Improvement of Bioethanol Productivity of Immobilized Saccharomyces Bayanus with Using Sodium Alginate-Graft-Poly(N-Vinyl-2-Pyrrolidone) Matrix

In this study, immobilization conditions and bioethanol production characteristics of immobilized Saccharomyces bayanus were investigated into sodium alginate-graft-poly(N-vinyl-2-pyrrolidone; NaAlg-g-PVP) matrix. The matrix that crosslinked with calcium clorid was used for immobilization of S. bayanus. Bioethanol productivity of the NaAlg-g-PVP matrix was found to increase from 4.21 to 4.84?gL^?1?h^?1 when compared with the convential sodium alginate matrix. The production of bioethanol was affected by initial glucose concentration and percentage of immobilized cell beads in fermentation medium. Bioethanol productivity was increased from 3.62 to 4.84?gL^?1?h^?1 while the glucose concentration increasing from 50 to 100?gL^?1. Due to the increase in percentage from 10 to 20?% of immobilized cell beads in the fermentation medium, bioethanol productivity was increased from 4.84 to 8.68?gL^?1?h^?1. The cell immobilized NaAlg-g-PVP beads were protected 92?% of initial activity after six repeated fermentation.     

Microbial Fed-batch Production of 1,3-Propanediol Using Raw Glycerol with Suspended and Immobilized Klebsiella pneumoniae

The production of 1,3-propanediol (1,3-PD) was investigated with Klebsiella pneumoniae DSM 4799 using raw glycerol without purification obtained from a biodiesel production process. Fed-batch cultures with suspended cells revealed that 1,3-PD production was more effective when utilizing raw glycerol than pure glycerol (productivity after 47 h of fermentation, 0.84 g?L^?1?h^?1 versus 1.51 g?L^?1?h^?1 with pure and raw glycerol, respectively). In addition, more than 80 g/L of 1,3-PD was produced using raw glycerol; this is the highest 1,3-PD concentration reported thus far for K. pneumoniae using raw glycerol. Repeated fed-batch fermentation with cell immobilization in a fixed-bed reactor was performed to enhance 1,3-PD production. Production of 1,3-PD increased with the cycle number (1.06 g?L^?1?h^?1 versus 1.61 g?L^?1?h^?1 at the first and fourth cycle, respectively) due to successful cell immobilization. During 46 cycles of fed-batch fermentation taking place over 1,460 h, a stable and reproducible 1,3-PD production performance was observed with both pure and raw glycerol. Based on our results, repeated fed batch with immobilized cells is an efficient fermentor configuration, and raw glycerol can be utilized to produce 1,3-PD without inhibitory effects caused by accumulated impurities.     

Development of continuous fermentation using immobilized yeast cells for wine cooler process development

The continuous wine fermentation process, which employs a newly designed tapered column type bioreactor and immobilized yeast cells (Montrachet 522), was studied and its fermentation performance was compared with batch and suspended cell continuous wine fermentation systems. It was found that a stable continuous culture fermentation process could be maintained for a period of 2–3 mo when the new bioreactor system packed with immobilized yeast cells was employed. The new bioreactor containing immobilized yeast cells performed significantly better than the suspended cell culture system or batch culture. The effluent wine from the continuous fermentor system contained 7.1% (v/v) ethanol and 0.18% (w/v) residual sugar at 0.01 h^-1 dilution rate. The new continuous bioreactor system also gave 17–34 times higher maximum ethanol productivity compared to the conventional batch wine fermentation. At a low dilution rate, 0.01^-1, as high as 92% sugar to ethanol yield was achieved. Based on the results obtained from this study, the possibility of developing a continuous wine cooler fermentation process was demonstrated. A two-stage continuous wine fermentation system may be designed and operated. The grape juice can be fed into the first-stage that is operated at about 0.2 h^-1 dilution rate and the effluent from the first-stage is fed into the second-stage continuous fermentor operated at about 0.01 h^-1 dilution rate. By doing so, a wine cooler can be produced continuously and efficiently, by employing the newly designed tapered column type bioreactor charged with the immobilized yeast cells.     

