Effective diffusivity of galactose in calcium alginate gels containing immobilized Zymomonas mobilis.

The effective diffusivity of galactose was measured for calcium alginate gel membranes containing immobilized live Zymomonas mobilis cells at concentrations ranging from 0 to 150 g dry wt/L of gel. Since galactose is not taken up by living Z. mobilis organisms, the diffusion of this representative six-carbon sugar could be studied independently of sugar consumption. Various immobilized biomass loadings were achieved by two different techniques: addition of biomass at known concentrations to the sodium alginate solution before membrane formation and growth of cells in the gel to various biomass concentrations. The highest immobilized cell concentration, attained by in situ growth, corresponds to the maximum of this system, as growth beyond this maximum concentration led to disintegration of the gel membrane. The galactose effective diffusivity measurements for both methods of immobilized cell loading overlap within experimental error and follow the same general monotonic decline with entrapped biomass concentration. Most of the data fall below the upper bound predicted by Hashin and Shtrikman (1962) and show good agreement with the random pore model of Wakao and Smith (1962, 1964). Available effective diffusivity data from the literature provide evidence that the random pore model is an excellent predictor of sugar effective diffusivity in gel immobilized cell systems in general.     

Cell immobilization technique for the enhanced production of alpha-galactosidase by Streptomyces griseoloalbus

Streptomyces griseoloalbus was immobilized in calcium alginate gel and the optimal immobilization parameters (concentrations of sodium alginate and calcium chloride, initial biomass and curing time) for the enhanced production of alpha-galactosidase were determined. The immobilization was most effective with 3% sodium alginate and 0.1M calcium chloride. The optimal initial biomass for immobilization was approximately 2.2g (wet wt.). The alginate-entrapped cells were advantageous because there was a twofold increase in the enzyme yield (55 U/ml) compared to the highest yield obtained with free cells (23.6 U/ml). Moreover, with immobilized cells the maximum yield was reached after 72 h of incubation in batch fermentation under optimal conditions, whereas in the case of free cells the maximum enzyme yield was obtained only after 96 h of incubation. The alginate beads had good stability and also retained 75% ability of enzyme production even after eight cycles of repeated batch fermentation. It is significant that this is the first report on whole-cell immobilization for alpha-galactosidase production.     

Influence of alginate properties and gel reinforcement on fermentation characteristics of immobilized yeast cells

The effect of alginate composition, gel concentration, gelation method, cell loading and surface area on fermentation characteristics of immobilized yeast cells have been investigated. Molecular weight and G/M ratio had only little effect on fermentation velocity and gel strength, while increasing the alginate concentration caused a sizeable decrease in fermentation velocity and an increase in gel strength. The internally gelled immobilizates generally showed a higher fermentation velocity for the same gel strength and no decrease in gel strength was seen during fermentation. With high initial cell loadings, the fermentation velocity per g of immobilizate was higher, but the productivity per cell was lower than with low initial cell loadings. The difference decreased with time. Specific surface area (surface/volume) was shown to be an important factor for the observed productivity per gram of immobilizate, with high S/V ratios giving the highest productivity. Gel shape had no influence on fermentation velocity for a given S/V ratio. Gelation behaviour of externally gelled beads was determined by estimating the amount of cells liberated during gel formation through measurement of invertase activity (yeast-bound) in the gelling solution. A method for reinforcement of internally gelled alginate slabs with a nylon mesh was developed and utilized for production of a continuous fermentation reactor with reinforced gels.     

pH-controlled cell release and biomass distribution of alginate-immobilized Lactococcus lactis subsp. lactis

AIMS: To investigate the growth and release of Lactococcus lactis subsp. lactis in gel beads and to affect rates of cell release by changing the growth conditions. METHODS AND RESULTS: The rate of release and the distribution of immobilized L. lactis subsp. lactis in alginate beads were studied in continuous fermentations for 48 h. A change in operating pH from 6.5 to 9.25 initially reduced the ratio of the rates of cell release to lactate production by almost a factor of 105. Compared with fermentations at pH 6.5, growth at pH 9.25 also increased the final internal bead biomass concentration by a factor of 5 and increased the final rate of lactate production by 25%. After 48 h, the ratio of the rates of cell release to lactate production was still 10 times lower than in fermentations at pH 6.5. CONCLUSIONS: A change in the operating pH from 6.5 to 9.25 reduced rates of cell release throughout 48 h of fermentation and increased the final rates of lactate production and internal bead biomass concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These data illustrate that diffusional limitations and corresponding pH gradients can be exploited in affecting the distribution of immobilized growing cells and their concomitant release.     

Production of l (+) lactic acid by Lactobacillus delbrueckii immobilized in functionalized alginate matrices

The role of functionalized alginate gels as immobilized matrices in production of l (+) lactic acid by Lactobacillus delbrueckii was studied. L. delbrueckii cells immobilized in functionalized alginate beads showed enhanced bead stability and selectivity towards production of optically pure l (+) lactic acid in higher yields (1.74Yp/s) compared to natural alginate. Palmitoylated alginate beads revealed 99% enantiomeric selectivity (ee) in production of l (+) lactic acid. Metabolite analysis during fermentation indicated low by-product (acetic acid, propionic acid and ethanol) formation on repeated batch fermentation with functionalized immobilized microbial cells. The scanning electron microscopic studies showed dense entrapped microbial cell biomass in modified immobilized beads compared to native alginate. Thus the methodology has great importance in large-scale production of optically pure lactic acid.     

