Search results for: Bacillus strain IIIJ3-1
Commenced in January 2007
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Edition: International
Paper Count: 1894

Search results for: Bacillus strain IIIJ3-1

454 The Use of Superplastic Tin-Lead Alloy as A solid Lubricant in Free Upsetting of Aluminum and Brass

Authors: Adnan I. O. Zaid, Hebah B. Melhem, Ahmad Qandil

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The main function of a lubricant in any forming process is to reduce friction between the work piece and the die set, hence reducing the force and energy requirement for forming process and to achieve homogeneous deformation. The free upsetting test is an important open forging test. In this paper, super plastic tin-lead alloy is used as solid lubricant in the free upsetting test of non-ferrous metals and compared with eight different lubricants using the following three criteria: one comparing the value of the reduction in height percentages, i.e. the engineering strain, in identical specimens of the same material under the effect of the same compressive force. The second is comparing the amount of barreling produced in each of the identical specimens, at each lubricant. The third criterion is using the specific energy, i.e. the energy per unit volume consumed in forming each material, using the different lubricants to produce the same reduction in height percentage of identical specimens from each of the two materials, namely: aluminum and brass. It was found that the super plastic tin-lead alloy lubricant has produced higher values of reductions in height percentage and less barreling in the two non-ferrous materials, used in this work namely: aluminum and brass. It was found that the super plastic tin-lead alloy lubricant has produced higher values of reductions in height percentage and less barreling in the two non-ferrous materials, used in this work, under the same compression force among the different used lubricants.

Keywords: aluminum, brass, different lubricants, free upsetting, solid lubricants, superplastic tin-lead alloy

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453 Modelling and Simulation Efforts in Scale-Up and Characterization of Semi-Solid Dosage Forms

Authors: Saurav S. Rath, Birendra K. David

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Generic pharmaceutical industry has to operate in strict timelines of product development and scale-up from lab to plant. Hence, detailed product & process understanding and implementation of appropriate mechanistic modelling and Quality-by-design (QbD) approaches are imperative in the product life cycle. This work provides example cases of such efforts in topical dosage products. Topical products are typically in the form of emulsions, gels, thick suspensions or even simple solutions. The efficacy of such products is determined by characteristics like rheology and morphology. Defining, and scaling up the right manufacturing process with a given set of ingredients, to achieve the right product characteristics presents as a challenge to the process engineer. For example, the non-Newtonian rheology varies not only with CPPs and CMAs but also is an implicit function of globule size (CQA). Hence, this calls for various mechanistic models, to help predict the product behaviour. This paper focusses on such models obtained from computational fluid dynamics (CFD) coupled with population balance modelling (PBM) and constitutive models (like shear, energy density). In a special case of the use of high shear homogenisers (HSHs) for the manufacture of thick emulsions/gels, this work presents some findings on (i) scale-up algorithm for HSH using shear strain, a novel scale-up parameter for estimating mixing parameters, (ii) non-linear relationship between viscosity and shear imparted into the system, (iii) effect of hold time on rheology of product. Specific examples of how this approach enabled scale-up across 1L, 10L, 200L, 500L and 1000L scales will be discussed.

Keywords: computational fluid dynamics, morphology, quality-by-design, rheology

Procedia PDF Downloads 269
452 Production of Mycelial Biomass, Exopolysaccharide, and Enzyme during Solid-State Fermentation of Plant Raw Materials by Medicinal Mushrooms

Authors: Tamar Khardziani, Violeta Berikashvili, Amrosi Chkuaseli, Vladimir Elisashvili

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The main objectives of this proposal are to develop low-cost, innovative, and competitive technologies for the production of mycelial biomass of medicinal mushrooms as a natural food supplement for poultry. To fulfill this task, industrial strains of Lentinus edodes, Ganoderma lucidum, and Pleurotus ostreatus were used in this study. The solid-state fermentation (SSF) of wheat grains, wheat bran, and soy flour was performed in flasks and bags. Among nine mushroom strains, P. ostreatus 2191 appeared to be the most productive in protein biomass accumulation in the SSF of wheat bran. All mushrooms produced exopolysaccharide with the highest yield of 5-8 mg/mL depending on fungal strain and growth substrate. Supplementation of medium with 1% glycerol and 2-4% peptone favored mushroom growth and protein accumulation. Among inorganic nitrogen sources, KNO₃ also provided high biomass and protein production. The SSF of all growth substrates was accompanied by the secretion of cellulase and xylanase activities. The highest CMCase activity (12-13 U/g) was revealed in the cultivation of P. ostreatus 2191 using wheat bran as a growth substrate and ammonium sulfate or yeast extract as a nitrogen source, whereas the highest xylanase activity was detected in the fermentation of soy flour supplemented with peptone. Acknowledgments: This work was supported by the Shota Rustaveli National Science Foundation of Georgia (Grant number STEM-22-2077).

