Cells In Calcium Alginate Beads Research Articles

Kinetics of dye decolorization using heterogeneous catalytic system with immobilized Achromobacter xylosoxidans DDB6

Textile effluents containing toxic dyes must be treated effectively before discharge to prevent adverse environmental impacts. Traditional physical and chemical treatment methods are costly and generate secondary pollutants. In contrast, biological treatment is a more suitable, clean, versatile, eco-friendly, and cost-effective technique for treating textile effluent. It is well established that indigenous microbial populations present in effluents can effectively degrade toxic dyes. In this regard, Achromobacter xylosoxidans DDB6 was isolated from the effluent sample to decolorize crystal violet (CV), Coomassie brilliant blue (CBB), and alizarin red (AR) by 67.20%, 28.58%, and 20.41%, respectively. The growth parameters of A. xylosoxidans DDB6 in media supplemented with 100 ppm of various dyes were determined using the modified Gompertz growth model. The immobilized cells in calcium alginate beads showed apparent decolorization rate constant of 0.27, 0.18, and 0.13 h−1 for CV, CBB, and AR, respectively. The immobilized cells in a packed bed reactor with an optimum flow rate of 0.5 mL/min were used to treat 100 ppm of CV with a percentage decolorization of 79.47% after three cycles. Based on the findings, A. xylosoxidans DDB6 could be effectively used for decolorization of various dyes.

Hydrogen Production by Immobilized Rhodopseudomonas sp. Cells in Calcium Alginate Beads

The present investigation concerns the potentiality of Rhodopseudomonas sp. cells to produce clean energy such as molecular hydrogen (H2). The abovementioned goal could be reached by improving the capability of purple non-sulfur bacteria to produce H2 via a photofermentative process through the enzyme nitrogenase. Rhodopseudomonas sp. cells were immobilized in calcium alginate gel beads and cultured in a cylindrical photobioreactor at a working volume of 0.22 L. The semi-continuous process, which lasted for 11 days, was interspersed with the washing of the beads with the aim of increasing the H2 production rate. The maximum H2 production rate reached 5.25 ± 0.93 mL/h with a total output of 505 mL. The productivity was 40.9 μL (of H2)/mg (of cells)/h or 10.2 mL (of H2)/L (of culture)/h with a light conversion efficiency of 1.20%.

Encapsulating IM7-Displaying Yeast Cells in Calcium Alginate Beads for One-Step Protein Purification and Multienzyme Biocatalysis.

There are several commercial chromatographic systems for protein purification; however, development of cost-effective 3H-grade (high yield, high purity, and high activity) purification approaches is highly demanded. Here, we establish a methodology for encapsulating the IM7-displaying yeast cells in calcium alginate beads. Taking advantage of this biomaterial-based affinity chromatography, rapid and cost-effective purification of proteins with over 90% purity in a single step is achieved. Moreover, our system enables coating the multienzyme complex to produce reusable immobilized cells for efficient cascade biotransformation. Together, the present method has great application potentials not only in the laboratory but also in the industry for production of protein products as well as biocatalysis.

Immobilization of Actinobacillus Succinigenes in Alginate Beads for Succinic Acid Production

Nowadays the succinic acid (SA) - a C-4 dicarboxylic acid - is considered as one of the most promising platform chemicals produced from renewable resources via fermentation. Enhancement of the SA fermentation efficiency and productivity may take advantages from immobilized cultures..This work reports the immobilization of Actinobacillus succinogenes cells in calcium alginate beads. Glucose was used as carbon source for the fermentation. A parallel fermentation test with free cells was carried out to assess the advantages of the immobilization technology.The immobilization in alginate beads was proved to be an effective technique for SA production by A. succinogenes. The fermentation performances of the immobilized cells were higher than those of the free cells. In particular, at initial glucose concentrations of 40 g/L the maximum productivity in immobilized cells fermentation (0.77 g/Lh) is more than twice that measured for free cells (0.32 g/Lh).

Ethanol production from coffee mucilage fermentation by S. cerevisiae immobilized in calcium-alginate beads

Soluble sugars in coffee mucilage were directly fermented into ethanol using immobilized Saccharomyces cerevisiae cells in calcium alginate beads. Several tests were performed via alginate-entrapped cells in different bead sizes (3 or 7 mm) combined to alginate concentrations (2–4%). The highest yield of 0.33 g ethanol/g sugar was achieved at 18 h using alginate-entrapped cells in 3 mm diameter and 2% (w/v) alginate, which corresponded to 64% of the theoretically achievable yield of ethanol. Tests with 7 mm beads took 6 h longer to reach maximum ethanol productions. The reduction of bead size increased mass transfer of substrates from the liquid to the immobilized cells, accelerating sugar consumption and ethanol production. Entrapment enabled reuse of the cells for up to 3 consecutive batches with stable ethanol production (up to 72 h). This work confirms the reuse of alginate-entrapped cells is feasible for ethanol fermentation of mucilage without supplement of nutrients.

