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.     

Multilocus enzyme typing of human and animal strains of Clostridium perfringens

Abstract The stability of plasmids introduced in Lactobacillus lactis IL1403 was followed in continuous cultures. Four strains with or without pIL205 and pIL252 or pIL253 (low or high copy number) were studied. pIL205 was remarkably stable even after 180 generations, suggesting the presence of a partitioning system which is still unidentified. In contrast, pIL252 and pIL253 were unstable, in the presence as well as in the absence of pIL205. The multicopy plasmid pIL253 was nevertheless more stable than pIL252 (90% loss after 180 and 60 generations, respectively). The instability was not related to an incompatibility between pIL205 and pIL252 (or pIL253). The segregational instability of pIL252 and pIL253 plasmids could be explained by the loss of the resolvase gene during their construction.     

Dynamic modeling of immobilized cell reactor: application to ethanol fermentation

A mathematical model has been developed for the unsteady-state operation of an immobilized cell reactor. The substrate solution flows through a mixed-flow reactor in which cells immobilized in gel beads are retained. The substrate diffuses from the external surface of the gel beads to some internal location where reaction occurs. The product diffuses from the gel beads into liquid medium which flows out of the reactor. The model combines simultaneous diffusion and reaction, as well as cell growth, and it can predict how the rates of substrate consumption, product formation, and cell growth vary with time and with initial conditions. Ethanol fermentation was chosen as a representative reaction in the immobilized cell reactor, and numerical calculations were carried out. Excellent agreement was observed between model predictions and experimental data available in the literature.     

Hydrodynamic characteristics of immobilized cell beads in a liquid-solid fluidized-bed bioreactor

This study examined the hydrodynamic characteristics of a liquid-solid fluidized-bed bioreactor using elastic particles (PVA gel beads) of various diameters as carriers. The drag coefficient-Reynolds number, velocity-voidage, and expansion index-Reynolds number relationships observed during fluidization of PVA gel beads in a fluidized bed in our experiments were compared with the published results. Predictions made from previous correlations were examined with our new experimental findings, revealing the inadequacy of most of these correlations. Thus, new correlations describing the above-mentioned relationships are suggested. The drag coefficient of immobilized cell beads is larger than that of free cell ones at the same Reynolds number because the surface of the immobilized cell beads is rougher. For multiparticle systems, the correction factor, f(epsilon), is a function of the falling gel bead properties (Reynolds number) as well as the fluidized gel bead properties (Archimedes number), and depend strongly on the bed voidage (epsilon). A new simple relation was developed to predict easily the epsilon value from 0.5-0.9 at 4,986 < A(r) < 40,745 or 34 < Re(t) < 186. For all the immobilized cell beads used in this study, the prediction error of the bed voidage was less than 5% at epsilon > 0.5. The prediction equations in this study can be further applied to analyzing the hydrodynamic characteristics of a fluidized-bed reactor using similar entrapped elastic particles as carriers.     

Development of simple methods for preparation of yeast and plant protoplasts immobilized in alginate gel beads

A simple method for preparation of yeast and plant protoplasts immobilized in alginate gel beads was developed. Yeast cells were first immobilized in strontium alginate gel beads and then treated with protoplast isolation enzyme so that the protoplasts are formed inside the beads. In the case of plant cells, degassing treatment was necessary in order to facilitate enzyme penetration into the cell aggregates. A mixture of the degassing treated plant cells and sodium alginate solution was dropped into SrCl2 containing the protoplast isolation enzymes. Thus protoplasts isolation and gel solidification proceeded simultaneously. With these methods, the required time was shorter while the viability of the immobilized protoplasts were higher than when the conventional method is used.     

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

The effect of plasmid introduction into Lactococcus lactis subsp. lactis IL2661 on the growth of this strain and on plasmid stability was studied in pure batch cultures. The plasmids used (coding for erythromycin or chloramphenicol resistance) were the following: pIL205 (42 kb), pIL252 (4.6 kb, 6-9 copies), pIL253 (4.8 kb, 45-85 copies) and pE194 (inserted in the chromosome). Growth and acidification of L. lactis subsp. lactis IL2661 were similar to those of the derived recombinant lactococci. The maximal population at the end of the fermentation (9 h) was about 1.1 +/- 0.3 x 10(10) cfu/ml, and maximal growth rate 0.92 +/- 0.07 h-1. Growth yield and lactic acid concentrations were 3.9 +/- 0.8 x 10(11) cfu/g lactose consumed and 25.6 +/- 2.3 g/l, respectively. Different levels of plasmid stability were detected. Plasmid pE194, and plasmids pIL252 and pIL253 in the absence of pIL205, were stable after 10 h of culture. A slight loss (1-2%) of pIL205 was observed in all strains. In the presence of pIL205, plasmids pIL252 and pIL253 were maintained in only 56-95% of the cells. This result suggested an incompatibility between pIL205 and pIL252 or pIL253.     

