Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 33

immobilization Related Abstracts

33 Magnetite Nanoparticles Immobilized Pectinase: Preparation, Characterization and Application for the Fruit Juices Clarification

Authors: Leila Mosafa, Majid Moghadam, Mohammad Shahedi


In this work, pectinase was immobilized on the surface of silica-coated magnetite nanoparticles via covalent attachment. The magnetite-immobilized enzyme was characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, scanning electron microscopy and vibrating sample magnetometry techniques. Response surface methodology using Minitab Software was applied for statistical designing of operating conditions in order to immobilize pectinase on magnetic nanoparticles. The optimal conditions were obtained at 30°C and pH 5.5 with 42.97 µl pectinase for 2 h. The immobilization yield was 50.6% at optimized conditions. Compared to the free pectinase, the immobilized pectinase was found to exhibit enhanced enzyme activity, better tolerance to the variation of pH and temperature, and improved storage stability. Both free and immobilized samples reduced the viscosity of apple juice from 1.12 to 0.88 and 0.92 mm2s-1, respectively, after 30 min at their optimum temperature. Furthermore, the immobilized enzyme could be reused six consecutive cycles and the efficiency loss in viscosity reduction was found to be only 8.16%.

Keywords: Enzyme Activity, magnetite nanoparticles, pectinase enzyme, immobilization, juice clarification

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32 Determining the Nitrogen Mineralization Rate by Industrially Manufactured Organic Fertilizers on Alfisol in Southwestern Nigeria

Authors: Ayeni Leye Samuel


Laboratory incubation study was carried out at Adeyemi College of Education, Ondo Southwestern Nigeria to determine the rate of NO3-N, NH4-N, total N, OC and available P released to the soil samples collected from Okitipupa mangrove forest. The soil samples were incubated with organic (OG), organomineral (OMF) and NPK 15:15:15 (NPKF) fertilizers. Organic and organomineral fertilizers were separately applied at the rate of 0, 0.25 and 0.5mg/100 g soil while NPKF was applied at the rate of 0.002g/100g soil. The treatments were replicated three times and arranged on CRD. The treatments were incubated for 90 days. Compared with control, OG and NPKF at all rates significantly increased (p<0.05) soil NH4-N, NO3-N, total N and available P. The order of increase in NH4-N were 10t/ha OMF> 5t/ha OMF> 5t/ha OG>10t/ha OG>control>400 kg/ha while the order of increase in NO3-N were 5t/ha OMF>10t/ha OMF>10t/ha OG>5t/ha OG>control>400 kg/ha NPKF. 5t/ha OMF had the highest, 5t/ha OMF recorded the highest pH, 5t/ha OG had the highest OC while 10t/ha OG had the highest available P.

Keywords: immobilization, c/n ratio, incubation study, organomineral fertilizer

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31 Functionalization of Carbon-Coated Iron Nanoparticles with Fluorescent Protein

Authors: A. G. Pershina, P. S. Postnikov, M. E. Trusova, D. O. Burlakova, A. E. Sazonov


Invention of magnetic-fluorescent nanocomposites is a rapidly developing area of research. The magnetic-fluorescent nanocomposite attractiveness is connected with the ability of simultaneous management and control of such nanocomposites by two independent methods based on different physical principles. These nanocomposites are applied for the solution of various essential scientific and experimental biomedical problems. The aim of this research is development of principle approach to nanobiohybrid structures with magnetic and fluorescent properties design. The surface of carbon-coated iron nanoparticles ([email protected]) were covalently modified by 4-carboxy benzenediazonium tosylate. Recombinant fluorescent protein TagGFP2 (Eurogen) was obtained in E. coli (Rosetta DE3) by standard laboratory techniques. Immobilization of TagGFP2 on the nanoparticles surface was provided by the carbodiimide activation. The amount of COOH-groups on the nanoparticle surface was estimated by elemental analysis (Elementar Vario Macro) and TGA-analysis (SDT Q600, TA Instruments. Obtained nanocomposites were analyzed by FTIR spectroscopy (Nicolet Thermo 5700) and fluorescence microscopy (AxioImager M1, Carl Zeiss). Amount of the protein immobilized on the modified nanoparticle surface was determined by fluorimetry (Cary Eclipse) and spectrophotometry (Unico 2800) with the help of preliminary obtained calibration plots. In the FTIR spectra of modified nanoparticles the adsorption band of –COOH group around 1700 cm-1 and bands in the region of 450-850 cm-1 caused by bending vibrations of benzene ring were observed. The calculated quantity of active groups on the surface was equal to 0,1 mmol/g of material. The carbodiimide activation of COOH-groups on nanoparticles surface results to covalent immobilization of TagGFP2 fluorescent protein (0.2 nmol/mg). The success of immobilization was proved by FTIR spectroscopy. Protein characteristic adsorption bands in the region of 1500-1600 cm-1 (amide I) were presented in the FTIR spectrum of nanocomposite. The fluorescence microscopy analysis shows that [email protected] nanocomposite possesses fluorescence properties. This fact confirms that TagGFP2 protein retains its conformation due to immobilization on nanoparticles surface. Magnetic-fluorescent nanocomposite was obtained as a result of unique design solution implementation – the fluorescent protein molecules were fixed to the surface of superparamagnetic carbon-coated iron nanoparticles using original diazonium salts.

Keywords: immobilization, carbon-coated iron nanoparticles, diazonium salts, fluorescent protein

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30 Immobilization of Lipase Enzyme by Low Cost Material: A Statistical Approach

Authors: Md. Z. Alam, Devi R. Asih, Md. N. Salleh


Immobilization of lipase enzyme produced from palm oil mill effluent (POME) by the activated carbon (AC) among the low cost support materials was optimized. The results indicated that immobilization of 94% was achieved by AC as the most suitable support material. A sequential optimization strategy based on a statistical experimental design, including one-factor-at-a-time (OFAT) method was used to determine the equilibrium time. Three components influencing lipase immobilization were optimized by the response surface methodology (RSM) based on the face-centered central composite design (FCCCD). On the statistical analysis of the results, the optimum enzyme concentration loading, agitation rate and carbon active dosage were found to be 30 U/ml, 300 rpm and 8 g/L respectively, with a maximum immobilization activity of 3732.9 U/g-AC after 2 hrs of immobilization. Analysis of variance (ANOVA) showed a high regression coefficient (R2) of 0.999, which indicated a satisfactory fit of the model with the experimental data. The parameters were statistically significant at p<0.05.