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Effect of cell immobilization and pH on Scheffersomyces stipitis growth and fermentation capacity in rich and inhibitory media

Abstract Background A wide range of value-added products can potentially be produced by bioprocessing hardwood spent sulfite liquors (HSSLs) that are by-products of pulp and paper industry with a high pentose sugar content. However, besides sugars, HSSLs contain considerable amounts of sulfonated lignin derivatives and acetic acid that inhibit the metabolic activity of most microorganisms. Scheffersomyces stipitis is a yeast with high capacity to ferment the pentose sugar xylose under appropriate microaerophilic conditions but it has limited tolerance to HSSL inhibitors. In the present study, cultivations of suspended and immobilized S. stipitis were compared in terms of growth capacity and by-product formation using rich medium and HSSL to investigate whether the immobilization of cells in calcium alginate beads could be a protection against inhibitors while favoring the presence of microaerophilic conditions. Results Whereas cell immobilization clearly favored the fermentative metabolism in rich medium, pH control was found to play a more important role than cell immobilization on the ethanol production efficiency from bio-detoxified HSSL (bdHSSL), leading to an improvement of 1.3-fold on the maximum ethanol productivity than using suspended cells. When immobilization and pH control were applied simultaneously, the ethanol yield improved by 1.3-fold with unchanged productivity, reaching 0.26 g ethanol.(g glucose + xylose)−1. Analysis of the immobilized beads inside revealed that the cells had grown in the opposite direction of the cortex. Conclusions Immobilization and pH control at 5.5, when applied simultaneously, have a positive impact on the fermentative metabolism of S. stipitis, improving the ethanol production efficiency. For the first time light microscopic analysis of the beads suggested that the nutrient and mass transfer limitations played a more important role in the fermentation than a possible protective role against inhibitors.

Effect of immobilized cells in calcium alginate beads in alcoholic fermentation

Saccharomyces cerevisiae cells were immobilized in calcium alginate and chitosan-covered calcium alginate beads and studied in the fermentation of glucose and sucrose for ethanol production. The batch fermentations were carried out in an orbital shaker and assessed by monitoring the concentration of substrate and product with HPLC. Cell immobilization in calcium alginate beads and chitosan-covered calcium alginate beads allowed reuse of the beads in eight sequential fermentation cycles of 10 h each. The final concentration of ethanol using free cells was 40 g L-1 and the yields using glucose and sucrose as carbon sources were 78% and 74.3%, respectively. For immobilized cells in calcium alginate beads, the final ethanol concentration from glucose was 32.9 ± 1.7 g L-1 with a 64.5 ± 3.4% yield, while the final ethanol concentration from sucrose was 33.5 ± 4.6 g L-1 with a 64.5 ± 8.6% yield. For immobilized cells in chitosan-covered calcium alginate beads, the ethanol concentration from glucose was 30.7 ± 1.4 g L-1 with a 61.1 ± 2.8% yield, while the final ethanol concentration from sucrose was 31.8 ± 6.9 g L-1 with a 62.1 ± 12.8% yield. The immobilized cells allowed eight 10 h sequential reuse cycles to be carried out with stable final ethanol concentrations. In addition, there was no need to use antibiotics and no contamination was observed. After the eighth cycle, there was a significant rupture of the beads making them inappropriate for reuse.

Repeated Batch Cell-Immobilized System for the Biotechnological Production of Xylitol as a Renewable Green Sweetener

The present paper studies the biotechnological production of xylitol using sugarcane bagasse hydrolysate in a repeated batch fermentation system with immobilized cells of Candida guilliermondii FTI20037. Immobilized cell system is considered as an attractive alternative to reuse the well-grown and adapted yeast cells in a new fresh fermentation media, without the need of the inoculum stage. In this work, seven repeated batches were performed in a fluidized bed bioreactor using immobilized cells in calcium alginate beads. According to the obtained results it was observed that the immobilized cells of C. guilliermondii can be reused for six successive batches maintaining an average xylitol yield (Y(p/s)) of 0.7 g/L and a volumetric productivity (Q(p)) of 0.42 g/Lh at the end of 432 h of fermentation. On the other hand, in the seventh batch (504 h), a decrease of 44 % in the final concentration of xylitol was observed. This reduction can be explained by the possible diffusion and accumulation of insoluble substances, found in the hemicellulosic hydrolysate, in the interior of the immobilization support resulting in substrate mass transfer limitations.

