacardium occidentale L. and its application as cutaneous dressing

Polysaccharide obtained from Anacardium occidentale L. gum was used for trypsin entrapment using cellulose (gaze) as a support and this preparation was applied as cutaneous wound healing. Trypsin release in vitro and the influence of pH and temperature on activity, stability and storage time of entrapped enzyme were evaluated. The preparation showed that it was still capable to release enzyme even after 48 h. Entrapped enzyme presented an optimal pH and temperature of 8.6 and 55 °C, respectively. Also, it was stable at high temperature (45 °C for 60 min) and wide range of pH, retaining 80% of its initial activity when stored for 28 days at 25 °C. Histopathological analysis of mice skin wound healing under the entrapped trypsin preparation treatment showed an acceleration of fibroblast proliferation, neovascularization of granulation tissue and stimulating effect on the epithelium formation compared to the skin wound under the treatment using preparations without trypsin. These results demonstrate that the trypsin–polysaccharide–cellulose preparation could be used in cutaneous dressing applications for wound healing.     

Cell partitioning during the directional solidification of trehalose solutions

Previous studies have demonstrated that ice/cell interaction influences post thaw viability and specific cryoprotective agents can affect those interactions. Trehalose, a disaccharide, has been shown to have a protective benefit during conventional slow freezing. Existing theories have been put forth to explain the protective benefit of trehalose during desiccation and vitrification, but these theories do not explain the protective benefit observed during conventional freezing protocols. The overall objective of this investigation was to characterize cell/ice interactions in the presence of trehalose using non-planar freezing conditions. To that end, lymphoblasts suspended in phosphate buffered saline solution with various levels of trehalose (0, 10, 100, and 300 mM) were frozen on a directional solidification stage. The partitioning of cells into the interdendritic space or engulfment by an advancing dendrite was determined as a function of velocity and solution composition. For a given temperature gradient, the fraction of cells entrapped into the interdendritic region increased with increasing velocity. With small additions of trehalose (10 mM), the velocity at which cells were entrapped in the interdendritic region increased. At high trehalose concentrations (100, 300 mM), interface morphology was significantly different and cells were engulfed by the advancing interface. Dehydration of cells in the region shortly before and after the interface was significant and depended upon of the type of interaction experienced by the cell (entrapped vs. engulfed). These studies suggest that one potential mechanism for the action of trehalose involves changing the ice/cell interactions during conventional slow freezing.     

Enhanced cell viability and cell adhesion using low conductivity medium for negative dielectrophoretic cell patterning

Negative dielectrophoretic (n-DEP) cell manipulation is an efficient way to pattern human liver cells on micro-electrode arrays. Maintaining cell viability is an important objective for this approach. This study investigates the effect of low conductivity medium and the optimally designed microchip on cell viability and cell adhesion. To explore the influence of conductivity on cell viability and cell adhesion, we have used earlier reported dielectrophoresis (DEP) buffer with a conductivity of 10.2 mS/m and three formulated media with conductivity of 9.02 (M1), 8.14 (M2), 9.55 (M3) mS/m. The earlier reported isotonic sucrose/dextrose buffer (DEP buffer) used for DEP manipulation has the drawback of poor cell adhesion and cell viability. A microchip prototype with well-defined positioning of titanium electrode arrays was designed and fabricated on a glass substrate. The gap between the radial electrodes was accurately determined to achieve good cell patterning performance. Parameters such as dimension of positioning electrode, amplitude, and frequency of voltage signal were investigated to optimize the performance of the microchip.     

Decolorization of azo dye using PVA-immobilized microorganisms

A microbial consortium having a high capacity for rapid decolorization of azo dye (RED RBN) was immobilized by a phosphorylated polyvinyl alcohol (PVA) gel. The immobilized-cell beads exhibited a color removal capability of 75%, even at a high concentration of RED RBN (500 mg l(-1)) within 12 h using flask culture. The continuous operation was conducted at a hydraulic retention time (HRT) of 5-20 h in which the dye loading rate ranged from 240 to 60 mg dye h(-1). A removal efficiency exceeding 90% was obtained at the HRT higher than 10 h. No recognizable destruction of bead appearance was observed in the 6-month operation. Examination of the mechanism of the decolorization process by cell beads indicated that it proceeded primarily by biological decolorization associated with partial adsorption of the dye onto the entrapped cells and gel matrix. Microscopic observation revealed that the microbial consortium contained in the gel beads was at least made up of three kinds of bacterial species. From the economical viewpoint, alternative cheaper nitrogen sources such as fish meal, soybean meal, pharmamedia and vita yeast powder were examined.     

