Amperometric Algal Chlorella vulgaris Cell Biosensors Based on Alginate and Polypyrrole‐Alginate Gels

The successful development and analytical performances of two biosensor configurations based on the entrapment of algal cells of Chlorella vulgaris into either a regular alginate gel or a newly synthesized pyrrole‐alginate matrix are reported. These biosensors were compared in terms of their amperometric current measurements to p‐nitrophenyl phosphate when used as substrate for the detection of an algal alkaline phosphatase activity. The high stability of the pyrrole‐alginate gel when compared to that of the alginate coating is herein demonstrated.     

Study of biocatalyst to produce ethanol from starch

This article presents a detailed study on the conditions for achieving a stable biocatalyst to be used in the production of ethanol from starch. Different pellets were used depending on which characteristic of the biocatalyst was being studied: (a) Saccharomyces cerevisiae entrapped in pectin or calcium alginate gel particles; (b) silica containing immobilized glucoamylase entrapped in pectin gel particles; or (c) pectin gel particles, with the silicaenzyme derivative and yeast coimmobilized. The influence of several variables on the mechanical resistance of the particle, on the viability of the microorganism, and on the rate of substrate hydrolysis was studied with biocatalyst. The best conditions found were 6% pectin gel, 2-mm particle diameter, and curein 0.2 M CaCl₂·2H₂O/60 mM acetate buffer, pH 4.2, for gel preparation; and 6.0 g/L of CaCl₂·2H₂O in the fermentation medium. Biocatalyst (c) was successfully tested for the production of ethanol from liquefield manioc flour syrup.     

Polyluminol/hydrogel composites as new electrochemiluminescent-active sensing layers

This paper reports on electrochemiluminescent sensors and biosensors based on polyluminol/hydrogel composite sensing layers using chemical or biological membranes as hydrogel matrices. In this work, luminol is electropolymerized under near-neutral conditions onto screen-printed electrode (SPE)-supported hydrogel films. The working electrode coated with a hydrogel film is soaked in a solution containing monomeric luminol units, allowing the monomeric luminol units to diffuse inside the porous matrix to the electrode surface where they are electropolymerized by cyclic voltammetry (CV). Sensors and enzymatic biosensors for H₂O₂ and choline detection, respectively, have been developed, using choline oxidase (ChOD) as a model enzyme. In this case, hydrogel is used both as the enzymatic immobilization matrix and as a template for the electrosynthesis of polyluminol. The enzyme was immobilized by entrapment in the gel matrix during its formation before electropolymerization of the monomer. Several parameters have been optimized in terms of polymerization conditions, enzyme loading, and average pore size. Using calcium alginate or tetramethoxysilane (TMOS)-based silica as porous matrix, H₂O₂ and choline detection are reported down to micromolar concentrations with three orders of magnitude wide dynamic ranges starting from 4?×?10^?7 M. Polyluminol/hydrogel composites appear as suitable electrochemiluminescence (ECL)-active sensing layers for the design of new reagentless and disposable easy-to-use optical sensors and biosensors, using conventional TMOS-based silica gel or the more original and easier to handle calcium alginate, reported here for the first time in such a configuration, as the biocompatible hydrogel matrix. Figure

Elaboration of electrochemiluminent polyluminol/hydrogel composite sensing layers     

Polysaccharide Hydrogels for the Protection of Dairy-Related Microorganisms in Adverse Environmental Conditions

Adverse environmental conditions are severely limiting the use of microorganisms in food systems, such as probiotic delivery, where low pH causes a rapid decrease in the survival of ingested bacteria, and mixed-culture fermentation, where stepwise changes and/or metabolites of individual microbial groups can hinder overall growth and production. In our study, model probiotic lactic acid bacteria (L. plantarum ATCC 8014, L. rhamnosus GG) and yeasts native to dairy mixed cultures (K. marxianus ZIM 1868) were entrapped in an optimized (cell, alginate and hardening solution concentration, electrostatic working parameters) Ca-alginate system. Encapsulated cultures were examined for short-term survival in the absence of nutrients (lactic acid bacteria) and long-term performance in acidified conditions (yeasts). In particular, the use of encapsulated yeasts in these conditions has not been previously examined. Electrostatic manufacturing allowed for the preparation of well-defined alginate microbeads (180–260 µm diameter), high cell-entrapment (95%) and viability (90%), and uniform distribution of the encapsulated cells throughout the hydrogel matrix. The entrapped L. plantarum maintained improved viabilities during 180 min at pH 2.0 (19% higher when compared to the free culture), whereas, L. rhamnosus appeared to be less robust. The encapsulated K. marxianus exhibited double product yields in lactose- and lactic acid-modified MRS growth media (compared to an unfavorable growth environment for freely suspended cells). Even within a conventional encapsulation system, the pH responsive features of alginate provided superior protection and production of encapsulated yeasts, allowing several applications in lacto-fermented or acidified growth environments, further options for process optimization, and novel carrier design strategies based on inhibitor charge expulsion.     