Mass transfer analysis for immobilized cells of Lactococcus lactis sp. using both simulations and in-situ pH measurements

Mathematical modeling and in-situ pH measurements were used to characterize the effects of the microenvironment on alginate gel beads immobilized cells of Lactococcus lactis. Mass transfer limitations led to a progressive pH acidification within gel beads which determined both the cell distribution and the cellular activity of entrapped cells. The dynamics of the system is discussed in relation to the overall activity of the immobilized cell reactor.     

Enhancing the Viability of Lactobacillus plantarum Inoculum by Immobilizing the Cells in Calcium-Alginate Beads Incorporating Cryoprotectants

Many literature reports have cited the importance of the rehydration conditions of lyophilized cultures in determining viability. The rate of rehydration and the volume of fluid used have been identified as two important factors. One possible means of controlling these is by immobilizing the cells before lyophilization within a gel matrix in which the subsequent rehydration rate and fluid volume would be controlled by the properties of the gel. In this study Lactobacillus plantarum was immobilized and lyophilized in Ca-alginate beads in which 1 M glycerol or 0.75 M adonitol with skim milk were incorporated as a cryoprotectant. The properties of these Ca-alginate beads were examined before and after lyophilization and rehydration. The beads incorporating glycerol were smaller and stronger than those with adonitol. After lyophilization, size decreased and strength increased but to a greater extent in the beads with glycerol, indicating that the microenvironment within the two bead types was probably different. The protective effect of the bead microenvironment on immobilized L. plantarum was also examined. Lyophilization and rehydration within the alginate beads with either polyol yielded higher survival rates than that attained with free cell cultures during rehydration in optimal or suboptimal conditions. During rehydration under suboptimal conditions, the immobilized cell survival was greatest when 0.75 M adonitol was the incorporated cryoprotectant.     

Determination of diffusion coefficients and diffusion characteristics for chlorferon and diethylthiophosphate in Ca-alginate gel beads

Diffusion characteristics of chlorferon and diethylthiophosphate (DETP) in Ca-alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients for chlorferon and DETP in Ca-alginate gel beads determined at conditions suitable for biodegradation studies were 2.70 x 10(-11) m(2)/s and 4.28 x 10(-11) m(2)/s, respectively. Diffusivities of chlorferon and DETP were influenced by several factors, including viscosity of the bulk solution, agitation speed, and the concentrations of diffusing substrate and immobilized cells. Diffusion coefficients increased with increasing agitation speed, probably due to poor mixing at low speed and some attrition of beads at high speeds. Diffusion coefficients also increased with decreasing substrate concentration. Increased cell concentration in the gel beads caused lower diffusivity. Theoretical models to predict diffusivities as a function of cell weight fraction overestimated the effective diffusivities for both chlorferon and DETP, but linear relations between effective diffusivity and cell weight fraction were derived from experimental data. Calcium-alginate gel beads with radii of 1.65-1.70 mm used in this study were not subject to diffusional limitations: external mass transfer resistances were negligible based on Biot number calculations and effectiveness factors indicated that internal mass transfer resistance was negligible. Therefore, the degradation rates of chlorferon and DETP inside Ca-alginate gel beads were reaction-limited.     