Steroselective Hydrolysis of DL‐Beta‐Acetylthioisobutyramide Catalyzed by Genetically Engineered E. Coli Immobilized on Celite 580 in a Packed Bed Bioreactor

Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl ‐β‐acetylthioisobutyramide (dl ‐ATIA) to form d ‐β‐acetylthioisobutyric acid (DAT), a key intermediate for synthesis of a series of angiotensin converting enzyme inhibitors. The esterase gene of Pseudomonas putida IFO12996 was cloned and expressed in Escherichia coli which was further immobilized and retained on a packed bed bioreactor filled with Celite 580. The packed bed bioreactor was used to conduct the stereoselective hydrolysis of dl ‐ATIA and to give DAT with a yield of 34.5%, enantiometric excess value of 97% and enantioselectivity value > 150. The optimal pH and temperature for the reaction were 9.0 and 57 °C ～ 67 °C, respectively. The kinetic constants (Km and Vmax) of immobilized cells were found to be 372.5 mM and 285.7 μmol min^?1 (g cell)^?1, respectively. The immobilized cells retained over 60% of the initial catalytic activity after 5 batch cycles of production. This paper presents a simple, practical and economical process of immobilization of genetically engineered E. coli on a novel packed bed bioreactor for production of DAT.     

Synthesis and characterization of carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrid for amylase immobilization

Carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrids have been synthesized in a modified Stöber process. The hybrid synthesis was optimized to obtain an efficient immobilization matrix for diastase alpha amylase, a multimeric enzyme of high technological significance. The synthesized hybrids were characterized using FTIR, XRD, SEM, TGA and BET studies. The enzyme immobilization was done by adsorption and using the immobilized enzyme, the hydrolysis of soluble starch has been optimized in comparison to free enzyme. The optimum usable pH for the immobilized enzyme ranged from pH 4 to 5, while pH 5 was optimum pH for the free enzyme activity. The kinetic parameters for the immobilized, (K _M = 3.4610 mg ml^?1; V _max = 6.3540 mg ml^?1 min^?1) and free enzyme (K _M = 4.1664 mg ml^?1; V _max = 4.291 mg ml^?1 min^?1) hydrolysis indicated that the immobilization at the nanohybrid has significantly improved the catalytic property of the enzyme. In the immobilized state, the enzyme remained usable for many repeated cycles like our previous material, gum acacia-gelatin-AgNp-silica. Storage experiments indicated that the immobilization has increased the stability of the enzyme and also that AgNps play a role in stabilizing the immobilized enzyme.     

Development and evaluation of microfluidic device for the determination of organophosphorus pesticide incorporating monolith based immobilized AChE with spectrophotometric detection

A spectrophotometric microfluidic bioreactor system is described for the determination of organophosphorus pesticides. The glass chip was designed and fabricated for in situ monolithic preparation and subsequently acetlycholineserase (AChE) immobilization via a covalent bonding method. The porous polymer monolith was prepared using glycidyl methacrylate, ethylenedimethacrylate and 2,2-dimethoxy-1,2-diphenylethan-1-one in binary porogenic solvents of cyclohexanol and dodecanol. The epoxide groups of monolith were reacted with ethylenediamine and gluteraldehyde to allow immobilization of the enzyme using their amine groups. Organophosphorus pesticides can be determined by measuring their inhibition effect on the enzyme AChE using Ellman's reaction. A linear relationship between the absorbance and percentage inhibitions was obtained over the concentration range of 0.25 to 2.50?mg?L^?1 paraoxon with a correlation coefficient (r ²) of 0.9974. The limit of detection (LOD) defined as 10% inhibition (I ₁₀) was 0.17?mg?L^?1 for paraoxon. The relative standard deviations (RSD) of 1.0?mg?L^?1 paraoxon was 3.73% (n?=?5). The proposed µFI system incorporates efficient enzyme immobilization and reduces reagent consumption and waste production and could thus be considered to be more environmentally friendly.     