Keywords: Adsorption, activated carbon, immobilization, POME based lipase

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29 Immobilization Strategy of Recombinant Xylanase from Trichoderma reesei by Cross-Linked Enzyme Aggregates

Authors: R. A. Rahman, S. Md. Shaarani, J. Md. Jahim, R. Md. Illias


Modern developments in biotechnology have paved the way for extensive use of biocatalysis in industries. Although it offers immense potential, industrial application is usually hampered by lack of operational stability, difficulty in recovery as well as limited re-use of the enzyme. These drawbacks, however, can be overcome by immobilization. Cross-linked enzyme aggregates (CLEAs), a versatile carrier-free immobilization technique is one that is currently capturing global interest. This approach involves precipitating soluble enzyme with an appropriate precipitant and subsequent crosslinking by a crosslinking reagent. Without ineffective carriers, CLEAs offer high enzymatic activity, stability and reduced production cost. This study demonstrated successful CLEA synthesis of recombinant xylanase from Trichoderma reesei using ethanol as aggregating agent and glutaraldehyde (2% (v/v); 100 mM) as crosslinker. Effects of additives including proteic feeder such as bovine serum albumin (BSA) and poly-L-Lysine were investigated to reveal its significance in enhancing the performance of enzyme. Addition of 0.1 mg BSA/U xylanase showed considerable increment in CLEA development with approximately 50% retained activity.

Keywords: immobilization, xylanase, cross-linked, recombinant

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28 Modeling of Physico-Chemical Characteristics of Concrete for Filling Trenches in Radioactive Waste Management

Authors: Ilija Plecas, Dalibor Arbutina


The leaching rate of 60Co from spent mix bead (anion and cation) exchange resins in a cement-bentonite matrix has been studied. Transport phenomena involved in the leaching of a radioactive material from a cement-bentonite matrix are investigated using three methods based on theoretical equations. These are: the diffusion equation for a plane source, an equation for diffusion coupled to a first order equation and an empirical method employing a polynomial equation. The results presented in this paper are from a 25-year mortar and concrete testing project that will influence the design choices for radioactive waste packaging for a future Serbian radioactive waste disposal center.

Keywords: Waste, Concrete, Radioactivity, Leaching, cement, Permeability, immobilization

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27 Enzyme Immobilization on Functionalized Polystyrene Nanofibersfor Bioprocessing Applications

Authors: Mailin Misson, Bo Jin, Sheng Dai, Hu Zhang


Advances in biotechnology have witnessed a growing interest in enzyme applications for the development of green and sustainable bio processes. While known as powerful bio catalysts, enzymes are no longer of economic value when extended to large commercialization. Alternatively, immobilization technology allows enzyme recovery and continuous reuse which subsequently compensates high operating costs. Employment of enzymes on nano structured materials has been recognized as a promising approach to enhance enzyme catalytic performances. High porosity, inter connectivity and self-assembling behaviors endow nano fibers as exciting candidate for enzyme carrier in bio reactor systems. In this study, nano fibers were successfully fabricated via electro spinning system by optimizing the polymer concentration (10-30 %, w/v), applied voltage (10-30 kV) and discharge distance (11-26 cm). Microscopic images have confirmed the quality as homogeneous and good fiber alignment. The nano fibers surface was modified using strong oxidizing agent to facilitate bio molecule binding. Bovine serum albumin and β-galactosidase enzyme were employed as model bio catalysts and immobilized onto the oxidized surfaces through covalent binding. Maximum enzyme adsorption capacity of the modified nano fibers was 3000 mg/g, 3-fold higher than the unmodified counterpart (1000 mg/g). The highest immobilization yield was 80% and reached the saturation point at 2 mg/ml of enzyme concentration. The results indicate a significant increase of activity retention by the enzyme-bound modified nano fibers (80%) as compared to the nascent one (60%), signifying excellent enzyme-nano carrier bio compatibility. The immobilized enzyme was further used for the bio conversion of dairy wastes into value-added products. This study demonstrates great potential of acid-modified electrospun polystyrene nano fibers as enzyme carriers.

Keywords: enzyme, Nanofibers, immobilization, nanocarrier

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26 Rule of Natural Synthetic Chemical on Lead Immobilization in Polluted Sandy Soils

Authors: Saud S. AL Oud


Soil contamination can have dire consequences, such as loss of ecosystem and agricultural productivity, diminished food chain quality, tainted water resources, economic loss, and human and animal illness. In recent years, attention has focused on the development of in situ immobilization methods that are generally less expensive and disruptive to the natural landscape, hydrology, and ecosystems than are conventional excavation treatments, and disposal methods. Soft, inexpensive, and efficient agents were used in the present research to immobilize Pb in polluted sandy soil. Five agents, either naturally occurring or chemically prepared, were used for this purpose. These agents include; iron ore (72% Fe2O3), cement, a mixture of calcite and shale rich in aluminum (CASH), and two chemically prepared amorphous materials of Al- and Fe-gel. These agents were selected due to their ability to specifically adsorb heavy metals onto their surface OH functional groups, which provide permanent immobilization of metal pollutants and reduce the fraction that is potentially mobile or bioavailable. The efficiency of these agents in immobilizing Pb were examined in a laboratory experiment, in which two rates (0.5 and 1.0 %) of tested agents were added to the polluted soils containing total contents of Pb ranging from 17.4-49.8 mg/kg. The results show that all immobilizing agents were succeed in minimizing the mobile form of Pb as extracted by 0.5 N HNO3. The extracted Pb decreased with increasing addition rate of immobilizing agents. At addition rate of 0.5%, HNO3 extractable-Pb varied widely depending on the agents type and were found to represent 21-67% of the initial values. All agents were able to reduce mobile Pb to levels lower than that (2.0 mg/kg) reported for non polluted soil, particularly for soils had initials of mobile Pb less than 10 mg/kg. Both iron oxide and CASH had the highest efficiency in immobilizing Pb, followed by cement, then amorphous materials of Fe and Al hydroxides.