Entrapment of lipid vesicles and membrane protein-lipid vesicles in gel bead pores

Phospholipid vesicles were entrapped in gel beads of Sepharose 6B and Sephacryl S-1000 during vesicle preparation by dialysis. Egg-yolk phospholipids solubilized with cholate or octyl glucoside were dialysed together with gel beads for 2.5 days in a flat dialysis bag. Some vesicles were formed in gel bead pores and vesicles of sufficient size became trapped. Red cell membrane protein-phospholipid vesicles could be immobilized in the same way. Non-trapped vesicles were carefully removed by chromatographic procedures and by centrifugation. The amount of entrapped vesicles increased with the initial lipid concentration and was dependent on the relative sizes of vesicles and gel pores. The largest amount of trapped vesicles, corresponding to 9.5 mumol of phospholipids per ml gel, was achieved when Sepharose 6B gel beads were dialysed with cholate-solubilized lipids at a concentration of 50 mM. In this case the vesicles had an average diameter of 60 nm and an internal volume of 15 microliters/ml gel. The amount of vesicles trapped in Sephacryl S-1000 gel beads upon dialysis under the same conditions was smaller: 2.2 mumol of phospholipids per ml gel. Probably most of the gel pores were too large to trap such vesicles. Larger vesicles, with an average diameter of 230 nm, were entrapped in the Sephacryl S-1000 matrix in an amount corresponding to 3.0 mumol phospholipids per ml gel upon dialysis of the gel beads and octyl glucoside-solubilized lipids at a concentration of 20 mM. The internal volume of these vesicles was 22 microliters/ml gel. The yield of immobilized phospholipids was up to 19%. The entrapped vesicles were somewhat unstable: 9% of the phospholipids were released during 9 days of storage at 4 degrees C. By the dialysis entrapment method vesicles can be immobilized in the gel beads without using hydrophobic ligands or covalent coupling.     

Plasmid inheritability and biomass production: comparison between free and immobilized cell cultures of Escherichia coli BZ18(pTG201) without selection pressure.

Maintenance of the plasmid pTG201 in Escherichia coli BZ18 was studied for both free and immobilized cells during chemostat culture, in the absence of the antibiotic against which resistance was plasmid encoded. Electron microscopic observations of immobilized proliferant cells within carrageenan gel beads showed high cell concentrations and growth into distinct cavities. The plasmid which coded for the catechol 2,3-dioxygenase activity was stably maintained during 80 generations in the case of immobilized cells. A theoretical analysis founded on the compartmentalization resulting from the immobilized growth conditions was described. However, the model still showed a plasmid stability inferior to that determined experimentally. Hypotheses dealing with physiological changes of immobilized cells were presented. In addition, the high cell concentrations obtained in the outer 50 microns of the carrageenan gel beads gave a biomass productivity within this useful volume which was 20 times higher than in free-cell cultures.     

固定化芽孢菌产碱性蛋白酶的研究

本文报导了用海藻酸钙固定地衣芽孢杆菌(Bacilluslicheniformis),发酵生产碱性蛋白酶的研究。固定化细胞颗粒在低浓度的玉米粉、黄豆饼粉为原料的培养基中发酵24h,酶活高达1724u/ml,充分利用并节省了原材料,缩短了发酵周期。发酵液菌浓度的测定结果表明,固定化细胞凝胶粒细菌的渗漏程度较低,有利于提取工艺的简化。     

Efficient production of celery embryos and plantlets released in culture of immobilized gel beads

Celery embryos and plantlets were found to be selectively released in a culture of immobilized Ca-alginate gel beads in which celery callus was entrapped under regeneration conditions. We studied the feasibility of use of this process for celery embryogenesis in an artificial seed system. The cells released from the gel beads were larger than those obtained in suspension culture. The optimal concentration of alginate gel for embryo and plantlet production was 2% for the immobilized cell culture. Considering the maintenance of the gel bead structure and detrimental effect of CaCl₂ on plantlet development, 5 mM CaCl₂ supplementation gave the best result in terms of the number of heart and torpedo embryos and plantlets. The ratio of the number of heart embryos, torpedo embryos and plantlets to total number of cells in the immobilized cell culture was higher than that in the suspension culture. Repeated batch culture with 5 mM CaCl₂ provided long-term (more than 154 d) embryo and plantlet production without gel beads disruption. Productivity of plantlets in the immobilized cell culture with 5 mM CaCl₂ was 2.2-fold as high as that in the suspension culture.     

Conjugative plasmid transfer between Pseudomonas strains within alginate bead microcosms: effect of the internal gel structure.

