Growth and sporulation of entrapped Bacillus subtilis cells

Growing Bacillus subtilis cells were immobilized in k-carrageenan gel beads. Growth and sporulation of entrapped cells were studied by two different methods: cell enumeration and transmission electron microscopy. Immobilized growing cells had a shorter generation time than free cells did, whereas sporulation timing was unchanged. When steady state was reached, cell density was very high in gel beads.     

Depolymerization of chondroitin C Sulfate by immobilized Proteus vulgaris cells

Chondroitinase ABC catalyzing the depolymerization of chondroitin sulfate was induced by incubating the Proteus vulgaris cells in a medium containing chondroitin C sulfate as an inducer. Incubation of P. vulgaris cells for 12 h in the presence of 0.3% inducer was optimal to obtain the cells with highly active chondroitinase ABC. Such cells were immobilized in k-carrageenan gel lattice, and some properties of chondroitinase ABC in immobilized cells were studied in comparison with those of the enzyme without immobilization (free enzyme). The stabilities of the enzyme toward heat and storage were remarkably improved by immobilizing the cells in k-carrageenan gel lattice. Optimal pH and temperature for activity of the enzyme were slightly shifted to the alkaline region and higher temperature by immobilization and were 9.0 and 35 degrees C, respectively.     

Dynamics of T cells and TCR excision circles differ after treatment of acute and chronic HIV infection

We quantified T cell proliferation and thymic function in primary HIV infection (PHI; n = 19) and chronic HIV infection (CHI; n = 14) by measuring Ki67 staining and TCR excision circle (TREC) number. After antiretroviral therapy of PHI there is a profound decrease in the number and percentage of Ki67(+) T cells (<6% Ki67(+)) with no significant increase in TREC per million cells and a transient increase in TREC per milliliter. In contrast, after antiretroviral therapy of CHI there is a reduction in the percentage but little change in the total number of Ki67(+)CD4(+) T cells associated with increases in both TREC per million cells and TREC per milliliter. Using a mathematical model that accounts for proliferation, death, and redistribution of T cells, we find that redistribution is consistent with the TREC changes observed during treatment of PHI and that an increase in thymic output is needed to explain the increase in TREC during treatment of CHI. Consideration of TREC per milliliter shows that changes in proliferation alone cannot explain the changes in TREC. In addition, although increased proliferation of memory cells in HIV infection has been established, we find no difference in TREC per million CD45RA(-) "memory" T cells between healthy and infected individuals (p = 0.154 for CD4(+); p = 0.383 for CD8(+)). Finally, although the number of TREC per million cells is always much lower in memory T cells than in naive T cells, in the setting of HIV infection, given that memory cells make up a larger proportion of total T cells, we find that 50% of TREC per milliliter in CD4(+) T cells is harbored in the CD45RA(-) "memory" subset of our infected subjects.     

Galactose oxidase production by immobilized cells ofDactylium dendroides

Summary Dactylium dendroides cells were immobilized with calcium alginate, calcium pectate and k-carrageenan. Alginate immobilized cells produced relatively small amounts of (D-galactose: O₂ oxidoreductase, EC 1.1.3.9, GOase). Pectate immobilized cells gave the best yield of GOase, which was comparable with that obtained with free cells, and productivity could be extended up to 28 days (7 cycles). Controlled dosage of phosphate to the medium markedly improved GOase production with higher yields per cycle than with free cells.     

k-Carrageenan Gel as Agent to Sequester Paralytic Shellfish Poison

The action of k-carrageenan gel to sequester paralytic shellfish poison (PSP) was tested and characterized. When an extract from a Philippine strain of Pyrodinium bahamense var. compressum was used as PSP solution, the PSP-sequestering property of k-carrageenan gel was found to be dependent on gel surface area, interaction time, and polysaccharide concentration. The interaction was also found to be affected by high concentrations of monovalent cations. The characteristics of k-carrageenan as a PSP-sequestering agent all point to cation exchange as its mechanism of action. It is also proposed that the polysaccharide gel can be utilized as an agent to alleviate PSP intoxication.     

