Batch fermentation with entrapped growing cells ofLactobacillus casei

Summary Growing cells ofLactobacillus casei were entrapped in-carrageenan/locust bean gum (LBG) (2:1 or 2.75%:0.25% w/w respectively) mixed gel beads (two ranges of diameter: 0.5–1.0 and 1.0–2.0 mm) to fermentLactobacillus Selection (LBS) medium and produce biomass. The results showed significant influence of initial cell loading of the beads and bead size on the fermentation rate. The highest cell release rates were obtained with 2.75%:0.25%-carrageenan/LBG small diameter gel beads. However, 17 h fermentation of LBS medium with immobilized cells resulted in substantial softening of the gel matrix, prohibiting reuse of immobilized biocatalysts as inoculum in subsequent batch fermentation. A dynamic shear rheological study showed that the gel weakness was related to chemical interactions with the medium. Results indicated that part of the matrix-stabilizing K⁺ ions diffused back to the medium. Stabilization of the gel was obtained by adding potassium ions to the LBS medium;L. casei growth was not altered by this supplementation. Fermentation of LBS medium supplemented with KCl byL. casei showed higher cell counts in the broth medium with immobilized cells than with free cells, reaching 10¹⁰ cells/ml after about 10 h with entrapped cells in 0.5–1.0 mm diameter beads and 17 h with free cells. Counts in the gel beads after fermentation were higher than 10¹¹ cells/ml and bead integrity was maintained throughout fermentation.     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

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.     

Batch fermentations with a mixed culture of lactic acid bacteria immobilized separately in κ-carrageenan locust bean gum gel beads

Summary Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus were immobilized separately in -carrageenan-locust bean gum gel beads. The beads were prepared by a dispersion process in a two-phase system (water in oil) and two ranges of bead diameter selected by sieving (0.5–1.0 mm and 1.0–2.0 mm). Fermentations with the two strains were conducted in bench bioreactors in a supplemented whey permeate medium. Free and entrapped cells (two ranges of bead diameter and two levels of initial bead cell load) were grown in mixed culture, and carbohydrate utilization, acid production and cell growth or cell release rate measured. Fermentation rates were influenced by bead diameter and initial cell load of the beads. Beads with high initial cell density increased fermentation rates compared to low cell density beads or free cells. Smaller diameter beads (0.5–1.0 mm) showed a stable tendency (not statistically significant p _a > 0.05) towards higher cell release rates, lactose utilization, galactose accumulation and lactic acid production than did larger diameter beads (1.0–2.0 mm). Immobilization of S. salivarius subsp. thermophilus and L. delbrueckii subsp. bulgaricus in separate beads did not seem to affect protocooperation during batch fermentation, and allowed for high cell release rates into the medium.     

Immobilized growing lactic acid bacteria with κ-carrageenan — locust bean gum gel

Summary A cell entrapment process using -carrageenan — locust bean gum gel is presented. Streptococcus thermophilus, Lactobacillus bulgaricus and S. lactis were immobilized in small gel beads (0.5–1.0 mm and 1.0–2.0 mm diameter) and fermentations in bench bioreactors were conducted. Viability of entrapped cells, lactose utilization, lactic acid production and cell release rates were measured during fermentation. The procedure was effective for S. thermophilus, L. bulgaricus and S. lactis, and the viability of these bacteria remained very high throughout entrapment steps and subsequent storage. Bead diameter influenced the fermentation rate: smaller beads (0.5–1.0 mm) permitted an increase in release rates, lactose utilization and acid production by entrapped cells, approximating values attained with free cells.     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli.

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

Immobilization of Infant Fecal Microbiota and Utilization in an <Emphasis Type="Italic">in vitro</Emphasis> Colonic Fermentation Model

Bacteria isolated from infant feces were immobilized in polysaccharide gel beads (2.5% gellan gum, 0.25% xanthan gum) using a two-phase dispersion process. A 52-day continuous culture was carried out in a single-stage chemostat containing precolonized beads and fed with a medium formulated to approximate the composition of infant chyme. Different dilution rates and pH conditions were tested to simulate the proximal (PCS), transverse (TCS), and distal (DCS) colons. Immobilization preserved all nine bacterial groups tested with survival rates between 3 and 56%. After 1 week fermentation, beads were highly colonized with all populations tested (excepted Staphylococcus spp. present in low numbers), which remained stable throughout the 7.5 weeks of fermentation, with variations below 1 log unit. However, free-cell populations in the circulating liquid medium, produced by immobilized cell growth, cell-release activity from gel beads, and free-cell growth, were altered considerably by culture conditions. Compared to the stabilization period, PCS was characterized by a considerable and rapid increase in Bifidobacterium spp. concentrations (7.4 to 9.6 log CFU/mL), whereas Bifidobacterium spp., Lactobacillus spp., and Clostridium spp. concentrations decreased and Staphylococcus spp. and coliforms increased during TCS and DCS. Under pseudo-steady-state conditions, the community structure developed in the chemostat reflected the relative proportions of viable bacterial numbers and metabolites generally encountered in infant feces. This work showed that a complex microbiota such as infant fecal bacteria can be immobilized and used in a continuous in vitro intestinal fermentation model to reproduce the high bacterial concentration and bacterial diversity of the feces inoculum, at least at the genera level, with a high stability during long-term experiment.     

