Immobilized Cell Concentration Research Articles

Kinetic characterization of tyrosinase containing mushroom (Agaricus bisporus) cells immobilized in silica alginate

A tyrosinase containing cell preparation from the edible mushroom Agaricus bisporus was immobilized in silica alginate matrix capsules. This catalyst system was characterized in terms of its reaction kinetics using the orthohydroxylation of bisphenol A (BPA) as a model system. The effect of BPA concentration on the reaction rate could be described with the Michaelis–Menten kinetics with an apparent Michaelis–Menten constant of Km=50.4μmol/l. The reaction rate was proportional to the immobilized cell concentration in the investigated range. The highest observed apparent maximal reaction rate vmax related to the wet weight of matrix capsules at 30°C was 51.5nmol/(ming)). The diffusion coefficient D of BPA within the matrix capsules was determined to 4.9×10−10m2/s ±10% at 30°C. The studies revealed that the observed reaction rate was not limited by the BPA diffusion rate in the matrix material. The immobilized cells exhibited tyrosinase activity over the whole examined pH range (pH 3–11) with a broad maximum between pH 6 and pH 10. The apparent activation energy EA for the BPA conversion was determined to 37.6kJ/mol, whereas the apparent activation energy EA,inact for the thermal inactivation of the catalyst was determined to 71.7kJ/mol. It was demonstrated that the determined parameter values were suitable to predict the change of the BPA concentration with respect to time under different reaction conditions and therefore could be useful for future applications of the catalyst, for example in synthesis of o-diphenols or bioremediation processes.

Hemicellulosic ethanol production by immobilized cells of Scheffersomyces stipitis: Effect of cell concentration and stirring

Bioconversion of hemicellulosic hydrolysate into ethanol plays a pivotal role in the overall success of biorefineries. For the efficient fermentative conversion of hemicellulosic hydrolysates into ethanol, the use of immobilized cells system could provide the enhanced ethanol productivities with significant time savings. Here, we investigated the effect of 2 important factors (e.g., cell concentration and stirring) on ethanol production from sugarcane bagasse hydrolysate using the yeast Scheffersomyces stipitis immobilized in calcium alginate matrix. A 22 full factorial design of experiment was performed considering the process variables- immobilized cell concentration (3.0, 6.5 and 10.0 g/L) and stirring (100, 200 and 300 rpm). Statistical analysis showed that stirring has the major influence on ethanol production. Maximum ethanol production (8.90 g/l) with ethanol yield (Yp/s) of 0.33 g/g and ethanol productivity (Qp) of 0.185 g/l/h was obtained under the optimized process conditions (10.0 g/L of cells and 100 rpm).

Ethanol production from concentrated food waste hydrolysates with yeast cells immobilized on corn stalk

The aim of the present study was to examine ethanol production from concentrated food waste hydrolysates using whole cells of S. cerevisiae immobilized on corn stalks. In order to improve cell immobilization efficiency, biological modification of the carrier was carried out by cellulase hydrolysis. The results show that proper modification of the carrier with cellulase hydrolysis was suitable for cell immobilization. The mechanism proposed, cellulase hydrolysis, not only increased the immobilized cell concentration, but also disrupted the sleek surface to become rough and porous, which enhanced ethanol production. In batch fermentation with an initial reducing sugar concentration of 202.64 ± 1.86 g/l, an optimal ethanol concentration of 87.91 ± 1.98 g/l was obtained using a modified corn stalk-immobilized cell system. The ethanol concentration produced by the immobilized cells was 6.9% higher than that produced by the free cells. Ethanol production in the 14th cycle repeated batch fermentation demonstrated the enhanced stability of the immobilized yeast cells. Under continuous fermentation in an immobilized cell reactor, the maximum ethanol concentration of 84.85 g/l, and the highest ethanol yield of 0.43 g/g (of reducing sugar) were achieved at hydraulic retention time (HRT) of 3.10 h, whereas the maximum volumetric ethanol productivity of 43.54 g/l/h was observed at a HRT of 1.55 h.