Because microorganisms frequently live in an immobilized state in natural habitats, a cell-confined system was used to study bacterial conjugation. Two Pseudomonas putida strains were introduced together within calcium alginate gels. Different alginate beads were designed by varying the polysaccharide and the gelation solution concentrations. Microscopic examinations showed that 2% gels were quite homogeneous, but that 1.5% and 1% gels were rather heterogeneous. In these two last cases, shaft-shaped macrostructures were present. They were colonized during the culture by great densities of highly motile bacteria. Gene transfers due to conjugation were investigated in such alginate gel bead microcosms, in batch and continuous cultures. High-initial transfer frequencies were detected whatever the gel, but no conjugation events seemed to occur with further growth in the beads. Transfer frequency values were roughly similar in the different tested systems. Alginate gels used as artificial microcosms may be valuable to study the effect of cell microenvironment on genetic transfers in complex systems.     

结冷胶固定化保加利亚乳杆菌及在玉米秸秆连续发酵d-乳酸中的应用

秸秆生物炼制化学品是解决秸秆资源利用附加值低、减轻秸秆焚烧带来的环境污染的主要方法之一。本研究制备了结冷胶固定化保加利亚乳酸杆菌(Lactobacillus bulgaricus)T15凝胶珠(结冷胶-T15凝胶珠),并对其性质进行表征,建立了结冷胶-T15凝胶珠固定化细胞循环连续发酵产D-乳酸发酵工艺。结冷胶-T15凝胶珠的断裂应力为(91.68±0.11) kPa,较海藻酸钙固定化T15凝胶珠(海藻酸钙-T15凝胶珠)提高了125.12%,表明结冷胶-T15凝胶珠的强度更强。以结冷胶-T15凝胶珠为出发菌株,葡萄糖为发酵基质,10批次循环(720h)发酵,其D-乳酸最高批次产量为(72.90±2.79)g/L,较海藻酸钙-T15凝胶珠提高了33.85%,较游离T15提高了37.70%。将葡萄糖更换为玉米秸秆酶解液,使用结冷胶-T15凝胶珠进行10批次循环(240 h)发酵,D-乳酸生产强度可达(1.74±0.79)g/(L·h),远高于游离菌。10批次循环发酵后结冷胶-T15凝胶珠磨损率小于5%,表明结冷胶是一种细胞固定化的良好载体,可广泛应用于细胞固定化工业发酵领域。本研究为细胞...     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Improved immobilized enzyme systems using spherical micro silica sol-gel enzyme beads

Spherical micro silica sol-gel immobilized enzyme beads were prepared in an emulsion system using cyclohexanone and Triton-X 114. The beads were used for thein situ immobilization of transaminase, trypsin, and lipase. Immobilization during the sol to gel phase transition was investigated to determine the effect of the emulsifying solvents, surfactants, and mixing process on the formation of spherical micro sol-gel enzyme beads and their catalytic activity. The different combinations of sol-gel precursors affected both activity and the stability of the enzymes, which suggests that each enzyme has a unique preference for the silica gel matrix dependent upon the characteristics of the precursors. The resulting enzyme-entrapped micronsized beads were characterized and utilized for several enzyme reaction cycles. These results indicated improved stability compared to the conventional crushed form silica sol-gel immobilized enzyme systems.     

Improvement of biodesulfurization activity of alginate immobilized cells in biphasic systems

The immobilization of Pseudomonas delafieldii R-8 in calcium alginate beads has been studied in order to improve biodesulfurization activity in oil/water (O/W) biphasic systems. A gas jet extrusion technique was performed to produce immobilized beads. The specific desulfurization rate of 1.5 mm diameter beads was 1.4-fold higher than that of 4.0 mm. Some nonionic surfactants can significantly increase the activity of immobilized cells. The desulfurization rate with the addition of 0.5% Span 80 increased 1.8-fold compared with that of the untreated beads. The rate of biodesulfurization was markedly enhanced by decreasing the size of alginate beads and adding the surfactant Span 80, most likely resulting from the increasing mass transfer of substrate to gel matrix.     

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.     

Gel beads composed of collagen reconstituted in alginate

Spherical gel beads of collagen/alginate were prepared by discharging droplets of a mixture containing collagen (1.07-1.9 mg/ml) and alginate (1.2-1.5% w/v) into 1.5% w/v CaCl2 solution at 4°C. Collagen in the gel beads was reconstituted by raising the temperature to 37°C after alginate was liquefied by citrate. Scanning electron microscopy of the beads revealed the characteristic fibrous structure of collagen. To demonstrate the application of this new technique in cell culture, GH3 rat pituitary tumor cells were entrapped and grown in the gel beads. The immobilized cells proliferated to a density of 1.95 x 106 cell/ml which is about an order of magnitude higher than that grown in the alginate beads.