Immobilization of enzymes and microbial cells using carrageenan as matrix.

Conditions for the gelation k-carrageenan, which is a new polymer for immobilization of enzymes and microbial cells, were investigated in detail. k-Carrageenan was easily induced to gel by contact with metal ions, amines, amino acid derivatives, and water-miscible organic solvents. By using this property of k-carrageenan, the immobilization of enzymes and microbial cells was investigated. Several kinds of enzymes and microbial cells were easily immobilized with high enzyme activities. Immobilized preparations were easily tailor-made to various shape such as cube, bead, and membrane. The obtained immobilized preparations were stable, and columns packed with them were used for continuous enzyme reaction for a long period. Their operational stabilities were enhanced by hardening with glutaraldehyde and hexamethylenediamine.     

Production of ethanol by immobilized Saccharomyces bayanus in an extractive fermentation system

An extractive fermentation system using immobilized yeast cells was developed to study the ethanol production at high sugar concentrations. Organic acids were used as extracting solvents of ethanol and their toxicity was tested in free and k-carrageenan entrapped cell preparations. Immobilization seems to protect cells against solvent toxicity, when long-chain organic acids, e.g., oleic acid, were used, probably due to steric and diffusional limitations, the free cells not being viable at high oleic acid concentrations. The entrapped cells also present a higher metabolic activity than their free counterparts at high glucose concentrations. A solution of 300 g/L of glucose was totally fermented by the immobilized yeast cells, which when free cannot normally convert more than 200 g/L. In situ recovery of ethanol by oleic acid in a batch immobilized cell system led to higher ethanol productivities and to the fermentation of 400 g/L, when an oleic acid/medium ratio of 5 was used.     

Cell-density-dependent Changes in the Metabolism of Chloronema Cell Cultures: I. Relationship between Cell Density and Enzymic Activities

In the growing chloronema cell suspension cultures of the moss Funaria hygrometrica Hedw., activities of several enzymes have been found to be cell-density-dependent. Cyclic nucleotide phosphodiesterase (cNPDE), nitrate reductase (NR), and protein kinase showed highest activity at a low cell density (1 to 2 milligrams per milliliter) while indoleacetic acid (IAA) oxidase and peroxidase were highest at a high cell density (>10 milligrams per milliliter). 3′-Nucleotidase and the glycolytic enzymes (aldolase, hexokinase, phosphofructokinase, phosphoglucoisomerase, pyruvate kinase, and triose phosphate isomerase) showed no significant dependence on the cell density. Alternatively, if the NR and peroxidase activities were determined as a function of time in batch cultures, their levels were maximal 60 to 70 and 320 hours after subculture, respectively, the corresponding cell densities being 1 to 2 and 23 milligrams per milliliter. The relationship between cell density and NR and peroxidase activities is the same, whether these enzymes are measured in batch cultures during a growth cycle or in the cells cultured at different initial inoculum densities for a constant time. Conventionally enzymic changes have been correlated with growth phases; however, it is felt that the pattern of enzymic activities can also be interpreted as cell-density-dependent.     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Influence of phosphate starvation on cultures of Pseudomonas aeruginosa

Hou, Cynthia I. (University of British Columbia, Vancouver, B.C., Canada), Audrey F. Gronlund, and J. J. R. Campbell. Influence of phosphate starvation on cultures of Pseudomonas aeruginosa. J. Bacteriol. 92:851-855. 1966.-The changes occurring in Pseudomonas aeruginosa during phosphate starvation in a phosphate-deficient medium were assessed by measuring alterations in optical density, viable-cell count, chemical composition, and ribosome patterns. After a 24-hr period of starvation, optical density, protein, and deoxyribonucleic acid per milliliter of culture increased, whereas ribonucleic acid decreased. Extensive ribosomal degradation was apparent from sucrose density gradient centrifugation patterns. The induction of an alkaline phosphomonoesterase during phosphate starvation was observed. A linear response of phosphate-starved cells to low levels of phosphate supplied exogenously was evident from optical-density measurements, and a threshold requirement for phosphate analogous to the "energy of maintenance" was not detected.     