Studies on the respiration rate of free and immobilized cells of Cephalosporium acremonium in cephalosporin C production

Bioprocesses using filamentous fungi immobilized in inert supports present many advantages when compared to conventional free cell processes. However, assessment of the real advantages of the unconventional process demands a rigorous study of the limitations to diffusional mass transfer of the reagents, especially concerning oxygen. In this work, a comparative study was carried out on the cephalosporin C production process in defined medium containing glucose and sucrose as main carbon and energy sources, by free and immobilized cells of Cephalosporium acremonium ATCC 48272 in calcium alginate gel beads containing alumina. The effective diffusivity of oxygen through the gel beads and the effectiveness factors related to the respiration rate of the microorganism were determined experimentally. By applying Monod kinetics, the respiration kinetics parameters were experimentally determined in independent experiments in a complete production medium. The effectiveness factor experimental values presented good agreement with the theoretical values of the approximated zero‐order effectiveness factor, considering the dead core model. Furthermore, experimental results obtained with immobilized cells in a 1.7‐L tower bioreactor were compared with those obtained in 5‐L conventional fermentor with free cells. It could be concluded that it is possible to attain rather high production rates working with relatively large diameter gel beads (ca. 2.5 mm) and sucrose consumption‐based productivity was remarkably higher with immobilized cells, i.e., 0.33 gCPC/kg sucrose/h against 0.24 gCPC/kg sucrose/h in the aerated stirred tank bioreactor process. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 593–600, 1999.     

An oxygen supply strategy for the large-scale production of tissue plasminogen activator by microcarrier cell culture

An oxygen supply strategy involving agitation speed and aeration method for the large-scale production of tissue plasminogen activator (TPA) by a microcarrier cell culture was investigated by small-scale model experiments. A preliminary calculation indicated that diffusion limitation of dissolved oxygen (DO) could be caused in a microcarrier sedimentation layer more than 0.5 mm in thickness. Within an agitation speed range above 70 rpm, which was the critical speed for all of the microcarrier beads to remain suspended and thus for avoiding a deficiency of DO, the TPA productivity was higher at a lower agitation speed, while the cell concentration was not affected by the agitation speed. The addition of soluble starch to the culture medium prevented sedimentation of the microcarrier beads, even at the low agitation speed of 20 rpm, resulting in a TPA productivity higher than that at 70 rpm, which was the optimum speed without soluble starch. Use of an air spray system with an optimized air flow rate resulted in a k_La 2.35 times higher than that with simple surface aeration. Increasing the internal pressure of the culture from 0.2 kg/cm² (1209 hPa) to 1.5 kg/cm² (2483 hPa) had no effect on the cell growth but slightly increased the TPA production rates. However, based on the glucose consumption, both the cell and TPA yields were much improved by pressurization. As an optimum mixing and oxygen supply strategy for the production of TPA on a large scale, it is recommended that soluble starch be added to the culture medium to allow the microcarrier suspension to be maintained at a low agitation speed, while keeping a high oxygen transfer rate by means of an air spray system and pressurization.     

Variation of intracellular sodium and potassium concentration with changes in agitation rate for chemostat-cultivatedEscherichia coli

Summary Escherichia coli was continuously cultivated in a disc turbine agitated laboratory fermenter at constant dilution rate under conditions of carbon limitation. Agitation rate (impeller speed) was varied over the range 600 to 1500 rev. min^-1. As previously reported, the mean cell volume was found to increase linearly with increase in agitation rate, whereas total cell counts and dry cell weights remained constant. Measurements of intracellular sodium and potassium concentration showed that these both increased as the cell volume increased: the potassium content was about twenty times the sodium content and the intracellular content of each ion doubled over the range of agitation rates tested.     

Enhancement and stabilization of the production of glucoamylase by immobilized cells of Aureobasidium pullulans in a fluidized-bed reactor

Summary Glucoamylase production by Aureobasidium pollulans A-124 was compared in free-living cells, cells immobilized in calcium alginate gel beads aerated on a rotary shaker (agitation rate 150 rpm), and immobilized cells aerated in an air bubble column reactor. Fermentation conditions in the bioreactor were established for bead concentration, substrate (starch) concentration, calcium chloride addition to the fermentation medium, and rate of aeration. Production of glucoamylase was optimized at approximately 1.5 units of enzyme activity/ml medium in the bioreactor under the following conditions: aeration rate, 2.0 vol air per working volume of the bioreactor (280 ml) per minute; gel bead concentration, 30% of the working volume; substrate (starch) concentration, at 0.3% (w/v); addition of calcium chloride to the medium at a final concentration of 0.01 M. Productivity levels were stabilized through the equivalent of ten batches of medium with the original inoculum of immobilized beads. Offprint requests to: M. Petruccioli     

Immobilization of Lactobacillus casei cells to ceramic material pretreated with polyethylenimine