Keywords: Soil, lead, immobilization, synthetic chemical, polluted

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25 Introduction of PMMA-Tag to VHH for Improving Recovery and Immobilization Rate of VHHS

Authors: Yuji Ito, Bongmun Kang, Kagnari Yamakawa, Yoshihisa Hagihara, Michimasa Kishimoto, Yoichi Kumada


The PMMA-tag was genetically fused with the C-terminal region of VHH molecules. This antibody, VHH, is known as a single-chain domain, which is devoid of light chains. The PMMA-tag, which could affect the isoelectric point (pI) changeable with a charge of amino acid in VHHs were closely related to the solubility of VHH molecules during refolding. The genetic fusion of PMMA-tag to C-terminal region of VHHs significantly affects the recovery of their soluble protein during refolding by 50 mM TAPS at pH 8.5. It could be refolded with a recovery of more than 95% by dialysis at pH 8.5. A marked difference in the antigen-binding activities in the adsorption state was significantly high in VHH-PM compared to the wild type of VHH. There are approximately 8-fold differences in the antigen-binding activities in the adsorption state between VHH-PM and VHH.

Keywords: solubility, ELISA, immobilization, VHH, PMMA-tag, isoelectric point, refolding

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24 New Heterogenous α-Diimine Nickel (II)/ MWCNT Catalysts for Ethylene Polymerization

Authors: Sasan Talebnezhad, Saeed Pormahdian, Naghi Assali


Homogeneous α-diimine nickel (II) catalyst complexes, with and without amino para-aryl position functionality, were synthesized. These complexes were immobilized on carboxyl, hydroxyl, and acyl chloride functionalized multi-walled carbon nanotubes to form five novel heterogeneous α-diiminonickel catalysts. Immobilization was performed by covalent or electrostatic bonding via methylaluminoxane (MAO) linker or amide linkage. Both the nature of α-diimine ligands and the kind of interaction between anchored catalyst complexes and multi-walled carbon nanotube surface influenced the catalytic performance, microstructure, and morphology of obtained polyethylenes. The catalyst prepared by amide bonding showed lowest relative weight loss in thermogravimetry analysis and highest activities up to 5863 gr PE This catalyst produced polyethylene with dense botryoidal morphology.

Keywords: immobilization, α-diimine nickel (II) complexes, multi-walled carbon nanotubes, ethylene polymerization

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23 New Heterogenous α-Diimine Nickel (II)/MWCNT Catalysts for Ethylene Polymerization

Authors: Sasan Talebnezhad, Naghi Assali, Saeed Pourmahdian


Homogeneous α-diimine nickel (II) catalyst complexes, with and without amino para-aryl position functionality, were synthesized. These complexes were immobilized on carboxyl, hydroxyl and acyl chloride functionalized multi-walled carbon nanotubes to form five novel heterogeneous α diiminonickel catalysts. Immobilization was performed by covalent or electrostatic bonding via methylaluminoxane (MAO) linker or amide linkage. Both the nature of α-diimine ligands and the kind of interaction between anchored catalyst complexes and multi-walled carbon nanotube surface influenced the catalytic performance, microstructure, and morphology of obtained polyethylenes. The catalyst prepared by amide bonding showed lowest relative weight loss in thermogravimetry analysis and highest activities up to 5863 gr PE This catalyst produced polyethylene with dense botryoidal morphology.

Keywords: immobilization, α-diimine nickel (II) complexes, multi-walled carbon nanotubes, ethylene polymerization

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22 Preparation of Low-Molecular-Weight 6-Amino-6-Deoxychitosan (LM6A6DC) for Immobilization of Growth Factor

Authors: Koo-Yeon Kim, Eun-Hye Kim, Tae-Il Son


Epidermal Growth Factor (EGF, Mw=6,045) has been reported to have high efficiency of wound repair and anti-wrinkle effect. However, the half-life of EGF in the body is too short to exert the biological activity effectively when applied in free form. Growth Factors can be stabilized by immobilization with carbohydrates from thermal and proteolytic degradation. Low molecular weight chitosan (LMCS) and its derivate prepared by hydrogen peroxide has high solubility. LM6A6DC was successfully prepared as a reactive carbohydrate for the stabilization of EGF by the reactions of LMCS with alkalization, tosylation, azidation and reduction. The structure of LM6A6DC was confirmed by FT-IR, 1H NMR and elementary analysis. For enhancing the stability of free EGF, EGF was attached with LM6A6DC by using water-soluble carbodiimide. EGF-LM6A6DC conjugates did not show any cytotoxicity on the Normal Human Dermal Fibroblast(NHDF) 3T3 proliferation at least under 100 ㎍/㎖. In the result, it was considered that LM6A6DC is suitable to immobilize of growth factor.