Immobilization of Bacillus amyloliquefaciens MBL27 cells for enhanced antimicrobial protein production using calcium alginate beads

Cell immobilization is one of the common techniques for increasing the overall cell concentration and productivity. Bacillus amyloliquefaciens MBL27 cells were immobilized in calcium alginate beads and it is a promising method for repeated AMP (antimicrobial protein) production. The present study aimed at determining the optimal conditions for immobilization of B. amyloliquefaciens MBL27 cells in calcium alginate beads and the operational stability for enhanced production of the AMP. AMP production with free and immobilized cells was also done. In batch fermentation, maximum AMP production (7300 AU (arbitrary units)/ml against Staphylococcus aureus) was obtained with immobilized cells in shake flasks under optimized parameters such as 3% (w/v) sodium alginate, 136mM CaCl2 with 350 alginate beads/flask of 2.7-3.0mm diameter. In repeated cultivation, the highest activity was obtained after the second cycle of use and approx. 94% production was noted up to the fifth cycle. The immobilized cells of B. amyloliquefaciens MBL27 in alginate beads are more efficient for the production of AMP and had good stability. The potential application of AMP as a wound healant and the need for development of economical methods for improved production make whole cell immobilization an excellent alternative method for enhanced AMP production.

Survival enhancement of probiotic Lactobacillus plantarum CMU-FP002 by granulation and encapsulation techniques.

Two processes of enclosure of probiotic Lactobacillus plantarum CMU-FP002, probiotic granules and calcium alginate beads, were studied. Sodium alginate solution at 1.0, 1.5 and 2.0 % (w/v) was used as a binder. The results showed that 20 log cfu/ml initial concentration of cells could be entrapped by the granules and beads with 12 to 13 log cfu/g and 16 cfu/g, respectively. The physical properties of granules and beads revealed that the strength increased when sodium alginate concentration was increased. On the other hand, the dissolution decreased. Probiotic granules completely released the cells within 60 min after being suspended in stimulate gastric fluid (SGF) pH 1.8 and had 2 to 3 log survival cells per gram. Calcium alginate beads, which were formulated from 1.0 and 1.5% (w/v) sodium alginate solution, gradually released bacterial cells and were completely released in SGF within 120 min. The beads formulated from 2.0 %(w/v) sodium alginate solution could not completely release the probiotics. The beads contained more survival cells than granules. Furthermore, the beads formulated from 1.5% (w/v) sodium alginate solution had the highest survival cells (9.30 log cfu/g). Probiotic cells in calcium alginate beads were still high (11 log cfu/g), although they were stored at 4EsC for 5 days alternating with room temperature for 5 days, for a total of 2 month. Further application in broilers will be studied. Key words: Probiotic, survival enhancement, granulation, encapsulation,Lactobacillus plantarum.

Limited beneficial effects of perfluorocarbon emulsions on encapsulated cells in culture: Experimental and modeling studies

Due to the high solubility of oxygen in perfluorocarbons (PFCs), these compounds have been explored for improved cell and tissue oxygenation. The goal of this study is to investigate the effects of a PFC emulsion on cellular growth and function in a tissue engineered construct. A perfluorotributylamine (PFTBA) emulsion was co-encapsulated at 10 vol% with mouse βTC-tet insulinoma cells in calcium alginate beads and cultured under normoxic and severely hypoxic conditions. The number of metabolically active cells and the induced insulin secretion rate were measured over time for up to 16 days. Results showed no significant effect of PFTBA relative to the PFTBA-free control. The alginate–PFC-cell system was also modeled mathematically, and simulations tracked the number of viable cells over time under the same conditions used experimentally. Simulations revealed only a small, likely experimentally undetectable difference in cell density between the PFC-containing and PFC-free control beads. It is concluded that PFTBA up to 10 vol% has no significant effect on the growth and function of encapsulated βTC-tet cells under normoxic and hypoxic conditions.

Decolorization of an industrial effluent by free and immobilized cells of Stenotrophomonas maltophilia AAP56. Implementation of efficient down flow column reactor

A bacterial strain AAP56, isolated from a polluted soil (from Kelibia city) and identified as Stentrophomonas maltophilia, was particularly interesting for its ability to decolorize recalcitrant dyes of an industrial effluent: SITEX Black. The final percentage decolorization 60% was shown by bacterial culture after incubation in LB medium at 30°C under shaking conditions. The decolorization was closely correlated with the metabolic bacterial growth. The replacement of yeast extract in LB medium composition by soya flour was clearly efficient to enhance the percentage decolorization by 20% and also to reduce the growth medium cost 60-fold. The bacteria were able to reduce 23% from the initial COD and 28% from the initial BOD5 of the effluent. The immobilization of bacterial cells in calcium alginate beads improved by 25% the effectiveness of the biotransformation within 24 h in batch conditions. The potential of a downflow fixed column reactor (DFCR) to decolorize SITEX Black was evaluated under dilution rate. The best decolorization percentage (82%) was recorded at 0.3 h−1. This bioprocess seems to be a potentially useful method to remediate the colored textile wastewater.