Immobilization of Acidithiobacillus ferrooxidans with complex of PVA and sodium alginate

A new Acidithiobacillus ferrooxidans cell immobilization technique utilizing the complex of PVA solution and sodium alginate solution crosslinked by Ca(NO₃)₂ as entrapment medium is reported. The mixture of A. ferrooxidans suspension and the entrapment complex were extruded into a solution of Ca(NO₃)₂ (1-5%) to form beads, then the beads were frozen at −20 °C for 1-2 days and thawed at room temperature. The forming mechanism, characteristic of this immobilized beads and the factors affecting activity of immobilized cells were also discussed. A maximum oxidation rate of 4.6 g Fe²⁺/(L h) was achieved in batch cultures by these immobilized cells. Precipitation formed during culture process was analyzed. The forming mechanism of this precipitation and how this precipitation affects the whole system were also discussed. In addition, the immobilization technique is operated simply, and the gel beads have high stability even under non-sterile conditions. So its application on an industrial scale would be more practicable.     

Mesoporous alginate/silica biocomposites for enzyme immobilisation

Alginate/silica biocomposites were synthesised by impregnation of mesoporous silica particles with alginic acid solution, followed by Ca²⁺-induced biopolymer gelation. They consist of alginate trapped in the open macroporosity of the silica network, whose porous structure is maintained, as indicated by X-ray diffraction and nitrogen sorption measurements. β-galactosidase enzymes incorporated in the initial biopolymer solution maintain their catalytic activity towards 4-nitrophenyl-β-d-galactopyranoside hydrolysis. When compared to the pure Ca-alginate gel, the biocomposites exhibit a better stability upon ageing, limiting enzyme leaching.     

Gentle cell trapping and release on a microfluidic chip by in situ alginate hydrogel formation

Microfluidic devices are increasingly used to perform biological experiments on a single-cell basis. However, long-term stability of cell positions is still an issue. A novel biocompatible method for cell entrapment and release on a microchip is presented. It is based on the controlled formation of an alginate hydrogel by bringing two laminar flows of alginate and calcium ions in the range of 2 mM to 40 mM into contact. The resulting growth of a gel bar is used to enclose and immobilize yeast cells. Adding ethylenediaminetetraacetic acid (EDTA) to the alginate solution allows for control of the hydrogel growth, and by varying the ratio of Ca(2+) to EDTA concentrations gel growth or gel shrinkage can be induced at will. Trapped cells are released during shrinkage of the gel. The trapping efficiency for different cell speeds is investigated and the properties of gel growth are discussed using a diffusion model. Precise positioning of a single cell is demonstrated. The technique presented allows not only the reversible immobilization of cells under gentle conditions but also offers the potential of long-term cell cultures as shown by on-chip incubation of yeast cells. The procedure may provide a simple and fully biocompatible technique for a multitude of innovative experiments on cells in microsystems.     

Stability of new optical pH sensing material based on cross-linked poly(vinyl alcohol) copolymer

Cross-linked poly(vinyl alcohol) (PVA)-silica gel copolymer has been employed as a optical pH sensor substrate for immobilisation of fluorescein. Cross-linking was carried out by the sol-gel process incorporating PVA in initial sol-gel solution of tetra-methoxysilane (TMOS) under acidic conditions. Three dimensional network formation could be achieved using compositions of PVA/TMOS=80-90/20-10 vol.% to result in crack-free films. The fluorescent sensor layers were prepared by dip-coating of gel solution onto glass slides. The dynamic fluorescence response towards different pH values was investigated in terms of the influence of sample ionic strength, membrane composition as well as age of sol-gel layers. Depending on the composition of the matrix pK_a values of 6.50, 6.68 and 7.06 were found 18 days after continues storage in buffer.     