Keywords: immobilization, epidermal growth factor (EGF), low-molecular-weight chitosan

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21 Removal of Lead (Pb) by the Microorganism Isolated from the Effluent of Lead Acid Battery Scrap

Authors: Narasimhulu Korrapati, Harikrishna Yadav Nanganuru


The demand for the lead (Pb) in the battery industry has been growing for last twenty years. On an average about 2.35 million tons of lead is used in the battery industry. According to the survey of supply and demand battery industry is using 75% of lead produced every year. Due to the increase in battery scrap, secondary lead production has been increasing in this decade. Europe and USA together account for 75% of the world’s secondary lead production. The effluent from used battery scrap consists of high concentrations of lead. Unauthorized disposal of spent batteries, which contain intolerable concentration of lead, into landfills or municipal water canals causes release of Pb into the environment. Lead is one of the toxic heavy metals that have large damaging effects on the human health. Due to its persistence and toxicity, the presence of Pb in drinking water is considered as a special concern. Accumulation of Pb in the human body for long period of time can result in the malfunctioning of some organs. Many technologies have been developed for the removal of lead using microorganisms. In this paper, effluent was taken from the spent battery scrap and was characterized by inductively coupled plasma atomic emission spectrometer. Microorganisms play an important role in removal of lead from the contaminated sites. So, the bacteria were isolated from the effluent. Optimum conditions for the microbial growth and applied for the lead removal. These bacterial cells were immobilized and used for the removal of Pb from the known concentration of metal solution. Scanning electron microscopic (SEM) studies were shown that the Pb was efficiently adsorbed by the immobilized bacteria. From the results of Atomic Absorption Spectroscopy (AAS), 83.40 percentage of Pb was removed in a batch culture.

Keywords: Adsorption, effluent, immobilization, lead (Pb)

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20 Optimization of Fermentation Parameters for Bioethanol Production from Waste Glycerol by Microwave Induced Mutant Escherichia coli EC-MW (ATCC 11105)

Authors: Refal Hussain, Saifuddin M. Nomanbhay


Glycerol is a valuable raw material for the production of industrially useful metabolites. Among many promising applications for the use of glycerol is its bioconversion to high value-added compounds, such as bioethanol through microbial fermentation. Bioethanol is an important industrial chemical with emerging potential as a biofuel to replace vanishing fossil fuels. The yield of liquid fuel in this process was greatly influenced by various parameters viz, temperature, pH, glycerol concentration, organic concentration, and agitation speed were considered. The present study was undertaken to investigate optimum parameters for bioethanol production from raw glycerol by immobilized mutant Escherichia coli (E.coli) (ATCC11505) strain on chitosan cross linked glutaraldehyde optimized by Taguchi statistical method in shake flasks. The initial parameters were set each at four levels and the orthogonal array layout of L16 (45) conducted. The important controlling parameters for optimized the operational fermentation was temperature 38 °C, medium pH 6.5, initial glycerol concentration (250 g/l), and organic source concentration (5 g/l). Fermentation with optimized parameters was carried out in a custom fabricated shake flask. The predicted value of bioethanol production under optimized conditions was (118.13 g/l). Immobilized cells are mainly used for economic benefits of continuous production or repeated use in continuous as well as in batch mode.

Keywords: Optimization, Bioethanol, immobilization, Escherichia coli

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19 Enhanced Enzymes Production through Immobilization of Filamentous Fungi

Authors: Zhanara B. Suleimenova, Zhazira K. Saduyeva


Filamentous fungi are major producers of enzymes that have important applications in the food and beverage industries. The overall objective of this research is a strain improvement technology for efficient industrial enzymes production. The new way of filamentous fungi cultivation method has been developed. Such technology prolong producers’ cultivation period up to 60 days and create the opportunity to obtain enzymes repeatedly in every 2-3 days of fungal cultivation. This method is based on immobilizing enzymes producers with solid support in submerged conditions of growth. Immobilizing has a range of advantages: Decreasing the price of the final product, absence of foreign substances, controlled process of enzyme-genesis, ability of various enzymes simultaneous production, etc. Design of proposed technology gives the opportunity to increase the activity of immobilized cells culture filtrate comparing to free cells, growing in periodic culture conditions. Thus, proposed research focuses on new, more versatile, microorganisms capable of squeezing more end-products as well as proposed cultivation technology led to increased enzymatic productivity by several times.

Keywords: Filamentous Fungi, immobilization, industrial enzymes production, strain improvement

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18 Cross-Linked Amyloglucosidase Aggregates: A New Carrier Free Immobilization Strategy for Continuous Saccharification of Starch

Authors: Shah Ali Ul Qader, Sidra Pervez, Afsheen Aman


The importance of attaining an optimum performance of an enzyme is often a question of devising an effective method for its immobilization. Cross-linked enzyme aggregate (CLEAs) is a new approach for immobilization of enzymes using carrier free strategy. This method is exquisitely simple (involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules) and amenable to rapid optimization. Among many industrial enzymes, amyloglucosidase is an important amylolytic enzyme that hydrolyzes alpha (1→4) and alpha (1→6) glycosidic bonds in starch molecule and produce glucose as a sole end product. Glucose liberated by amyloglucosidase can be used for the production of ethanol and glucose syrups. Besides this amyloglucosidase can be widely used in various food and pharmaceuticals industries. For production of amyloglucosidase on commercial scale, filamentous fungi of genera Aspergillus are mostly used because they secrete large amount of enzymes extracellularly. The current investigation was based on isolation and identification of filamentous fungi from genus Aspergillus for the production of amyloglucosidase in submerged fermentation and optimization of cultivation parameters for starch saccharification. Natural isolates were identified as Aspergillus niger KIBGE-IB36, Aspergillus fumigatus KIBGE-IB33, Aspergillus flavus KIBGE-IB34 and Aspergillus terreus KIBGE-IB35 on taxonomical basis and 18S rDNA analysis and their sequence were submitted to GenBank. Among them, Aspergillus fumigatus KIBGE-IB33 was selected on the basis of maximum enzyme production. After optimization of fermentation conditions enzyme was immobilized on CLEA. Different parameters were optimized for maximum immobilization of amyloglucosidase. Data of enzyme stability (thermal and Storage) and reusability suggested the applicability of immobilized amyloglucosidase for continuous saccharification of starch in industrial processes.