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。     

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.     

Mathematical model for evaluation of mass transfer limitations in phenol biodegradation by immobilized Pseudomonas putida

A mathematical model is proposed to analyze the mass transfer limitations in phenol biodegradation using Pseudomonas putida immobilized in calcium alginate. The model takes into account internal and external mass transfer limitations, substrate inhibition kinetics and the dependence of the effective diffusivity of phenol in alginate gel on cell concentration. The model is validated with the experimental data from batch fermentation. The effect of various operating conditions such as initial phenol concentration, initial cell loading, alginate gel loading on the biodegradation of phenol is experimentally demonstrated. Phenol degradation time is found to decrease initially and reach stationary value with increase in cell loading as well as gel loading. The model predicts these trends reasonably well and shows the presence of external mass transfer limitations. A new concept of effectiveness factor is introduced to analyze the overall performance of batch fermentation.     

Characterization of Lactobacillus carbohydrate fermentation activity using immobilized cell technique

A microbial bioreactor based on calcium alginate immobilized Lactobacillus cells coupled to a pH electrode was developed for quantitative determination of carbohydrate fermentation activity. A high biomass (10(10) cfu mL(-)(1)) and particular pregrowth conditions were needed. Reduction of catabolite repression by monosaccharides was achieved by pregrowth in lactose. The evolution of acid production in a continuous flow-stopped flow bioreactor was monitored for different sugar solutions in contact with the immobilized bacteria. The resulting slopes (DeltamV/Deltat) were used to quantify the fermentation capability for a defined sugar related to that of glucose, which was taken as 100%. The procedure is simple, being based on pH variation that can give quantitative results compared to other reported techniques for carbohydrate fermentation pattern from which only qualitative results are obtained. In addition, it offers reduction in time and costs and is a suitable tool for the rapid analysis of isolated strains and in studies of modifications of sugar metabolism in mutants.     

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.     

微生物发酵-膜分离法制备褐藻胶寡糖及其产物分析

比较褐藻胶裂解酶产生菌Alteromonassp .在摇瓶和发酵罐培养过程中生物量、褐藻胶寡糖含量以及褐藻胶裂解酶活性的变化 ,根据其变化确立了通过微生物发酵 膜分离技术结合制备褐藻胶寡糖的条件 ,并对寡糖进行凝胶过滤色谱和薄层色谱分析。用组成为每升含酵母粉 5g、蛋白胨 10g、FeSO4 0 1g、褐藻酸钠 12g、NaCl 1 5g ,pH为7 5的培养基 ,在 2 8℃培养褐藻胶裂解酶产生菌 ,结果表明 ,发酵罐培养 30h ,发酵液寡糖含量达到最大。发酵液通过超滤 纳滤两级膜分离 ,得到褐藻胶寡糖 ,寡糖的回收率和脱盐率分别为 94 0 %和 93 3%。通过凝胶柱分离和TLC分析 ,得到 5个褐藻胶寡糖组分。     

Diffusion coefficients of glucose and ethanol in cell-free and cell-occupied calcium alginate membranes

The diffusivities of glucose and ethanol in cell-free and cell-occupied membranes of calcium alginate were measured in a diffusion cell. The lag time analysis was used. Diffusivities decreased with increasing alginate concentration and were comparable with those in water for a 2% alginate membrane. Glucose and ethanol concentrations had no effect on the respective diffusion coefficients. The ratio of ethanol diffusivity to glucose diffusivity in 2 and 4% alginate agreed closely with the inverse ratio of the hydrodynamic raii for the two molecules in water, indicating that the hydrodynamic theory of diffusion in liquids may be applicable to diffusion in dilute alginate gels. Also, the presence of 20% dead yeast cells had no effect on the diffusivities. The data reported can be used to study reaction and diffusion in immobilized cell reactors and cell physiology under immobilized conditions.     

Properties and application of immobilized beta-D-glucosidase coentrapped with Zymomonas mobilis in calcium alginate

The enzyme beta-D-glucosidase has been immobilized on concanavalin A-Sepharose to give a maximum loading of 2050 units/g dry weight of support material. The immobilized beta-D-glucosidase was also entrapped within calcium alginate gel spheres with apparently only 35% retention of activity when assayed with 10mM cellobiose. However, it was discovered that, unlike the immobilized enzyme, the entrapped immobilized enzyme was not subject to substrate inhibition up to 100mM cellobiose, suggesting that a concentration gradient of cellobiose existed between the bulk solution and the interior of the gel sphere. Thus, the activity of the entrapped immobilized enzyme was almost twice as high as that of the immobilized enzyme when assayed with 100mM cellobiose. Concanavalin A-Sepharose-immobilized beta-D-glucosidase and the bacterium Zymomonas mobilis coimmobilized in calcium alginate gel spheres converted cellobiose to ethanol in both batch and continuous-flow fermentation systems.     