Valorization of olive husk flour as a filler for biocomposites based on poly(3-hydroxybutyrate-co-3-hydroxyvalerate): Effects of silane treatment

The paper deals with the influence of surface treatment of olive husk flour (OHF) by trimethoxyoctadecylsilane (TMOS) on the morphology and physical properties of bio-composites based on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Biocomposites based on PHBV/OHF: 80/20 (w/w) before and after filler treatment were prepared by melt compounding, while the modification of OHF surface by TMOS was performed by thermo-chemical vapor deposition. Scanning electron microscopic observations showed that modification of OHF by TMOS led to homogeneous and finer dispersion of the filler particles in PHBV matrix, indicating improved compatibility between the two components. Further, TGA data revealed an increase in thermal stability of treated PHBV/OHF biocomposites. An increase in the crystalline index determined by DSC was also observed due to the nucleating effect of OHF in the polymer matrix, however more pronounced for the treated biocomposites. The latter exhibited better tensile properties, as confirmed by DMA results, showing higher reinforcing effect of OHF for the treated biocomposites. This study highlighted significant improvements of the properties of PHBV/OHF biocomposites through silane treatment of OHF.     

Determination of effective diffusion coefficients in calcium alginate gel plates with varying yeast cell content

Effective diffusion coefficients (D_e) have been determined for lactose, glucose, galactose, and ethanol in calcium alginate gel with varying yeast cell concentration. The measurements have been performed in a diffusion cell, and the results evaluated with the quasisteady-state method. An ultrasonic meter was used for gel thickness determination with an accuracy of 1.5% and a new method for the reproducible preparation of gel plates was developed. It was found thatD _e in pure alginate gel decreased to about 90% of the diffusivity in water and did not vary with alginate concentration.D _e decreased considerably with increasing yeast cell concentration. For the solutes studied, the effective diffusion coefficient can be estimated according to the equationD _e =D _eo (1 - ?)/[1+(?/2)], whereD _eo is the effective diffusivity in pure gel and ? is the volume fraction of yeast cells.     

Fucoidan and Alginate from the Brown Algae Colpomenia sinuosa and Their Combination with Vitamin C Trigger Apoptosis in Colon Cancer

Brown seaweeds are producers of bioactive molecules which are known to inhibit oncogenic growth. Here, we investigated the antioxidant, cytotoxic, and apoptotic effects of two polysaccharides from the brown algae Colpomenia sinuosa, namely fucoidan and alginate, in a panel of cancer cell lines and evaluated their effects when combined with vitamin C. Fucoidan and alginate were isolated from brown algae and characterized by HPLC, FTIR, and NMR spectroscopy. The results indicated that highly sulfated fucoidans had higher antioxidant and cytotoxic effects than alginate. Human colon cancer cells were the most sensitive to the algal treatments, with fucoidan having an IC₅₀ value (618.9 µg/mL⁻¹) lower than that of alginate (690 µg/mL⁻¹). The production of reactive oxygen species was increased upon treatment of HCT-116 cells with fucoidan and alginate, which suggest that these compounds may trigger cell death via oxidative damage. The combination of fucoidan with vitamin C showed enhanced effects compared to treatment with fucoidan alone, as evidenced by the significant inhibitory effects on HCT-116 colon cancer cell viability. The combination of the algal polysaccharides with vitamin C caused enhanced degeneration in the nuclei of cells, as evidenced by DAPI staining and increased the subG1 population, suggesting the induction of cell death. Together, these results suggest that fucoidan and alginate from the brown algae C. sinuosa are promising anticancer compounds, particularly when used in combination with vitamin C.     