A milliliter-scale yeast-based fuel cell with high performance

Microbial fuel cells are attracting attention as one of the systems for producing electrical energy from organic compounds. We used commercial baker's yeast (Saccharomyces cerevisiae) for a glucose fuel cell because the yeast is a safe organism and relatively high power can be generated in the system. In the present study, a milliliter (mL)-scale dual-chamber fuel cell was constructed for evaluating the power generated by a variety of yeasts and their mutants, and the optimum conditions for high performance were investigated. When carbon fiber bundles were used as an electrode in the fuel cell, high volumetric power density was obtained. The maximum power produced per volume of anode solution was 850 W/m³ under optimum conditions. Furthermore, the power was examined using seven kinds of yeast. In Kluyveromyces marxianus, not only the power but also the power per consumed glucose was high. Moreover, it was suggested that xylose is available as fuel for the fuel cell. The fuel cell powered by K. marxianus may prove to be helpful for the effective utilization of woody biomass.     

Continuous production ofL-isoleucine using immobilized growing Serratia marcescens cells

An immobilized growing cell system was applied to the continuous L -isoleucine production by Serratia marcescens. In the new immobilized-cell systems using the carrageenan gel method. S. marcescens cells in the gel required nutrients and oxygen for growth, and the numbers of living cells per milliliter of gel increased to the levels of that of free cells in the liquid medium. This immobilized growing cell system exhibited high and stable activity for isoleucine production under steady-state conditions. Continuous isoleucine production was carried out by feeding the nutrient medium under aeration into a fluidized bed reactor containing the immobilized cells. In the continuous operation, an efficient production was maintained by automatically controlling the pH of the reaction mixture at 7.5. The productivity of isoleucine increased using multibed reactors. In a two-bed reactor system, the effluent L -isoleucine concentration reached 4.5 mg/ml at a retention time of 10 hr, and a steady state was maintained for longer than 30 days.     

Regulatory mechanisms of cellular respiration; the role of cell membranes; uranium inhibition of cellular respiration

Uranium as UO(2)(NO(3))(2) combines reversibly with proteins. The degree of dissociation of this combination depends, among other factors, on the H(+) concentration. At pH 7.3 the U-albumin complex was easily dissociated on addition of citrate, while at pH 3.8 it was not. Uranium inhibited reversibly a number of enzyme systems. Uranium enzyme inhibitions could be reversed on addition of certain hydroxypolycarboxylic acids (citric acid, alpha-hydroxyaspartic acid, malic acid); in no case, however, did phosphate have any effect. In cell-free yeast juice, the fermentation of glucose-hexosediphosphate was inhibited by UO(2)(NO(3))(2). Slight reactivation occurred on addition of phosphate. In living yeast cells, the fermentation and oxidation of glucose was inhibited by small amounts of UO(2)(NO(3))(2) (7,7 micrograms per mg. dry weight), while the oxidation of acetic acid, ethyl alcohol, malic and citric acids, was not affected at all. U inhibition in living yeast cells at pH 7.3 was completely released on addition of small amounts of phosphate, adenosinetriphosphate, and citrate, while at pH 3.8 U inhibition was not released by phosphate and citrate. At saturation, one yeast cell contained 7.06 x 10(6) molecules of uranium. Lactic dehydrogenase was not inhibited by U while the oxidation of lactic acid by gonococci was inhibited. Addition of phosphate released this inhibition. The U inhibition of liver succinoxidase was unaffected by phosphate, while the U inhibition of the oxidation of succinate by E. coli was released by phosphate. It has been concluded from these experiments that U inhibition of cell metabolism is due to combination of the metal with the protein portion of the cell membrane. Uranium is presented as an example of surface inhibition.     