Production of High Hydroxytyrosol Yields via Tyrosol Conversion by Pseudomonas aeruginosa Immobilized Resting Cells

An immobilized whole cell system was successfully performed to produce the most powerful antioxidant, hydroxytyrosol. Bioconversion of tyrosol into hydroxytyrosol was achieved via the immobilization of Pseudomonas aeruginosa resting cells in calcium alginate beads. Immobilization was advantageous as it allows immobilized cells to tolerate a greater tyrosol concentration than free cells. The bioconversion yield reached 86% in the presence of 5 g L-1 of tyrosol when cells immobilized in alginate beads were carried out in single batches. Evaluation of kinetic parameters showed the maintenance of the same catalytic efficiency expressed as Kcat/Km for both free and immobilized cells. The use of immobilized cells in repeated batches demonstrated a notable activity stabilization since the biocatalyst reusability was extended for at least four batches with a molar yield greater than 85%.

Encapsulation of RIN-m5F cells within Ba2+ cross-linked alginate beads affects proliferation and insulin secretion

The viability, proliferation and insulin production of RIN-m5F cells when loaded into alginate beads to form a 3D culture system has been investigated. The mechanism of alginate cross-linking (calcium ions vs barium ions), the addition of poly(L-lysine) (PLL) and poly(L-ornithine (PLO) and presence of different extra-cellular matrix proteins (ECM) influence the RIN-m5F cell behaviour. Cells in calcium alginate beads (CAB) proliferated and produced more insulin per cell than monolayer culture, but the physical properties of the beads were poor and they ruptured within a few days of culture. Barium alginate beads (BABs) provided a stable encapsulation method. Addition of PLL and PLO at concentrations above 0.1% w/v with the culture medium increased cell proliferation. With the addition of ECMs after bead formation there was a further increase in cell proliferation for certain combinations of ECM and PLO. It was concluded that RIN-m5F-loaded Ba-alginate beads (BABs), when incorporated with varying concentrations of poly (L) lysine (PLL), poly (L) ornithine (PLO) in the presence of extra-cellular matrix proteins (ECMs) were superior to both tissue culture and RIN-m5F-loaded Ca-alginate beads (CABs) in terms of physical stability, cell proliferation and insulin production.

Culture of Neural Stem Cells in Calcium Alginate Beads

Culture of Neural Stem Cells in Calcium Alginate Beads

Biodegradation of polyphenols with immobilized Candida tropicalis under metabolic induction

During olive oil production, large quantities of olive mill wastewater (OMW) are produced. This wastewater material, containing a high level of phenolic compounds, poses a serious environmental problem in almost all Mediterranean countries. Candida tropicalis YMEC14 was used as an extremophile strain to design an aerobic biotreatment process to detoxify OMW and reduce its polluting organic load. The process was enhanced by directing yeast metabolism towards biodegradation pathways using hexadecane as co-metabolite and by immobilizing yeast cells in calcium alginate beads. Under immobilization conditions, C. tropicalis YMEC14 grown at 40°C in OMW supplemented with hexadecane resulted in 69.7%, 69.2% and 55.3% reduction of chemical oxygen demand, monophenols and polyphenols, respectively, after a 24-h fermentation cycle.

Fermentation of xylose and rice straw hydrolysate to ethanol by Candida shehatae NCL-3501.

Candida shehatae NCL-3501 utilized glucose and xylose efficiently in batch cultures. The specific rate of ethanol production was higher with mixtures of glucose and xylose (0.64-0.83 g g-1 cells d-1) compared to that with individual sugars (0.38-0.58 g g-1 cells d-1). Although the optimum temperature for growth was 30 degrees C, this strain grew and produced appreciable levels of ethanol at 45 degrees C. A stable ethanol yield (0.40-0.43 g g-1 substrate utilized) was obtained between 10 g L-1 and 80 g L-1 of initial xylose concentration. Conversion efficiency was further improved by immobilization of the cells in calcium alginate beads. Free or immobilized cells of C. shehatae NCL-3501 efficiently utilized sugars present in rice straw hemicellulose hydrolysate, prepared by two different methods, with 48 h. Ethanol yields of 0.45 g g-1 and 0.5 g g-1 from autohydrolysate, and 0.37 g g-1 from acid hydrolysate were produced by free and immobilized cells, respectively.

Effectiveness factor calculation for immobilised growing cell systems

Gel immobilised living cell systems represent a special form of heterogeneous catalysis. Due to mass transfer limitations on substrate delivery and product removal, time-dependent spatial variations in growth rate and biomass densities are created. A dynamic mathematical model has been developed which was used to simulate the start-up dynamics of growing yeast cells in calcium alginate beads. By coupling the transient model with the effectiveness factor calculation, the “dynamic effectiveness factor” could be calculated. The influence of the bead radius and the initial biomass concentration on the dynamic effectiveness factor have been investigated.