Permeabilization of Kluyveromyces marxianus with Mild Detergent for Whey Lactose Hydrolysis and Augmentation of Mixed Culture

Cheese whey is a by-product of cheese-manufacturing industries, and the utilization of whey is a challenging problem either to use it or dispose it, because only few microorganisms can metabolize the whey lactose. Enzymatic hydrolysis of whey lactose to glucose and galactose by β-galactosidase is the approach for biotechnological application. Kluyveromyces marxianus cells were permeabilized with non-toxic, biodegradable, anionic detergent N-lauroyl sarcosine (N-LS) for the enzyme activity. The permeabilization process parameters (N-LS concentration, solvent volume, temperature and incubation time) were optimized. The maximum β-galactosidase activity of 1,220 IU/g dry weight was obtained using permeabilized cells under optimized conditions. Moreover, viability of the permeabilized cells was also evaluated, which showed that cells were alive; however, viability was reduced by two log cycles. The permeabilized cells were evaluated for whey lactose hydrolysis. The maximum lactose hydrolysis of 91 % was observed with 600 mg (dry cell weight/100 mL) in whey powder (5 % w/v) solution at 180-min incubation, pH 6.5 and 30 °C. Further, the hydrolyzed whey was evaluated for amelioration of growth of non-lactose-consuming yeast Saccharomyces cerevisiae. S. cerevisiae was able to grow in hydrolyzed whey simultaneously with K. marxianus. The study confirmed that N-LS could be used to permeabilize K. marxianus cells to make available the enzyme activity.     

Synthesis and Characterization of Alginate/Silica Biocomposites

We have investigated the formation of silica/alginate biocomposites. The silica gel was first obtained by the sol-gel route using aqueous precursors, in the presence of the polymer. Gelation of the trapped alginate could be consequently performed by addition of a divalent metal cation. Thermogravimetric analysis, SEM and nitrogen adsorption-desorption experiments indicated that the polymer is located in the macroporosity of the biocomposites so that it can be partially removed by citrate treatment. Even though only weak hydrogen bonds are expected, the morphological features of the composites revealed to depend on the biopolymer content of the initial solution.     

Screening of matrix suitable for immobilization of microbial cells

To find a suitable matrix for immobilization of microbial cells, synthetic and natural polymers were screened. As a result,kappa-carrageenan,iota-carrageenan, furcellaran, sodium alginate, ethyl succinylated cellulose, succinylated zein, and 2-methyl-5-vinyl-pyridine-methylacrylate-methacrylic acid copolymer were studied. These polymers were induced to gel under mild conditions.Streptomyces phaeochromogenes cells having glucose isomerase activity were successfully immobilized in these polymer matrices. If a gelinducing reagent were added to a substrate solution, these gel matrices could be stabilized. The microbial cells did not leak out from the gel lattice. When these immobilized cells were treated with hardening reagents such as glutaraldehyde or tannins, the gel matrices were strengthened, and the glucose isomerase activity became stable for a long period even in the absence of gel-inducing reagents. Among these polymer matrices tested,kappa -carrageenan was most suitable for immobilization of microbial cells.     

Fluorescence monitoring of immobilzed microorganisms in cultures

An experimental reactor system for monitoring the fluorescence of suspended and immobilized cells is described. The growth of S. cerevisiae was monitored during batch fermentations by fluorescence of the culture. Thus, it was possible to use this intracellular parameter to study the influence of immobilization on cells. The fermentations were done under aerobic conditions with suspended and immobilized cells. A comparison of these two systems showed that the rate of ethanol consumption was significantly slower for the cells immobilized in calcium alginate. This reduced rate of oxidative decomposition may be due to mass-transfer limitations of oxygen. Pulse experiments with different substrates (glucose and ethanol) were made to monitor the changes in cell metabolism. The reactor system presented is also suitable as a “toxin guard system”, because substances toxic to cells, such as 2,4-dinitrophenol, cause clearly visible changes in the fluorescence of the immobilized cells.     

A highly enantioselective chemoenzymatic synthesis of syn-3-amino-2-hydroxy esters: key intermediates for taxol side chain and phenylnorstatine

Starting from the bromination of α-ketoesters to obtain 3-bromo-2-oxoalkanoates and bioreduction with Saccharomyces cerevisiae entrapped in calcium alginate pellets with double gel layers, syn-(2R,3S)-β-bromo-α-hydroxy esters were obtained regioselectively in high yields and high ee. These chiral bromohydrins were cyclized to epoxides that were transformed into oxazolidines and finally opened by acidic hydrolysis to give syn-(2S,3S)-β-amino-α-hydroxy esters in high overall yields and high ee. The enantiomeric excesses of all the intermediates were maintained during the reaction sequence.     