Summary The cells of Lactobacillus casei were adsorbed to Poraver, foam glass particles pretreated with polyethylenimine (PEI). Exposure of cells for a relatively short period to Poraver beads coated with a high concentration of PEI resulted in maximal adsorption with good retention of metabolic activity. The immobilized cells were tested in packed-bed and stirred-tank reactors for lactic acid production. Stirred-tank operations were more effective in terms of productivity but the support was sensitive to attrition. The beads exhibited good mechanical stability to withstand pressure in the packed-bed reactor. Correspondence to: Bo Mattiasson     

Strain identification of probiotic Lactobacillus casei-related isolates with randomly amplified polymorphic DNA and pulsed-field gel electrophoresis methods

Typing of reference strains and isolates identified as Lactobacillus casei, Lactobacillus paracasei or Lactobacillus rhamnosus was carried out using randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) analyses. Strains of L. paracasei were mainly grouped in the same cluster as those of L. casei. The RAPD fingerprints of strains ATCC 393 and ATCC 15820 differ from those of the L. rhamnosus and L. paracasei/casei strains further supporting classification of these strains as a separate group. The RAPD profiling could be used for classification and discrimination of isolates belonging to the L. casei group.     

Continuous fermentation of a supplemented milk with immobilized Bifidobacterium infantis

Bifidobacterium infantis immobilized in -carrageenan - locust bean gum gel beads (1.0–2.0 mm diameter) was used to ferment. 10% reconstituted skim milk supplemented with 1% yeast extract in a continuous stirred tank reactor. Cell release rate from the gel beads into the milk and growth of free cells in the bioreactor allowed for a steady inoculation of the feed, with cell counts in the outflow varying from 1.0 to 2.2 × 10⁹ CFU/mL for dilution rates in the range 0,5 to 1,0 h^-1. High mechanical stability of the gel beads was observed in milk.     

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.     

Measurement of oxygen concentration gradients in gel-immobilized recombinantEscherichia coli

Summary In this study, an oxygen microsensor was used to measure oxygen concentration profiles in carrageenan gel particles containing growing, immobilizedEcherichia coli B (pTG201). Profiles, which were measured at intervals during continuous culture of gel slabs and beads, became increasingly steep with time. The oxygen penetration depth in the gel decreased with time, eventually reaching a steady state value of approximately 100 m for both gel beads and slabs. A reaction-diffusion model employing zero-order cell growth kinetics was found to provide an excellent fit to the experimental concentration data. Growth rates estimated from profiles obtained during the first few hours of culture were 0.24h^–1 (gel slabs) and 0.18 h^–1 (beads), compared to a value of 0.30 h^–1 measured in free-cell suspensions at 25° C.     

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...     

Improvement of poly(γ-glutamic acid) biosynthesis and quantitative metabolic flux analysis of a two-stage strategy for agitation speed control in the culture of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> NX-2

In this study, the production of poly(γ-glutamic acid) by Bacillus subtilis NX-2 (PGA) at different agitation speeds was investigated. Based on the analysis of specific cell growth rate (μ) and specific PGA formation rate (q _p ), a two-stage strategy for agitation speed control was proposed. During the first 24 h, an agitation speed of 600 rpm was used to maintain a high μ for better cell growth, which then reduced to 400 rpm after 24 h to maintain a high q _p to enhance PGA production. Using this method, the maximum concentration of PGA reached 40.5 ± 0.91 g/L and the PGA productivity was 0.56 ± 0.012 g/L/h, which was 17.7 and 9.8% higher, respectively, than the best results obtained when a constant agitation speed was used. The flux distributions and the related enzymes of 2-oxoglutarate could be affected by this two-stage strategy for agitation speed. The activity of isocitrate dehydrogenase and glutamate dehydrogenase at the key node of 2-oxoglutarate increased, and more flux distribution was directed to glutamate. The flux distribution from extracellular to intracellular glutamate also increased and improved PGA production as the glutamate uptake rates increased using the agitation-shift control method.     

Poly(carbamoylsulphonate) as a matrix for whole cell immobilization - biological characterization

Summary Poly(carbamoylsulphonate) (PCS), a hydrogel matrix of low toxicity for the immobilization of microorganisms was characterized with respect to physiological properties. The survival rates of immobilized Paracoccus denitrificans in PCS were greater than 99 %, the initial division rate of the bacterium inside the gel was the same as of free suspended cells and decreases with increasing cell density within the first days. The polymer has good resistance to microbial degradation and excellent mechanical stability. First results of long term behaviour and kinetic data of nitrifying bacteria entrapped within PCS gel beads are shown.     

Determination of the radial distribution of Saccharomyces cerevisiae immobilised in calcium alginate gel beads

Summary The dissolution of alginate gel beads in 20 g sodium citrate /l produces a linear decrease in bead diameter. The rate of dissolution is dependent on the concentration of CaCl₂ within the gel beads. This method allows the controlled release of Saccharomyces cerevisiae from alginate gel beads and permits the simple and rapid determination of the radial distribution of cell concentration.