Evaluating the potential of wine-making residues and corn cobs as support materials for cell immobilization for ethanol production

Three wine-making residues (grape seeds, skins and stems), and corn cobs were evaluated as support material for immobilization of Saccharomyces cerevisiae and the ethanol production by the immobilized cells was assessed. The main objective of this study was to find an abundant and low cost material suitable for the cells immobilization and able to be used in a next step of wine production by immobilized yeast cells. The four natural materials were used as support in two different forms: untreated, and treated by a sequence of acid and basic reactions. Untreated grape skin and corn cobs provided the highest cell immobilization results (25.1 and 22.2 mg cells/g support, respectively). The maximum ethanol production yield (about 0.50 g/g) was also obtained when the cells were immobilized in these untreated materials. It was also found that the support materials released nutrients to the medium, which favored the yeast development and the ethanol production. The use of immobilized cells systems under agitated conditions gave ethanol yields similar to those obtained by the static fermentations, but the immobilized cell concentration was significantly lower. In brief, static fermentation with cells immobilized on grape skins or corn cobs appear to be an interesting alternative for use on wine-making. The use of grape skins, particularly, which is a by-product of the wine elaboration, could be of larger interest to obtain an integrated wine production process with by-product reuse.

Review of Gardasil

A single vertical rotating immobilized cell reactor (VRICR) with the bacterium R. erythropolis, as a biocatalyst, was developed and used for investigation of biodesulfurization process with its two successive stages of cell growth and desulfurization activity. With a rotation speed of 15 rpm and oxygen transfer rate of 90 mM O2.l-1.h-1, immobilized cell concentration of up to 70.0 g.l-1 was achieved during the first stage and further used, in the second, to carry out a stable continuous desulfurization of model oil (dibenzothiophene in hexadecane). A steady state with specific desulfurization rate as high as 167 mM 2HBP.Kg-1.h-1 and sulfur removal efficiency of 100% were maintained for more than 120 h. The proposed integrated biodesulfurization process utilizing the VRICR has the potential to lower operating costs and support possibilities of commercial application at the expense of Hydrodesulfurization process currently employed.

Repeated fed-batch lactic acid production in a packed bed-stirred fermentor system using a pH feedback feeding method

Lactic acid production by repeated fed-batch fermentation using free and immobilized cells of Lactobacillus lactis-11 in a packed bed-stirred fermentor (PBSF) system filled with different support materials including ceramic beads, macro-activated carbon cylinders and glass fiber balls was investigated. The results showed that the optimal support materials were the ceramic beads with diameters of 1-2 mm. Compared with the free cell fermentation system, lactic acid production and volumetric productivity in the PBSF system increased by 16.6 and 12.5%, respectively. Though the concentration of free cells decreased sharply, lactic acid production remained stable in five consecutive fed-batch runs using the PBSF system. pH gradients, immobilized cell concentration and mass diffusion in the packed bed were all affected by the recirculation rate of the culture broth. Maximum lactic acid production, productivity and yield occurred at a recirculation rate of 50 mL min(-1).

Immobilization of Saccharomyces cerevisiae to modified bagasse for ethanol production

Ethanol production was carried out by growing yeast cells immobilized on bagasse carriers. The effects of cellulase hydrolysis of carriers on cell immobilization and ethanol production were investigated. The electrical conductivity of carriers after three days cellulase hydrolysis was 3.7 times higher than that of basic bagasse carrier and the immobilized cell concentration of modified carriers was 1.46 times higher than that of unmodified ones. The scanning electron microscopy (SEM) of the carriers indicated that cellulase hydrolysis disrupted the sleek surface into rough and porous, which promoted the mass transfer and resulted in high specific ethanol production rate. Cellulase hydrolysis of carriers has positive effects on both of cell immobilization and ethanol productivity.

Optimization of lactic acid production by immobilized Lactococcus lactis IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l(-1), the optimal production medium had the following initial concentrations of nutrients (g l(-1)): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 degrees C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l(-1) h(-1). Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l(-1) h(-1). The packed bed could be used in repeated batch runs to produce lactic acid.

Cell immobilization of Trichosporon cutaneum strain with phenol degradation ability on new modified polymer carriers

Chemical immobilization of Trichosporon cutaneum R57 cells onto modified polyacrylonitrile granules and fibres was carried out. The polymer carriers were modified in water solutions of NaOH and 1,2-diaminoethane. The concentration of amino groups obtained by the modification was determined. The optimal conditions for chemical immobilization of cells of T. cutaneum R57 with glutaraldehyde were found to be: 40% NaOH concentration, 46.8 mg/ml initial cell suspension, 24 h immobilization time and pH 6. The change in protein concentration of free and immobilized cells was studied during a full biodegradation cycle. The rate of phenol biodegradation by T. cutaneum R57 cells immobilized on porous polyacrylonitrile (PAN) fibres was higher than that obtained using modified PAN beds.