Keywords: Industrial Processes, immobilization, aspergillus, starch saccharification

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17 ENDO-β-1,4-Xylanase from Thermophilic Geobacillus stearothermophilus: Immobilization Using Matrix Entrapment Technique to Increase the Stability and Recycling Efficiency

Authors: Shah Ali Ul Qader, Zainab Bibi, Afsheen Aman


Introduction: Xylan is a heteropolysaccharide composed of xylose monomers linked together through 1,4 linkages within a complex xylan network. Owing to wide applications of xylan hydrolytic products (xylose, xylobiose and xylooligosaccharide) the researchers are focusing towards the development of various strategies for efficient xylan degradation. One of the most important strategies focused is the use of heat tolerant biocatalysts which acts as strong and specific cleaving agents. Therefore, the exploration of microbial pool from extremely diversified ecosystem is considerably vital. Microbial populations from extreme habitats are keenly explored for the isolation of thermophilic entities. These thermozymes usually demonstrate fast hydrolytic rate, can produce high yields of product and are less prone to microbial contamination. Another possibility of degrading xylan continuously is the use of immobilization technique. The current work is an effort to merge both the positive aspects of thermozyme and immobilization technique. Methodology: Geobacillus stearothermophilus was isolated from soil sample collected near the blast furnace site. This thermophile is capable of producing thermostable endo-β-1,4-xylanase which cleaves xylan effectively. In the current study, this thermozyme was immobilized within a synthetic and a non-synthetic matrice for continuous production of metabolites using entrapment technique. The kinetic parameters of the free and immobilized enzyme were studied. For this purpose calcium alginate and polyacrylamide beads were prepared. Results: For the synthesis of immobilized beads, sodium alginate (40.0 gL-1) and calcium chloride (0.4 M) was used amalgamated. The temperature (50°C) and pH (7.0) optima of immobilized enzyme remained same for xylan hydrolysis however, the enzyme-substrate catalytic reaction time raised from 5.0 to 30.0 minutes as compared to free counterpart. Diffusion limit of high molecular weight xylan (corncob) caused a decline in Vmax of immobilized enzyme from 4773 to 203.7 U min-1 whereas, Km value increased from 0.5074 to 0.5722 mg ml-1 with reference to free enzyme. Immobilized endo-β-1,4-xylanase showed its stability at high temperatures as compared to free enzyme. It retained 18% and 9% residual activity at 70°C and 80°C, respectively whereas; free enzyme completely lost its activity at both temperatures. The Immobilized thermozyme displayed sufficient recycling efficiency and can be reused up to five reaction cycles, indicating that this enzyme can be a plausible candidate in paper processing industry. Conclusion: This thermozyme showed better immobilization yield and operational stability with the purpose of hydrolyzing the high molecular weight xylan. However, the enzyme immobilization properties can be improved further by immobilizing it on different supports for industrial purpose.

Keywords: reusability, immobilization, xylanase, thermozymes

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16 Separation of Chlorinated Plastics and Immobilization of Heavy Metals in Hazardous Automotive Shredder Residue

Authors: Srinivasa Reddy Mallampati, Chi-Hyeon Lee, Nguyen Thi Thanh Truc, Byeong-Kyu Lee


In the present study, feasibility of the selective surface hydrophilization of polyvinyl chloride (PVC) by microwave treatment was evaluated to facilitate the separation from automotive shredder residue (ASR), by the froth flotation. The combination of 60 sec microwave treatment with PAC, a sharp and significant decrease about 16.5° contact angle of PVC was observed in ASR plastic compared with other plastics. The microwave treatment with the addition of PAC resulted in a synergetic effect for the froth flotation, which may be a result of the 90% selective separation of PVC from ASR plastics, with 82% purity. While, simple mixing with a nanometallic Ca/CaO/PO4 dispersion mixture immobilized 95-100% of heavy metals in ASR soil/residues. The quantity of heavy metals leached from thermal residues after treatment by nanometallic Ca/CaO/PO4 was lower than the Korean standard regulatory limit for hazardous waste landfills. Microwave treatment can be a simple and effective method for PVC separation from ASR plastics.

Keywords: separation, Heavy Metals, Hazardous waste, immobilization, automotive shredder residue, chlorinated plastics

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15 Effect of Anion and Amino Functional Group on Resin for Lipase Immobilization with Adsorption-Cross Linking Method

Authors: Heri Hermansyah, Annisa Kurnia, A. Vania Anisya, Adi Surjosatyo, Yopi Sunarya, Rita Arbianti, Tania Surya Utami


Lipase is one of biocatalyst which is applied commercially for the process in industries, such as bioenergy, food, and pharmaceutical industry. Nowadays, biocatalysts are preferred in industries because they work in mild condition, high specificity, and reduce energy consumption (high pressure and temperature). But, the usage of lipase for industry scale is limited by economic reason due to the high price of lipase and difficulty of the separation system. Immobilization of lipase is one of the solutions to maintain the activity of lipase and reduce separation system in the process. Therefore, we conduct a study about lipase immobilization with the adsorption-cross linking method using glutaraldehyde because this method produces high enzyme loading and stability. Lipase is immobilized on different kind of resin with the various functional group. Highest enzyme loading (76.69%) was achieved by lipase immobilized on anion macroporous which have anion functional group (OH). However, highest activity (24,69 U/g support) through olive oil emulsion method was achieved by lipase immobilized on anion macroporous-chitosan which have amino (NH2) and anion (OH-) functional group. In addition, it also success to produce biodiesel until reach yield 50,6% through interesterification reaction and after 4 cycles stable 63.9% relative with initial yield. While for Aspergillus, niger lipase immobilized on anion macroporous-kitosan have unit activity 22,84 U/g resin and yield biodiesel higher than commercial lipase (69,1%) and after 4 cycles stable reach 70.6% relative from initial yield. This shows that optimum functional group on support for immobilization with adsorption-cross linking is the support that contains amino (NH2) and anion (OH-) functional group because they can react with glutaraldehyde and binding with enzyme prevent desorption of lipase from support through binding lipase with a functional group on support.