Short-term treatment with cell wall degrading enzymes increases the activity of the inositol phospholipid kinases and the vanadate-sensitive ATPase of carrot cells

Treating carrot (Daucus carota L.) suspension culture cells with a mixture of cell wall degrading enzymes, Driselase, resulted in an increase in the percentage of [³H]phosphatidylinositol bisphosphate. Analysis of the lipid kinase activities in the isolated plasma membranes after whole cell treatment indicated that treatment with Driselase (2% weight/volume; the equivalent of 340 units per milliliter of hemicellulase and 400 units per milliliter of cellulase activity) or treatment with hemicellulase (31.7% weight/volume, 20.7 units per milliliter) resulted in an increase in the inositol phospholipid kinase activity. However, treatment with cellulase alone had no effect at 0.5% (weight/volume, 17.2 units per milliliter) or inhibited the kinase activity at 1% (weight/volume, 34.4 units per milliliter). The active stimulus in Driselase was heat sensitive. The plasma membrane vanadate-sensitive ATPase activity also increased when the cells were treated with Driselase. A time course study indicated that both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase responded to as little as 5 seconds of treatment with 2% Driselase. However, at the lowest concentration of Driselase (0.04%, weight/volume) that resulted in an increase in inositol phospholipid kinase activity, the ATPase activity was not affected. Because inositol phospholipids have been shown to activate the vanadate-sensitive ATPase from plants (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301), a stimulus-response pathway involving both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase activity is discussed.     

Suppressive effect of delta 9-tetrahydrocannabinol in vitro on phagocytosis by murine macrophages

Incubation of normal mouse peritoneal cells consisting of over 90% phagocytizing macrophages with delta 9-tetrahydrocannabinol (THC) resulted in a inhibition of phagocytic function. The THC in a dose-related manner suppressed the percentage of macrophages per culture which ingested yeast and the average number of yeast particles ingested by the phagocytizing macrophages. The vehicle used to suspend the THC in vitro, i.e., DMSO, had no detectable effect on macrophage function. Suppression of phagocytosis with no effects on viability or cell number occurred with doses of 10 micrograms or less THC per milliliter culture medium. Measurable suppression also occurred after 24- to 48-hr treatment of the macrophages with the THC. This compound had little if any detectable effect on phagocytosis when added directly to the cultures shortly before testing for phagocytosis. Further studies concerning the effects of THC on macrophage function appear warranted.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

Yeast Cell Microcapsules as a Novel Carrier for Cholecalciferol Encapsulation: Development,Characterization and Release Properties

In this study Saccharomyces cerevisiae yeast cells was used as a novel vehicle for encapsulation of vitamin D₃. The effects of initial cholecalciferol concentration (100,000 and 500,000 IU/g yeast), yeast cell pretreatment (plasmolysis with NaCl) and drying method (spray or freeze drying) on microcapsules properties were investigated. It was found that the vitamin concentration and drying method had significant influence on encapsulation efficiency (EE) and size of yeast microcapsules. Furthermore, EE values were more increased by the plasmolysis treatment. The highest EE was obtained for plasmolysed and spray dried yeast cells prepared using initial cholecalciferol concentration of 2.5 mg per gram of yeast cells (76.10?±?6.92%). The values of mean particle size were 3.43–7.91 μm. The presence of cholecalciferol in yeast microcapsules was confirmed by X-ray diffraction (XRD) and Fourier transform-infrared (FT-IR) analyses. The in vitro cholecalciferol release from yeast microcapsules in phosphate buffer saline solution (PBS) followed a controlled release manner consistent with a Fickian diffusion mechanism. In addition, the release studies in simulated gastrointestinal tract showed sustained release of cholecalciferol in the stomach condition and significant release in intestinal medium.     

Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Saccharomyces cerevisiae

The development of a coimmobilized mixed culture sys tem of aerobic and facultative anaerobic microorganisms in Ca-alginate gel beads and the production of useful metabolites by the system were investigated. A coimmobilized mixed culture system of Aspergillus awamori (obligate aerobe) and Saccharomyces cerevisiae (facultative anaerobe) in Ca-alginate gel beads was used as a model system, and ethanol production from starch by the system was used as a model production. Mold Asp. awamori is an amylolytic microorganism while yeast S. cerevisiae is an ethanol producer. The two microorganisms grew competitively in the oxygen-rich surface area of the gel beads because they had similar oxygen demands in aerobic culture conditions. Neither microorganism exhibited "habitat segregation" in the gel beads and leaked yeast cells grew aerobically without ethanol production in the broth. Ethanol productivity was low under these conditions.A more desirable coimmobilized mixed culture system of Asp. awamori and S. cerevisiae was established by adding Vantocil IB (a biocidal compound) to the production medium. The antimicrobial activity of Vantocil IB was more effective with S. cerevisiae than with Asp. awamori, so that a dense mycelial layer of Asp. awamori formed in the surface of the gel beads While S. cerevisiae grew densely in the more inner areas of the gel beads. Also, yeast cell leakace was repressed and ethanol productivity was improved. The system with Vantocil IB produced ethanol of 4.5 and 12.3 g/L from 16 and 40 g/L starch, respectively. A continuous culture using this system with Vantocil IB was also carried out, and a stable steady state could be maintained for six days without leakage of yeast cells and contamination. The selection of a factor suitable for producing "habitat segregation" enabled the development of a coimmobilized mixed culture system of an aerobe and a facultative anaerobe. In this study, total habitat segregation was used to denote a tendency to exhibit denser growth in different parts of one gel bead.     

Sexual Interaction in Heterothallic Strains of Closterium peracerosum-strigosum-littorale: Partial Characterization of a Male-Specific Sexual Pheromone, Protoplast-Releasing Substance

A sexual pheromone, named the protoplast releasing substance (PRS), was formed by mating type minus cells. PRS activates mating type plus cells and results in the release and fusion of protoplasts within distended conjugation-papilla in paired cells and in the release and disruption of protoplasts in unpaired mating type plus cells. In an agar barrier system, the activation of mating type plus cells was markedly inhibited by treatment with pronase (5-10 micrograms per milliliter), proteinase (100 micrograms per milliliter), and α-mannosidase (10 micrograms per milliliter). Trypsin (10-100 micrograms per milliliter) had no effect on the activation in the agar barrier system. The results suggest that PRS is a glycoprotein with pronase-sensitive and trypsin-insensitive structure.     

Increase in efficiency of media utilization for recombinant protein production in Chinese hamster ovary culture through dilution

Animal cells are extensively used for the large-scale production of recombinant proteins. Processes and genetically engineered cell lines have been developed to enhance longevity of the culture and increase protein productivity. In this study, we tested the effect of diluting a culture of Chinese hamster ovary (CHO) cells with phosphate-buffered saline (PBS) on cell growth and efficiency of media utilization. An immunoglobulin G-expressing CHO cell line was cultured in CD CHO media followed by dilution of the culture with PBS after the end of the exponential phase. A 28% and 61% increase in protein yield per milliliter of media was observed in the diluted culture in the batch and fed-batch mode with glucose and protein hydrolysate feeding, respectively. To aid in analyzing the potential causes of this observed increase, an unstructured mathematical model was constructed using previously reported kinetics to simulate cell growth, nutrient utilization, and protein production. The model predicts an increase in recombinant protein yield per milliliter of media in PBS diluted cultures under both batch and fed-batch conditions, and suggests that this observed increase could at least partly be due to a decrease in inhibitor concentration in the diluted culture.