Effects of cell entrapment on nucleic acid content and microbial diversity of mixed cultures in biological wastewater treatment

The effects of entrapment on nucleic acid content and microbial diversity of mixed cultures in biological municipal wastewater treatment were investigated. Deoxyribonucleic acid content increased 1.6-5.5 times more in alginate entrapped cells than in free and polyvinyl alcohol (PVA) entrapped cells. PVA entrapment resulted in 1.1- to 5.9-fold more increases in ribonucleic acid content compared to that experienced by free and alginate entrapped cells. Entrapment in carrageenan changed the bacterial community structure more than the alginate and PVA entrapments (35-80% versus 0-35%) as determined by single-strand conformation polymorphism analyses. The change in the bacterial community structure of alginate entrapped cells was less time dependent than that of PVA entrapped cells. This study enhances understandings on the physiology of entrapped cells and their community evolution in wastewater treatment environments.     

Effect of heating temperature and time on pharmaceutical characteristics of albumin microspheres containing 5-fluorouracil

Conclusion  In this study, we found that both heating temperature and heating time affect mean particle size, particle size distribution, and drug entrapment efficiency of albumin microspheres. The change in heating temperature may affect the particle size of the product, especially when heating is carried out at a lower temperature (90°C–120°C). Hence the temperature should be selected on the basis of desired size range. Given that it is desirable for a maximum amount of the drug used in the preparation to become entrapped in microspheres, heating temperature and heating time for denaturation of albumin should be selected cautiously, as both have a significant effect on drug entrapment efficiency. In the present case, the highest entrapment was found in batches prepared by heating at 90°C for 5 minutes. However, the extent of stabilization at the selected temperature and the time of heating should also be taken into consideration, as they may affect the release of drugs to target tissue.     

Survival of Bifidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Studies on the accumulation of heavy metal elements in biological systems

Summary Cells of a Daucus carota suspension culture were entrapped in a matrix of calcium alginate. The immobilised cells, incubated in a buffer mixture of sucrose, nitrate, KCl, CaCl₂, 2-(N-morpholino)-ethane sulphonic acid at pH 5.5, hydroxylated digitoxigenin. When compared under the same incubation conditions, freely suspended cells biotransformed digitoxigenin at a faster rate. Periplogenin formation was maximal at pH 5.3 and temperatures of 26°–34°C. The hydroxylase activity of the entrapped cells adapted to the presence of 20 mM CaCl₂ over a 12 day incubation. The diffusion barrier established on entrapment of the cells could not be overcome by addition of detergents or methanol. Controlled addition of chloroform (at 1/4 and 1/2 saturation) did stimulate hydroxylation of digitoxigenin without adversely affecting cell viability. The rate of hydroxylation of digitoxigenin was linear over an immobilised cell concentration of 0–7 mg dry weight and a digitoxigenin concentration of 0–20 mg/L. Five consecutive batch bioconversions at a rate greater than 60% could be achieved before the biocatalyst was inactivated. The results are discussed in relation to improving the hydroxylation reaction by immobilised D. carota and other reactions performed by immobilised plant cells.     

Immobilized catalase-containing yeast cells: Preparation and enzymatic properties

The cell of Saccharomyces cerevisiae previously induced for catalase (EC 1.11.1.6) activity were immobilized by entrapment of intact cells in acrylamide polymerized by γ irradiation (100 kR). Yeast cells showed an enhancement in catalase activity on entrapment, an effect similar to that observed on treatment with organic solvents like toluene. The cells pretreated with toluene, however, showed complete loss of catalase activity on entrapment. The entrapped enzyme exhibited a narrow pH optimum, reduced K_m for H₂O₂, and a decrease in thermostability. The temperature optimum of catalase was also decreased from 60 to 40°C on immobilization. A tenfold decrease in the activation energy was also observed. The enzyme in the entrapped cells was, however, stable toward inactivation by γ irradiation. Unlike the intact cells, the entrapped yeast cells did not have the ability to induce catalase.     

Entrapment of a Trigonopsis variabilis D‐amino acid oxidase variant F54Y for oxidative deamination of cephalosporin C

Trigonopsis variabilis D ‐amino acid oxidase (TvDAAO) is an enzyme used in the industrial bioconversion of cephalosporin C (CPC) into 7‐aminocephalosporanic acid, a crucial biosynthetic nucleus for a wide spectrum of semi‐synthetic cephem antibiotics. Using homology modeling and site‐directed mutagenesis, we have previously shown that the TvDAAO variant F54Y possesses improved catalytic activity and thermostability. To further explore its industrial application, the conditions for immobilization of the enzyme were examined in the present investigation. The results showed that entrapment in a calcium alginate (Ca‐alginate) matrix using 2% alginate, 500 mM CaCl₂, and 15 min stabilization appeared to be optimal for the immobilization of F54Y. The entrapped enzyme allowed complete CPC conversion. The entrapped enzyme also showed good operational stability and retained at least 90% of its original activity after 20 reaction cycles. To conclude, the entrapment of F54Y in Ca‐alginate appeared to be a simple and efficient biocatalysis system with potential application in the antibiotics industry.     