Plant Cell Immobilization in Loofa Sponge Using Two-Way Bubble Circular System.

In this study, we investigated the immobilization of loofa sponge as a carrier for plant cells using a novel immobilization reactor, namely, a two-way bubble circular (TBC) reactor, for analyzing the effects of medium flow pattern, A TBC reactor, in which two-way circular flow prevailed and was controlled by bubble flow and sparger adjustment, was developed as a suitable system for plant cell immobilization using loofa sponge. In this system, relatively small aggregates of cultured plant cells could be immobilized, and the entrapped cells grew quickly, avoiding cell leakage from the sponge throughout 31 days of incubation. The optimum immobilization conditions and immobilization mechanism in this system were investigated using Coffea arabica cells as a model cell line. By changing the flow direction of culture medium in the circular reactor periodically between downflow and up-flow, cell immobilization efficiency of the loofa sponge was improved as compared with reactors using one-directional flows. The specific respiration activity of cells immobilized in loofa sponge was also studied. Uniform distribution of cell density and specific respiration activity were obtained along the axial direction of the loofa sponge. In the same reactor system, loofa sponge exhibited high immobilized cell concentration and specific respiration activity not less than those of polyurethane foam.

Development of a method for immobilization of non-flocculating cells in loofa ( Luffa cylindrica) sponge

Both the matrix structure of loofa sponge and the flocculating property of cells were necessary for efficient immobilization. The addition of chitosan to a reactor containing a bed of loofa sponge and a Candida brassicae cell suspension induced cell flocculation and the cells were efficiently immobilized. During ethanol production by the immobilized cells, the free cell concentration in the broth was controlled at the desired level by intermittent addition of chitosan to the reactor. The immobilized cell concentration increased but their specific ethanol productivity decreased with an increase in the chitosan concentration. The maximum ethanol productivity was obtained at a low chitosan concentration of 0·03 g/litre. With this optimal concentration, the cell concentration, ethanol yield and productivity were, respectively, 2, 1·3 and 3 times higher than those of the suspension culture.

Effect of Cell Concentration on the Kinetics of Whole‐Cell Enzyme Entrapped within Calcium Alginate

AbstractA method for determining the kinetics of whole‐cell enzymes entrapped within calcium alginate gel beads is presented. Diffusivities of the substrate and the product and kinetic parameters, i.e., the Michaelis constant and the maximum velocity, could be estimated by fitting experimental data to a proposed mathematical model. Immobilized cell concentration was found to be the main factor causing a decrease in maximum velocity. The influence of cell concentration in the gel beads on the variation in the maximum velocity could be isolated from the effect of cell concentration on diffusional limitation and evaluated separately. The whole‐cell enzymes became partially difficult to access by substrate when the cell population increased. The loss of enzyme accessibility resulted in a decrease in the maximum velocity and apparent initial velocity. This phenomenon of the maximum velocity varying with immobilized cell concentration has not been reported in the literature.

Phosphate uptake and release activity in immobilized polyphosphate-accumulating bacterium Microlunatus phosphovorus strain NM-1

A polyphosphate-accumulating Microlunatus phosphovorus bacterium, strain NM-1, with the abilities of accumulating polyphosphate under aerobic conditions and releasing phosphate under anaerobic conditions, was successfully immobilized in polyacrylamide gel. Strain NM-1 still exhibited the phosphate uptake and release activities after immobilization as observed in activated sludge with enhanced phosphate removal activity. Under anaerobic conditions, the immobilized cells could take up substrates by deriving energy from the hydrolysis of polyphosphate to orthophosphate. The ratio of the amount of phosphorus released to that of TOC (total organic carbon) taken up was approximately 1 g-P/g-TOC in a synthetic medium. The initial phosphate release rate varied between 10 and 30 mg-P/g-cell·h depending on the initial TOC concentration. The TOC concentration required to induce sufficient phosphate release was about 1,000 mg/ l for a 12,500 mg/ l immobilized cell concentration. Under aerobic conditions, the immobilized cells rapidly took up the phosphate previously released. The phosphate uptake rate was about 10–20 mg-P/g-cell·h. The optimum pH and temperature for phosphate release and uptake were 8 and 25°C, respectively.