Keywords: immobilization, lipase, adsorption-cross linking, resin

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14 Simultaneous Nitrification and Denitrification in Suspended Activated Sludge Process Augmented with Immobilized Biomass: A Pilot Study

Authors: Haon-Yao Chen, Cheng-Fang Lin, Pui-Kwan Andy Hong, Ping-Yi Yang, Kok Kwang Ng, Sheng-Fu Yang


Simultaneous nitrification and denitrification (SND) are a natural phenomenon in the soil environment that can be applied in wastewater treatment. At a domestic wastewater treatment plant, we performed a pilot test of installing bioplates with entrapped biomass into a conventional aeration basin for SND, and investigated the effects of bioplate packing ratio, hydraulic retention time, dissolved oxygen level, on/off aeration mode, and supplemental carbon and alkalinity on nitrogen removal. With the pilot aeration basin of 1.3 m3 loaded with mixed liquor suspended solids of 1500-2500 mg/L and bioplates at PR of 3.2% (3.2% basin volume) operated at HRT of 6 h and DO of 4-6 mg/L without supplemental carbon or alkalinity, nitrogen in the wastewater was removed to an effluent total nitrogen (TN) of 7.3 mg/L from an influent TN of 28 mg/L. The bioplate robust cellulose triacetate structure carrying the biomass shows promise in retrofitting conventional aeration basins for enhanced nutrient removal.

Keywords: immobilization, nutrient removal, nitrification/denitrification, total nitrogen

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13 The Effect of Immobilization Conditions on Hydrogen Production from Palm Oil Mill Effluent

Authors: A. W. Zularisam, Lakhveer Singh, Mimi Sakinah Abdul Munaim


In this study, the optimization of hydrogen production using polyethylene glycol (PEG) immobilized sludge was investigated in batch tests. Palm oil mill effluent (POME) is used as a substrate that can act as a carbon source. Experiment focus on the effect of some important affecting factors on fermentative hydrogen production. Results showed that immobilized sludge demonstrated the maximum hydrogen production rate of 340 mL/L-POME/h under follow optimal condition: amount of biomass 10 mg VSS/ g bead, PEG concentration 10%, and cell age 24 h or 40 h. More importantly, immobilized sludge not only enhanced hydrogen production but can also tolerate the harsh environment and produce hydrogen at the wide ranges of pH. The present results indicate the potential of PEG-immobilized sludge for large-scale operations as well; these factors play an important role in stable and continuous hydrogen production.

Keywords: immobilization, dark fermentation, palm oil mill effluent, bioydrogen, polyethylene glycol

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12 Production of Biodiesel Using Tannery Fleshing as a Feedstock via Solid-State Fermentation

Authors: C. Santhana Krishnan, A. M. Mimi Sakinah, Lakhveer Singh, Zularisam A. Wahid


This study was initiated to evaluate and optimize the conversion of animal fat from tannery wastes into methyl ester. In the pre-treatment stage, animal fats feedstock was hydrolysed and esterified through solid state fermentation (SSF) using Microbacterium species immobilized onto sand silica matrix. After 72 hours of fermentation, predominant esters in the animal fats were found to be with 83.9% conversion rate. Later, esterified animal fats were transesterified at 3 hour reaction time with 1% NaOH (w/v %), 6% methanol to oil ratio (w/v %) to produce 89% conversion rate. C13 NMR revealed long carbon chain in fatty acid methyl esters at 22.2817-31.9727 ppm. Methyl esters of palmitic, stearic, oleic represented the major components in biodiesel.

Keywords: solid state fermentation, immobilization, tannery wastes, fatty animal fleshing, trans-esterification

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11 The Methods of Immobilization of Laccase for Direct Transfer in an Enzymatic Fuel Cell

Authors: Afshin Farahbakhsh, Hoda Khodadadi


In this paper, we compare five methods of biological fuel cell fabrication by combining a Shewanella oneidensis microbial anode and a laccase-modified air-breathing cathode. As a result of biofuel cell laccase with graphite nanofibers, carbon surface (PAMAN) on the pt/hpg electrode, graphite sheets MWCNT and with (PG) and (MWCNT) showed, respectively. Describes methods for creating controllable and reproducible bio-anodes and demonstrates the versatility of hybrid biological fuel cells. The laccase-based biocathodes prepared either with the crude extract or with the purified enzyme can provide electrochemically active and stable biomaterials. The laccase-based biocathodes prepared either with the crude extract or with the purified enzyme can provide electrochemically active and stable biomaterials. When the device was fed with transdermal extracts, containing only 30μM of glucose, the average peak power was proportionally lower (0.004mW). The result of biofuel cell with graphite nanofibers showed the enzymatic fuel cell reaches 0.5 V at open circuit voltage with both, ethanol and methanol and the maximum current density observed for E2electrode was 228.94mAcm.

Keywords: Fuel Cell, immobilization, laccase, enzymatic electrode

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10 Magnetic Silica Nanoparticles as Viable Support for the Immobilization of Oxidative Enzymes

Authors: Y. Moldes-Diz, M. Gamallo, G. Eibes, C. Vazquez-Vazquez, G. Feijoo, J. M. Lema, M. T. Moreira


Laccases (benzenediol oxygen oxidoreductases, EC are excellent biocatalysts for biotechnological and environmental applications because of their high activity, selectivity, and specificity. Specifically, these characteristics allow them to perform the oxidation of recalcitrant compounds with simple requirements for the catalysis (presence of molecular oxygen). Nevertheless, the low stability under unfavorable conditions (pH, inactivating agents or temperature) and high production costs still limits their use for practical applications. Immobilization of enzymes has proven particularly valuable to avoid some of the aforementioned drawbacks. Magnetic nanoparticles (MNPs) have received increasing attention as carriers for enzyme immobilization since they can potentially provide an easy recovery of the biocatalyst from the reaction medium under an external magnetic field. In the present work, silica-coated magnetic nanoparticles ([email protected]) were prepared, characterized and used for laccase immobilization by covalent binding. The synthesis of [email protected] was performed in a two-step procedure: co-precipitation and reverse microemulsion. The influence of immobilization conditions: concentrations of the functionalization agent (3-aminopropyl-triethoxy-silane) and the cross-linker (glutaraldehyde) as well as the influence of pH, T or inactivating agents were evaluated. In general, immobilized laccase showed superior stability compared to that of free enzyme. The reusability of the biocatalyst was demonstrated in successive batch reactions, where enzyme activity was maintained above 65% after 8 cycles of oxidation of the substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate).