Optimization of Biodiesel Production Catalyzed by Fungus Cells Immobilized in Fibrous Supports

A circulating packed-bed bioreactor system using fibrous nonwoven fabric as the immobilization matrix was suitable for simultaneous cell growth and immobilization of Rhizopus oryzae fungus cells, which could be used for lipase-mediated production of biodiesel by methanolysis of soybean oil. Response surface methodology and 5-level-5-factor central composite rotatable design was proved to be a powerful tool for the optimization of methanolysis conditions catalyzed by immobilized R. oryzae whole cell biocatalyst. A quadratic polynomial regression model was used to analyze the relationship between the yield and the significant reaction parameters. The analysis confirmed that water content, molar ratio of methanol to oil, cell weight, and reaction time were the significant factors affecting the yield at a 95% confidence level (p?<?0.05). Under the optimum condition at 10.97% (w/w) water content, 0.64 molar ratio of methanol to oil, 2.25% (w/w) cell weight, and 23.3 h reaction time, the predicted value of yield was 72.6%. Validation experiments with yields of 70.77?±?2.46% verified the availability and the accuracy of the model.     

Enzyme electrodes for the sugar substitute aspartame

A sensor of aspartame (l-aspartyl-l-phenylalanine methyl ester) is prepared by chemical immobilization of l-aspartase on an ammonia-selective electrode. Semi logarithmic response (E vs. log C) was observed in the 1 × 10^?3?1 × 10^?2 M range with a slope of ?30 mV/decade. The sensor is stable for more than eight days. The probe is successfully used for the assay of aspartame in commercially available sweeteners.     

Characteristics of an Immobilized Yeast Cell System Using Very High Gravity for the Fermentation of Ethanol

The characteristics of ethanol production by immobilized yeast cells were investigated for both repeated batch fermentation and continuous fermentation. With an initial sugar concentration of 280?g/L during the repeated batch fermentation, more than 98% of total sugar was consumed in 65?h with an average ethanol concentration and ethanol yield of 130.12?g/L and 0.477?g ethanol/g consumed sugar, respectively. The immobilized yeast cell system was reliable for at least 10 batches and for a period of 28?days without accompanying the regeneration of Saccharomyces cerevisiae inside the carriers. The multistage continuous fermentation was carried out in a five-stage column bioreactor with a total working volume of 3.75?L. The bioreactor was operated for 26?days at a dilution rate of 0.015?h^?1. The ethanol concentration of the effluent reached 130.77?g/L ethanol while an average 8.18?g/L residual sugar remained. Due to the high osmotic pressure and toxic ethanol, considerable yeast cells died without regeneration, especially in the last two stages, which led to the breakdown of the whole system of multistage continuous fermentation.     

Immobilization of Aspergillus nidulans SU04 cellulase on modified activated carbon

The present study deals with the immobilization of Aspergillus nidulans SU04 cellulase onto modified activated carbon (MAC). The effect of contact time, cellulase concentration, MAC dosage, and temperature for maximum immobilization percentage and immobilization capacity is investigated. The equilibrium nature of immobilization is described by Langmuir and Freundlich isotherms. The kinetic data were tested using the pseudo first order. The activation energy of immobilization was evaluated to be 11.78?J?mol^?1. Results of the thermodynamic investigation indicate the spontaneity (?G <0), slightly endothermic (?H >0), and irreversible (?S >0) nature of the sorption process. Entropy and enthalpy were found to be 41.32 J?mol^?1?mg^?1 and 10.99?kJ?mol^?1, respectively. The Gibbs free energy was found to be ?22.79?kJ?mol^?1. At 80?rpm, 323?K, 2?h, 5?mg of MAC, immobilization capacity was 4.935?mg cellulase per mg of MAC from an initial cellulase concentration of 16?mg?ml^?1 with retention of 70% of native cellulase activity up to 10 cycles of batch hydrolysis experiments. The diffusion studies that were carried out revealed the reaction rate as ??mol?min^?1. At optimized conditions, immobilized cellulase had a higher Michaelis?CMenten constant, K _m of 1.52?mmol and a lower reaction rate, V _max of 42.2???mol?min^?1, compared with the free cellulase, the K _m and V _max values of which were 0.52?mmol and 18.9???mol?min^?1, respectively, indicating the affinity of cellulase for MAC matrix.     