Regeneration of NADH in a bioreactor using yeast cells immobilized in alginate fiber: I. Method and effect of reactor variables

Saccharomyces cerevisiae cells immobilized in a calcium alginate fiber reactor were used as a source of alcohol dehydrogenase for the NAD(+)-to-NADH reaction. The reaction was catalyzed by enzyme in cells on the surface of the fiber. Internal diffusional effects were present. The enzyme cell concentration was optimized by harvesting cells finally grown under anaerobic conditions. The results were expressed as an apparent reaction rate constant that was independent of NAD(+) and excess ethanol concentration, was slightly affected by flow rate above a minimum value, and increased with immobilized cell concentration in the fiber. The reaction was complete after 6 to 7 h under optimal conditions of 36 degrees C and 9.5 pH. The latter was 0.5 pH units above the free enzyme optimum, indicating that microenvironmental effects were in evidence.

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.

Immobilized cell biocatalyst activation and pseudo‐steady‐state behavior: Model and experiment

An intrinsic, unstructured model has been utilized to describethe startup dynamics of a continuous Caalginate-immobilized Zymomonas mobilis (ATCC 10988) fermentation. This model predicts, at least qualitatively, transients in the fermenter effluent glucose, ethanol, and biomass concentrations as well as radial gradients in immobilized-cell concentration and activity within the gelbiocatalysts. Predicted intrabiocatalyst gradients in immobilized-cell specific growth rate were used to calculate the corresponding gradients in intracellular RNA level based on a reported linear relationship between the two. Mathematical simulations of immobilized biomass concentration profiles and RNA content were verified using a novel, scanning microfluorimetry technique.

Vertical Rotating Immobilized Cell Reactor of the bacteriumZymomonas mobilis for stable long-term continuous ethanol production

Vertical Rotating Immobilized Cell Reactor was designed and built for glucose conversion into ethanol. Immobilized biomass units with Z.mobilis cells attached into polyurethane foam discs were fixed along a rotating shaft inside the bioreactor. The effect of rotation speed on the concentration of immobilized biomass was studied. Stability of the bioreactor over long-term operation was dependent on the concentration of the immobilized biomass. With fermentation carried out at 6 rpm a constant active immobilized cell concentration of only 34.5 g/l was maintained and used to convert up to 140 g glucose/l into more than 70 g ethanol/l with a volumetric ethanol productivity of 63 g/l/h.

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

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.

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 2. Application for high fructose syrup production

Batch and continuous production of high fructose syrup from Jerusalem artichoke tubers has been studied using yeast cells immobilized in open pore gelatin matrix. In a batch reactor, the hydrolysis was 93% ( d-fructose/ d-glucose = 90/10 ) and 42 mg d-fructose per ml was produced from the artichoke tuber extract by immobilized cells in 3 h. The same immobilized cells were recycled and used repeatedly for 10 batch cycles starting with fresh juice at the beginning of each cycle. It was found that immobilized cells were extremely stable and the percent hydrolysis was almost constant for all 10 batch cycles. In a continuous reactor using an immobilized cell concentration of 65.7 g (dry wt) l −1 of total working bioreactor volume, the percent hydrolysis was found to remain constant at ∼100% at dilution rates <1.26 h −1, but beyond that it decreased. Volumetric productivity attained its maximum value at D = 2.08 h −1 and was found to be 100 g l −1 h −1. This was achieved at a feed sugar conversion of 80%. At 90% conversion and D = 1.66 h −1, the productivity was found to be 90 g l −1 h −1. Continuous operation of the immobilized cell bioreactor at a constant dilution rate of 1.65 h −1 for 240 h resulted in only 2% loss of original activity.

Kinetics of ethanol production by immobilized cells of Zymomonas mobilis at varying d-glucose concentrations

Ethanol fermentation by cells of Zymomonas mobilis immobilized in calcium alginate gel has been studied using 5 to 30 wt% initial d-glucose in the medium. Up to 27% d-glucose was completely fermented and the maximum ethanol concentration of 12.6% (w/v) was obtained using an immobilized cell concentration of 58 g dry wt l −1 of bead volume. The ethanol yield coefficient was almost unaffected by initial d-glucose concentration and its value was >95% of theoretical. The rates of ethanol production and d-glucose utilization first increased, with an increase in initial d-glucose concentration up to 13.6%, and then started to decrease upon a further increase in initial d-glucose concentration. Cell leakage from the calcium alginate beads was very low.