Keywords: Regeneration, immobilization, laccase, silica-coated magnetic nanoparticles

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9 Sorbitol Galactoside Synthesis Using β-Galactosidase Immobilized on Functionalized Silica Nanoparticles

Authors: Milica Carević, Katarina Banjanac, Marija ĆOrović, Ana Milivojević, Nevena Prlainović, Aleksandar Marinković, Dejan Bezbradica


Nowadays, considering the growing awareness of functional food beneficial effects on human health, due attention is dedicated to the research in the field of obtaining new prominent products exhibiting improved physiological and physicochemical characteristics. Therefore, different approaches to valuable bioactive compounds synthesis have been proposed. β-Galactosidase, for example, although mainly utilized as hydrolytic enzyme, proved to be a promising tool for these purposes. Namely, under the particular conditions, such as high lactose concentration, elevated temperatures and low water activities, reaction of galactose moiety transfer to free hydroxyl group of the alternative acceptor (e.g. different sugars, alcohols or aromatic compounds) can generate a wide range of potentially interesting products. Up to now, galacto-oligosaccharides and lactulose have attracted the most attention due to their inherent prebiotic properties. The goal of this study was to obtain a novel product sorbitol galactoside, using the similar reaction mechanism, namely transgalactosylation reaction catalyzed by β-galactosidase from Aspergillus oryzae. By using sugar alcohol (sorbitol) as alternative acceptor, a diverse mixture of potential prebiotics is produced, enabling its more favorable functional features. Nevertheless, an introduction of alternative acceptor into the reaction mixture contributed to the complexity of reaction scheme, since several potential reaction pathways were introduced. Therefore, the thorough optimization using response surface method (RSM), in order to get an insight into different parameter (lactose concentration, sorbitol to lactose molar ratio, enzyme concentration, NaCl concentration and reaction time) influences, as well as their mutual interactions on product yield and productivity, was performed. In view of product yield maximization, the obtained model predicted optimal lactose concentration 500 mM, the molar ratio of sobitol to lactose 9, enzyme concentration 0.76 mg/ml, concentration of NaCl 0.8M, and the reaction time 7h. From the aspect of productivity, the optimum substrate molar ratio was found to be 1, while the values for other factors coincide. In order to additionally, improve enzyme efficiency and enable its reuse and potential continual application, immobilization of β-galactosidase onto tailored silica nanoparticles was performed. These non-porous fumed silica nanoparticles (FNS)were chosen on the basis of their biocompatibility and non-toxicity, as well as their advantageous mechanical and hydrodinamical properties. However, in order to achieve better compatibility between enzymes and the carrier, modifications of the silica surface using amino functional organosilane (3-aminopropyltrimethoxysilane, APTMS) were made. Obtained support with amino functional groups (AFNS) enabled high enzyme loadings and, more importantly, extremely high expressed activities, approximately 230 mg proteins/g and 2100 IU/g, respectively. Moreover, this immobilized preparation showed high affinity towards sorbitol galactoside synthesis. Therefore, the findings of this study could provided a valuable contribution to the efficient production of physiologically active galactosides in immobilized enzyme reactors.

Keywords: β-galactosidase, immobilization, silica nanoparticles, transgalactosylation

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8 High Catalytic Activity and Stability of Ginger Peroxidase Immobilized on Amino Functionalized Silica Coated Titanium Dioxide Nanocomposite: A Promising Tool for Bioremediation

Authors: Misha Ali, Qayyum Husain, Nida Alam, Masood Ahmad


Improving the activity and stability of the enzyme is an important aspect in bioremediation processes. Immobilization of enzyme is an efficient approach to amend the properties of biocatalyst required during wastewater treatment. The present study was done to immobilize partially purified ginger peroxidase on amino functionalized silica coated titanium dioxide nanocomposite. Interestingly there was an enhancement in enzyme activity after immobilization on nanosupport which was evident from effectiveness factor (η) value of 1.76. Immobilized enzyme was characterized by transmission electron microscopy, scanning electron microscopy and Fourier transform infrared spectroscopy. Immobilized peroxidase exhibited higher activity in a broad range of pH and temperature as compared to free enzyme. Also, the thermostability of peroxidase was strikingly improved upon immobilization. After six repeated uses, the immobilized peroxidase retained around 62% of its dye decolorization activity. There was a 4 fold increase in Vmax of immobilized peroxidase as compared to free enzyme. Circular dichroism spectroscopy demonstrated conformational changes in the secondary structure of enzyme, a possible reason for the enhanced enzyme activity after immobilization. Immobilized peroxidase was highly efficient in the removal of acid yellow 42 dye in a stirred batch process. Our study shows that this bio-remediating system has remarkable potential for treatment of aromatic pollutants present in wastewater.

Keywords: immobilization, decolorization, acid yellow 42, ginger peroxidase

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7 Detoxification of Hazardous Organic/Inorganic Contaminants in Automobile Shredder Residue by Multi-Functioned Nano-Size Metallic Calcium Composite

Authors: Srinivasa Reddy Mallampati, Byoung Ho Lee, Yoshiharu Mitoma, Simion Cristian


In recent years, environmental nanotechnology has risen to the forefront and the new properties and enhanced reactivates offered by nanomaterial may offer a new, low-cost paradigm to solving complex environmental pollution problems. This study assessed the synthesis and application of multi-functioned nano-size metallic calcium (nMC) composite for detoxification of hazardous inorganic (heavy metals (HMs)/organic chlorinated/brominated compound (CBCs) contaminants in automobile shredder residue (ASR). ASR residues ball milled with nMC composite can achieve about 90-100% of HMs immobilization and CBCs decomposition. The results highlight the low quantity of HMs leached from ASR residues after treatment with nMC, which was found to be lower than the standard regulatory limit for hazardous waste landfills. The use of nMC composite in a mechanochemical process to treat hazardous ASR (dry conditions) is a simple and innovative approach to remediate hazardous inorganic/organic cross-contaminates in ASR.