The Effects of Photosensitizing Dyes Fagopyrin and Hypericin on Planktonic Growth and Multicellular Life in Budding Yeast

Naphthodianthrones such as fagopyrin and hypericin found mainly in buckwheat (Fagopyrum spp.) and St. John’s wort (SJW) (Hypericum perforatum L.) are natural photosensitizers inside the cell. The effect of photosensitizers was studied under dark conditions on growth, morphogenesis and induction of death in Saccharomyces cerevisiae. Fagopyrin and hypericin induced a biphasic and triphasic dose response in cellular growth, respectively, over a 10-fold concentration change. In fagopyrin-treated cells, disruptions in the normal cell cycle progression were evident by microscopy. DAPI staining revealed several cells that underwent premature mitosis without budding, a striking morphological abnormality. Flow Cytometric (FC) analysis using a concentration of 100 µM showed reduced cell viability by 41% in fagopyrin-treated cells and by 15% in hypericin-treated cells. FC revealed the development of a secondary population of G1 cells in photosensitizer-treated cultures characterized by small size and dense structures. Further, we show that fagopyrin and the closely related hypericin altered the shape and the associated fluorescence of biofilm-like structures. Colonies grown on solid medium containing photosensitizer had restricted growth, while cell-to-cell adherence within the colony was also affected. In conclusion, the photosensitizers under dark conditions affected culture growth, caused toxicity, and disrupted multicellular growth, albeit with different efficiencies.     

Efficiency of a fixed-bed and a gas-lift three-column reactor for continuous production of ethanol by pectate-and alginate-immobilized Saccharomyces cerevisiae cells

Ethanol-tolerant and thermo-tolerant yeast strain Saccharomyces cerevisiae C11-3 cells immobilized in calcium pectate and calcium alginate gels were used for ethanol fermentation in a three-reactor system with a gradient temperature control. The fermentation process has been tested in a fixed-bed and a gas-lift arrangement. The gas-lift system was more efficient due to a better mass transport between the phases. Abrasion was more evident in calcium alginate particles, while calcium pectate beads were not significantly damaged. Two different concentrations of alginate were tested and calcium pectate gel was demonstrated to be more suitable as an immobilization material in comparison with calcium alginate due to its mechanical resistance and favourable diffusion parameters, providing an ethanol production of more than 7.5 g dm⁻³ h⁻¹ over a period of 630 h.     

Silica hybrid nanocomposites

In this work we present experimental results about the formation, properties and structure of sol — gel silica based biocomposite containing Calcium alginate as an organic compound. Two different types of silicon precursors have been used in the synthesis: tetramethylortosilicate (TMOS) and ethyltrimethoxysilane (ETMS). The samples have been prepared at room temperature. The hybrids have been synthesized by replacing different quantitis of the inorganic precursor with alginate. The structure of the obtained hybrid materials has been studied by XRD, IR Spectroscopy, EDS, BET and AFM. The results proved that all samples are amorphous possessing a surface area from 70 to 290 m²/g. It has also been established by FT IR spectra that the hybrids containing TMOS display Van der Walls and Hydrogen bonding or electrostatic interactions between the organic and inorganic components. Strong chemical bonds between the inorganic and organic components in the samples with ETMS are present. A self-organized nanostructure has been observed by AFM. In the obtained hybrids the nanobuilding blocks average in size at about 8–14 nm for the particles.     

Matrix molecularly imprinted mesoporous sol–gel sorbent for efficient solid-phase extraction of chloramphenicol from milk

Highly selective and efficient chloramphenicol imprinted sol–gel silica based inorganic polymeric sorbent (sol–gel MIP) was synthesized via matrix imprinting approach for the extraction of chloramphenicol in milk. Chloramphenicol was used as the template molecule, 3-aminopropyltriethoxysilane (3-APTES) and triethoxyphenylsilane (TEPS) as the functional precursors, tetramethyl orthosilicate (TMOS) as the cross-linker, isopropanol as the solvent/porogen, and HCl as the sol–gel catalyst. Non-imprinted sol–gel polymer (sol–gel NIP) was synthesized under identical conditions in absence of template molecules for comparison purpose. Both synthesized materials were characterized by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FT-IR) and nitrogen adsorption porosimetry, which unambiguously confirmed their significant structural and morphological differences.