Bacterial Reduction of Cr(VI) at Technical Scale—The Malaysian Experience

The bacterial reduction of Cr(VI) from industrial wastewater was evaluated using a 2.0-m³ bioreactor. Liquid pineapple waste was used as a nutrient for the biofilm community formed inside the bioreactor. The use of rubber wood sawdust as packing material was able to immobilize more than 10⁶?CFU?mL^?1 of Acinetobacter haemolyticus cells after 3?days of contact time. Complete reduction of 15?C240?mg?L^?1 of Cr(VI) was achieved even after 3?months of bioreactor operation. Cr(VI) was not detected in the final effluent fraction indicating complete removal of Cr from solution from the flocculation/coagulation step and the unlikely re-oxidation of Cr(III) into Cr(VI). Impatiens balsamina L. and Gomphrena globosa L. showed better growth in the presence of soil?Csludge mixture compared to Coleus scutellarioides (L.) Benth. Significant amounts of Cr accumulated at different sections of the plants indicate its potential application in Cr phytoremediation effort. The bacterial-based system was also determined not to be detrimental to human health based on the low levels of Cr detected in the hair and nail samples of the plant operators. Thus, it can be said that bacterial-based Cr(VI) treatment system is a feasible alternative to the conventional system especially for lower Cr(VI) concentrations, where sludge generated can be used as growth supplement for ornamental plant as well as not detrimental to the health of the workers.     

Homogeneous immunoassay for human IgG using oriented hen egg IgY immobilized on gold sol nanoparticles

Homogeneous immunoassays using (red) gold nanoparticles represent an attractive detection scheme because of the option of photometric readout. We have applied oriented immobilization of hen egg immunoglobulin Y (IgY) on gold nanoparticles when developing a homogeneous immunoassay for human IgG. In oriented immobilization, as opposed to random immobilization, the antigen binding capabilities of the antibodies are retained. It is shown that such immunoassay has significantly better sensitivity in comparison with methods based on conventional immobilization of affinity-purified antibodies. It is also shown that hen egg IgY is better suited than rabbit antibodies, because much more antibody can be immobilized on gold nanoparticles without any destabilization, probably because of the more acidic nature of these antibodies. In addition, hen egg IgY can be supplied in higher quantity and can be prepared more easily than IgG from rabbits. Bleeding and slaughtering of animals is not needed. The assay presented here has a wide detection range (30–500?ng?.mL^?1) and a limit of detection as low as 30?ng.mL^?1 of human IgG.

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.     

Characterization and application of silylated substrates for the preconcentration of cations

Chelating functional groups immobilized on silica and controlled pore glass beads are used to preconcentrate cations from aqueous solution in the concentration region of ng ml^?1. The functional groups are chemically bonded to the substrates by silylation reagents which may be employed as received or further modified. For example, commercially available 1,2-diamines may be used or converted to the dithiocarbamate after immobilization. This report includes studies of ionic strength and sodium chloride effects on the recovery of transition metal ions. Batch extraction of transition metal ions is accomplished with silica as the substrate for solutions containing transition metal ions at μg ml^?1 concentrations. Column extraction of solutions pumped at 50 ml min^?1 is accomplished at concentrations of 25 ng ml^?1. Recovery is good in both cases. The cations were determined directly on pelletized substrates by x-ray fluorescence. However, the ions may be eluted from the substrate and determined by other methods. Examples of determinations of several cations in lake water and high-purity industrial water are given.     

Efficient Immobilization of Porcine Pancreatic α-Amylase on Amino-Functionalized Magnetite Nanoparticles: Characterization and Stability Evaluation of the Immobilized Enzyme

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe₃O₄) nanoparticles (NPs) which were subsequently coated with silica through sol–gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the α-amylase retained more than 50 % of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45 °C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of K_m and V_max. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The V_max values for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg^?1 min^?1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9 cycles while retaining 68 % of its initial activity.     

A Label Free Electrochemical Nanobiosensor Study

Abstract

Nano-porous silicon (PS) is an attractive material for incorporation into biosensors, because it has a large surface area combined with the ability to generate both optical and electrical signals. In this paper, we describe a label-free nanobiosensor for bovine serum albumin (BSA). Nano-porous silicon produced in our laboratory was functionalized prior to immobilization of anti-BSA antibody on the surface. Reaction with BSA in phosphate buffered saline (PBS) buffer resulted in an impedance change which was inversely proportional to the concentration of the analyte. The system PBS buffer/antigen-antibody/PS constitutes an electrolyte-insulator-semiconductor (EIS) structure, thus furnishing an impedance EIS nanobiosensor. The linear range of the sensor was 0–0.27 mg mL^?1 and the sensitivity was less than 10 µg mL^?1.     