Keywords: Detoxification, immobilization, nano-sized metallic calcium, automobile shredder residue, organic/inorganic contaminants

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6 Fluoride Immobilization in Plaster Board Waste: A Safety Measure to Prevent Soil and Water Pollution

Authors: Venkataraman Sivasankar, Kiyoshi Omine, Hideaki Sano


The leaching of fluoride from Plaster Board Waste (PBW) is quite feasible in soil and water environments. The Ministry of Environment, Japan recommended the standard limit of 0.8 mgL⁻¹ or less for fluoride. Although the utilization of PBW as a substitute for cement is rather meritorious, its fluoride leaching behavior deteriorates the quality of soil and water and therefore envisaged as a demerit. In view of this fluoride leaching problem, the present research is focused on immobilizing fluoride in PBW. The immobilization experiments were conducted with four chemical systems operated by DAHP (diammonium hydrogen phosphate) and phosphoric acid carbonization of bamboo mass coupled with certain inorganic reactions using reagents such as calcium hydroxide, sodium hydroxide, and aqueous ammonia. The fluoride immobilization was determined after shaking the reactor contents including the plaster board waste for 24 h at 25˚C. In the DAHP system, the immobilization of fluoride was evident from the leaching of fluoride in the range 0.071-0.12 mgL⁻¹, 0.026-0.14 mgL⁻¹ and 0.068-0.12 mgL⁻¹ for the reaction temperatures at 30˚C, 50˚C, and 90˚C, respectively, with final pH of 6.8. The other chemical systems designated as PACCa, PACAm, and PACNa could immobilize fluoride in PBW, and the resulting solution was analyzed with the fluoride less than the Japanese environmental standard of 0.8 mgL⁻¹. In the case of PACAm and PACCa systems, the calcium concentration was found undetectable and witnessed the formation of phosphate compounds. The immobilization of fluoride was found inversely proportional to the increase in the volume of leaching solvent and dose of PBW. Characterization studies of PBW and the solid after fluoride immobilization was done using FTIR (Fourier transform infrared spectroscopy), Raman spectroscopy, FE-SEM ( Field Emission Scanning Electron Microscopy) with EDAX (Energy Dispersive Spectroscopy), XRD (X-ray diffraction), and XPS (X-ray photoelectron spectroscopy). The results revealed the formation of new calcium phosphate compounds such as apatite, monetite, and hydroxylapatite. The participation of such new compounds in fluoride immobilization seems indispensable through the exchange mechanism of hydroxyl and fluoride groups. Acknowledgment: First author thanks to Japanese Society for the Promotion of Science (JSPS) for the award of the fellowship (ID No. 16544).

Keywords: Characterization, immobilization, fluoride, plaster board waste

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5 Performance Evaluation of a Spouted Bed Bioreactor (SBBR) for the Biodegradation of 2, 4 Dichlorophenol

Authors: Muftah El-Naas, Taghreed Al-Khalid


As an economical and environmentally friendly technology, biological treatment has been shown to be one of the most promising approaches for the removal of numerous types of organic water pollutants such as Chlorophenols, which are hazardous pollutants commonly encountered in wastewater generated by the petroleum and petrochemical industries. This study aimed at evaluating the performance of a spouted bed bioreactor (SBBR) for aerobic biodegradation of 2, 4 dichlorophenol (DCP) by a commercial strain of Pseudomonas putida immobilized in polyvinyl alcohol (PVA) gel particles. The SBBR is characterized by systematic intense mixing, resulting in improvement of the biodegradation rates through reducing the mass transfer limitations. The reactor was evaluated in both batch and continuous mode in order to evaluate its hydrodynamics in terms of stability and response to shock loads. The SBBR was able to maintain a stable operation and recovered quickly to its normal operating mode once the shock load had been removed. In comparison to a packed bed reactor bioreactor, the SBBR proved to be more efficient and more stable, achieving a removal percentage and throughput of 80% and 1414 g/m3day, respectively. In addition, the biodegradation of chlorophenols was mathematically modeled using a dynamic modeling approach in order to assess reaction and mass transfer limitations. The results confirmed the effectiveness of the use of the PVA immobilization technique for the biodegradation of phenols.

Keywords: biodegradation, immobilization, polyvinyl alcohol (PVA) gel

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4 Biodegradation of Chlorpyrifos in Real Wastewater by Acromobacter xylosoxidans SRK5 Immobilized in Calcium Alginate

Authors: Imran Hashmi, Saira Khalid


Agrochemical industries produce huge amount of wastewater containing pesticides and other harmful residues. Environmental regulations make it compulsory to bring pesticides to a minimum level before releasing wastewater from industrial units.The present study was designed with the objective to investigate biodegradation of CP in real wastewater using bacterial cells immobilized in calcium alginate. Bacterial strain identified as Acromobacter xylosoxidans SRK5 (KT013092) using 16S rRNA nucleotide sequence analysis was used. SRK5 was immobilized in calcium alginate to make calcium alginate microspheres (CAMs). Real wastewater from industry having 50 mg L⁻¹ of CP was inoculated with free cells or CAMs and incubated for 96 h at 37˚C. CP removal efficiency with CAMs was 98% after 72 h of incubation, and no lag phase was observed. With free cells, 12h of lag phase was observed. After 96 h of incubation 87% of CP removal was observed when inoculated with free cells. No adsorption was observed on vacant CAMs. Phytotoxicity assay demonstrated considerable loss in toxicity. Almost complete COD removal was achieved at 96 h with CAMs. Study suggests the use of immobilized cells of SRK5 for bioaugmentation of industrial wastewater for CP degradation instead of free cells.

Keywords: wastewater, biodegradation, immobilization, chlorpyrifos

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