Continuous penicillin production by Penicillium chrysogenum immobilized in calcium alginate beads

Summary A high penicillin-producing Penicillium chrysogenum strain immobilized in calcium alginate beads was used for continuous penicillin fermentation in a bubble column and in a conical bubble fermentor. The fermentation was limited by the growth rate, dilution rates and the stability of the alginate beads. The immobilized cells lost their ability to produce penicillin in the bubble column after 48 h from beginning of the continuous fermentation. In the conical bubble fermentor the immobilized cells remained active for more than 7 days. This bioreactor ensured a good distribution of nutrients and oxygen as well as a higher mechanical stability of the alginate beads.     

Batch production of propionic acid by immobilizedPropionibacterium sp

Whole cells ofPropionibacterium freudenreichii subsp.shermanii (two strains) were immobilized in a living state in 2 and 4% alginate gel and 2, 4 and 6% carrageenan gel. Production of propionic acid, acetic acid, and vitamin B₁₂ were examined. The best results were obtained in the fermentation with strains immobilized in 4% alginate gel when applied for the third time.     

A comparative study of gel entrapped and membrane attached microbial reactors for biodegrading phenol

A comparative study between two reactors, one using microorganisms entrapped in calcium alginate gel, and the other using microorganisms attached on the surface of a membrane (polymeric microporous sheeting, MPS^TM) to biodegrade phenol is performed. Results indicate that the alginate bead bioreactor is efficient at higher phenol concentrations while the membrane bioreactor shows better performance at lower phenol concentrations. This unique response is primarily attributed to the different techniques by which the microorganisms are immobilized in the two reactors.In batch mode, below a starting concentration of 100 ppm phenol, biodegradation rates in the membrane bioreactor are (7.58 to 12.02 mg phenol/h · g dry biomass) atleast 10 times the rates in alginate bead bioreactor (0.74 to 1.32 mg phenol/h · g dry biomass). Biodegradation rates for the two reactors match at a starting concentration of 250 ppm phenol. Above 500 ppm phenol, the rates in the alginate bead bioreactor are (7.3 to 8.1 mg phenol/h · g dry biomass) on an average 5.5 times the corresponding rates in the membrane bioreactor (2.18 to 1.03 mg phenol/h · g dry biomass).In continuous feed mode the steady state degradation rates in the membrane bioreactor are one to two orders of magnitude higher than the alginate bead bioreactor below 150 ppm inlet phenol concentration. At an inlet concentration around 250 ppm phenol the rates are comparable. Above 500 ppm of phenol the rates in the alginate bioreactor are an order of magnitude high than the membrane bioreactor.Due to substrate inhibition, and its inability to sustain a high biomass concentration, the membrane bioreactor shows poor efficiencies at phenol concentrations above 250 ppm. At low phenol concentrations the apparent reaction rates in the alginate bead bioreactor decrease due to the diffusional resistance of the gel matrix, while biodegradation rates in the membrane bioreactor remain high due to essentially no external diffusional resistance.Results indicate that a combined reactor system can be more effective for bioremediation than either separate or attached microbial reactors.     

A novel method for immobilization of yeast cells in alginate gels of various shapes by internal liberation of Ca-ions

Summary In situ gelation of alginate, in which Ca-ions are liberated internally in the gel, was used for immobilization of yeast cells. Compared to the traditional alginate gelation method, internal gelling gave immobilized yeast particles of higher strength, without reduction in fermentation rate.     

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     

Co-immobilization effect on H2 production by a chloroplast membranes-hydrogenase system

Summary Chloroplast membranes immobilized within a BSA-GA matrix or within an alginate gel have been associated with native or immobilized hydrogenase in order to produce hydrogen gas through biophotolysis of water. Due to the reaction geometry, co-immobilization of chloroplast membranes with the enzyme inside the same matrix considerably improved the amount of H₂ produced and the initial activity. The use of entrapment methods such as alginate gel allowed diffusion of proteins through the matrix. Electron microscopic observations illustrated these results.     

Kinetics of ethanol production by immobilized Kluyveromyces marxianus cells at varying sugar concentrations of Jerusalem artichoke juice

Summary Kinetics of ethanol fermentation at varying sugar concentrations of Jerusalem artichoke tuber extract has been studied using Kluyveromyces marxianus cells immobilized in calcium alginate gel beads. A maximum ethanol concentration of 111 g/l was achieved at an initial sugar concentration of 260 g/l in 20 hours, when the immobilized cell concentration in the calcium alginate beads was 53.3 g dry wt./l bead volume. Ethanol yield remained almost unaffected by initial sugar concentration up to 250 g/l and was found to be about 88% of the theoretical. Maximum rate of ethanol production decreased from 22.5 g ethanol/l/h to 10.5 g ethanol/l/h while the maximum rate of total sugars utilization decreased from 74.9 g sugars/l/h to 28.5 g sugars/l/h as the initial substrate concentration was increased from 100 to 300 g/l. The concentration of free cells in the fermentation broth was low.