Optimization and characterization of immobilized E. coli for engineered thermostable xylanase excretion and cell viability

There are many parameters that may have influenced the properties of cell during immobilization process. Particularly, the immobilization methods, carrier materials, and enzyme loading amount that have been proved to be important for immobilization process. The physiological responses of microorganisms are depending on the immobilization technique used. Typical alterations to the micro-environment of the immobilized cell involved the altered water activity, presence of ionic charges, cell confinement and modified surface tension. In this study, the graphene oxide was selected as a suitable carrier for immobilization process of recombinant E.coli and adsorption was chosen as an appropriate method to improve the production of engineered thermostable xylanase. High level production of thermostable xylanase by immobilized recombinant cell in the 5 L bioreactor was studied by using optimum research surface methodology (RSM) conditions was studied. The immobilization of E. coli onto nanoparticle matrix manages to improve the cell performance by improving the protein expression, reduced the occurrences of cell lysis as well as improved the plasmid stability of the host cell. Thus, immobilization contributes a physical support for both whole cells as well as enzymes to develop a better operative achievement system for industrialized fields and give rise to the biological advancement existing enzyme for instance xylanase.     

Multi-walled carbon nanotubes used as support for lipase from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

Commercial lipase from Burkholderia cepacia is immobilized on functionalized multi-walled carbon nanotubes (MWNT-COOH and MWNT-OH) provided by a physical adsorption. The immobilization processes for the carbon nanotubes are defined using immobilization time (0–30 min) and distinct adsorbent:adsorbate ratios (1:4, 1:7, and 1:10) with lipase loading of 100, 175, and 250 mg, respectively. The characterization of the immobilized preparations, the free lipase, and the pure nanotubes (MWNT-COOH and MWNT-OH) indicate that the lipase adsorption is increased. Thermogravimetric analysis, differential scanning calorimetry, and scanning electron microscopy are used. The specific surface area, pore volumes, and average pore diameters are determined by nitrogen adsorption–desorption isotherms. For the pure lipase, in the range between 40 and 300 °C, the micrograph is acquired. Experimental results clearly show an effective lipase adsorption in a lower period of time (5 min) in MWNT-COOH and MWNT-OH as well as a decrease in the surface area (98.30–45.9(86)?±?2.5 and 97.61–37.71?±?3.3(7) m² g^?1) and the pore volume (0.48–0.25?±?0.01 and 0.39–0.24?±?0.05 cm³ g^?1), indicating that functionalized multi-walled carbon nanotubes can be successfully used as enzyme support.     

Cell Disruption Optimization and Covalent Immobilization of β-D-Galactosidase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YW-1 for Lactose Hydrolysis in Milk

β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40 °C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.     

Extraction and Immobilization of SA-α-2,6-Gal Receptors on Magnetic Nanoparticles to Study Receptor Stability and Interaction with Sambucus nigra Lectin

The interaction between influenza virus hemagglutinins and host cell with terminal sialic acid linked receptors, SA-α-2,6-Gal for human strains is important to obtain insights into this infectious disease. Sambucus nigra lectin has high affinity for SA-α-2,6-Gal receptors. The goals of this work were: to extract the SA-α-2,6-Gal receptors from porcine airways; to perform receptors immobilization and study their storage stability; and to determine some parameters of interaction between the receptor and S. nigra lectin. The receptor isolation was monitored by means of bound sialic acid (BSAc) detection. A major band of protein at 66.7 kDa was clearly visible in SDS-PAGE assay. Eighty-one percent of isolated glycoproteins were immobilized on magnetic nanoparticles. The kinetics of BSAc storage stability at 4 °C was approximated as the first order reaction with kinetic constant and half-life estimated as 0.062 day^?1 and 11.2 days, respectively. The dissociation constant (K _d) calculated from Scatchard's plot was 2.47?×?10^?7 M, and the receptor concentration was equal to 7.92?×?10^?5 M. Procedure for N-SA-α-2,6-Gal -receptors extraction based on their affinity to S. nigra lectin with magnetic nanoparticles, and their immobilization in active form, was not described previously, and may have wide application in designing biosensors or virus removal from areas or contaminated samples.