Single-cell trapping utilizing negative dielectrophoretic quadrupole and microwell electrodes

The handling of individual cells, which has attracted increasing attention, is a key technique in cell engineering such as gene introduction, drug injection, and cloning technology. Alternating current (AC) electrokinetics has shown great potential for microfluidic functions such as pumping, mixing, and concentrating particles. The non-uniform electric field gives rise to Joule heating and dielectrophoresis (DEP). The motion of particles suspended in the medium can be influenced directly, by means of dielectrophoretic effects, and indirectly, via fluid flow through a viscous drag force that affects the particles. Thus alternating current electrothermal effect (ACET) induced flow and DEP force can be combined to manipulate and trap single particles and cells. This study presents a microfluidic device which is capable of specifically guiding and capturing single particles and cells by ACET fluid flow and the negative dielectrophoretic (nDEP) trap, respectively. The experiment was operated at high frequencies (5–12 MHz) and in a culture medium whose high conductivity (σ = 1.25 S/m) is of interest to biochemical analysis and environmental monitoring, which are both prone to producing ACET and nDEP. Manipulation of particle motion using ACET-induced fluid flow to the target trap is modeled numerically and is in good agreement with the experimental results.     

Enzymatic methods for in situ cell entrapment and cell release

We report an enzyme-based method for the in situ entrapment of cells within a biopolymeric hydrogel matrix. Specifically, we used a calcium-independent microbial transglutaminase that is known to cross-link proteins and observed that it catalyzes the formation of gels from a pre-gel solution containing 10% gelatin and E. coli cells. Hydrogel formation occurs 2-3 h after adding transglutaminase, and no additional external intervention is required to initiate gel formation. The in situ entrapped cells grow rapidly and to high cell densities within the gelatin hydrogel. Additionally, the entrapped cells respond to isopropylthiogalactoside induction. The cross-linked gelatin network can be rapidly hydrolyzed (within 1 h) by the protease, proteinase K. Treatment of the network by this protease releases the entrapped E. coli cells. These cells appear unharmed by proteinase K; they can grow and be induced after protease treatment. The ability to in situ entrap, grow, and release cells under mild conditions provides unique opportunities for a range of applications and should be especially useful for microfluidic biosensor systems.     

Ultrasound‐assisted swelling of bacterial cellulose

Bacterial cellulose (BC) was obtained by static cultivation using commercial BC gel from scoby. BC membranes (oven dried and freeze‐dried) were swelled with 8% NaOH, in the absence and in the presence of ultrasound (US), for 30, 60, and 90 min. The influence of swelling conditions on both physico‐chemical properties and molecules entrapment was evaluated. Considering the highest levels of entrapment, an optimum swelling procedure was established: 8% NaOH for 30 min at room temperature in the presence of US. Native and PEGylated laccase from Myceliophthora thermophila was immobilized on BC membranes and a different catalytic behaviour was observed after immobilization. Native laccase presented activity values similar to published reports (5–7 U/gBC) after immobilization whereas PEGylated enzymes showed much lower activity (1–2 U/gBC). BC swelled membranes are presented herein as a potential support for the preparation of immobilized enzymes for industrial applications, like phenolics polymerization.     

Matrix resistance stress: A key parameter for immobilized cell growth regulation

Microenvironmentally restricted yeast cell growth within Ca-alginate beads with and without entrapped gas bubbles was considered based on experimental data. Cell growth dynamics was described by (1) the dimensionless cell number density as a function of the cell growth time and (2) the cell distribution per bead cross sections. One of the key control parameters for bioprocess optimization is the matrix resistance stress generated during immobilized cell expansion. The dynamics of the increase in matrix stress was described theoretically based on a multi-scale mathematical model. In order to estimate and reduce the accumulation of matrix stress we considered repeated stress relaxation cycles in separate rheological experiments without immobilized cells.The results revealed that the increase in resistance stress within the Ca-alginate matrix was significant (∼7 kPa) after 10 repeated cycles, even under a low compression strain of 2% per cycle. The stress could be reduced by using the Ca-alginate matrix with entrapped gas bubbles. The final cell concentration within the beads with entrapped bubbles was 3.3 times higher in comparison with the beads without bubbles. The bubbles could locally amortize the compression effects within the surrounding cell clusters.