Keywords: mushrooms, plant raw materials, fermentation, biomass protein, cellulase

Procedia PDF Downloads 78
451 Orthogonal Metal Cutting Simulation of Steel AISI 1045 via Smoothed Particle Hydrodynamic Method

Authors: Seyed Hamed Hashemi Sohi, Gerald Jo Denoga

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Machining or metal cutting is one of the most widely used production processes in industry. The quality of the process and the resulting machined product depends on parameters like tool geometry, material, and cutting conditions. However, the relationships of these parameters to the cutting process are often based mostly on empirical knowledge. In this study, computer modeling and simulation using LS-DYNA software and a Smoothed Particle Hydrodynamic (SPH) methodology, was performed on the orthogonal metal cutting process to analyze three-dimensional deformation of AISI 1045 medium carbon steel during machining. The simulation was performed using the following constitutive models: the Power Law model, the Johnson-Cook model, and the Zerilli-Armstrong models (Z-A). The outcomes were compared against the simulated results obtained by Cenk Kiliçaslan using the Finite Element Method (FEM) and the empirical results of Jaspers and Filice. The analysis shows that the SPH method combined with the Zerilli-Armstrong constitutive model is a viable alternative to simulating the metal cutting process. The tangential force was overestimated by 7%, and the normal force was underestimated by 16% when compared with empirical values. The simulation values for flow stress versus strain at various temperatures were also validated against empirical values. The SPH method using the Z-A model has also proven to be robust against issues of time-scaling. Experimental work was also done to investigate the effects of friction, rake angle and tool tip radius on the simulation.

Keywords: metal cutting, smoothed particle hydrodynamics, constitutive models, experimental, cutting forces analyses

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450 Pre-Transformation Phase Reconstruction for Deformation-Induced Transformation in AISI 304 Austenitic Stainless Steel

Authors: Manendra Singh Parihar, Sandip Ghosh Chowdhury

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Austenitic stainless steels are widely used and give a good combination of properties. When this steel is plastically deformed, a phase transformation of the metastable Face Centred Cubic Austenite to the stable Body Centred Cubic (α’) or to the Hexagonal close packed (ԑ) martensite may occur, leading to the enhancement in the mechanical properties like strength. The work was based on variant selection and corresponding texture analysis for the strain induced martensitic transformation during deformation of the parent austenite FCC phase to form the product HCP and the BCC martensite phases separately, obeying their respective orientation relationships. The automated method for reconstruction of the parent phase orientation using the EBSD data of the product phase orientation is done using the MATLAB and TSL-OIM software. The method of triplets was used which involves the formation of a triplet of neighboring product grains having a common variant and linking them using a misorientation-based criterion. This led to the proper reconstruction of the pre-transformation phase orientation data and thus to its microstructure and texture. The computational speed of current method is better compared to the previously used methods of reconstruction. The reconstruction of austenite from ԑ and α’ martensite was carried out for multiple samples and their IPF images, pole figures, inverse pole figures and ODFs were compared. Similar type of results was observed for all samples. The comparison gives the idea for estimating the correct sequence of the transformation i.e. γ → ε → α’ or γ → α’, during deformation of AISI 304 austenitic stainless steel.

Keywords: variant selection, reconstruction, EBSD, austenitic stainless steel, martensitic transformation

Procedia PDF Downloads 497
449 Seismic Loss Assessment for Peruvian University Buildings with Simulated Fragility Functions

Authors: Jose Ruiz, Jose Velasquez, Holger Lovon

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Peruvian university buildings are critical structures for which very little research about its seismic vulnerability is available. This paper develops a probabilistic methodology that predicts seismic loss for university buildings with simulated fragility functions. Two university buildings located in the city of Cusco were analyzed. Fragility functions were developed considering seismic and structural parameters uncertainty. The fragility functions were generated with the Latin Hypercube technique, an improved Montecarlo-based method, which optimizes the sampling of structural parameters and provides at least 100 reliable samples for every level of seismic demand. Concrete compressive strength, maximum concrete strain and yield stress of the reinforcing steel were considered as the key structural parameters. The seismic demand is defined by synthetic records which are compatible with the elastic Peruvian design spectrum. Acceleration records are scaled based on the peak ground acceleration on rigid soil (PGA) which goes from 0.05g to 1.00g. A total of 2000 structural models were considered to account for both structural and seismic variability. These functions represent the overall building behavior because they give rational information regarding damage ratios for defined levels of seismic demand. The university buildings show an expected Mean Damage Factor of 8.80% and 19.05%, respectively, for the 0.22g-PGA scenario, which was amplified by the soil type coefficient and resulted in 0.26g-PGA. These ratios were computed considering a seismic demand related to 10% of probability of exceedance in 50 years which is a requirement in the Peruvian seismic code. These results show an acceptable seismic performance for both buildings.

Keywords: fragility functions, university buildings, loss assessment, Montecarlo simulation, latin hypercube

Procedia PDF Downloads 144
448 Cars in a Neighborhood: A Case of Sustainable Living in Sector 22 Chandigarh

Authors: Maninder Singh

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The Chandigarh city is under the strain of exponential growth of car density across various neighborhood. The consumerist nature of society today is to be blamed for this menace because everyone wants to own and ride a car. Car manufacturers are busy selling two or more cars per household. The Regional Transport Offices are busy issuing as many licenses to new vehicles as they can in order to generate revenue in the form of Road Tax. The car traffic in the neighborhoods of Chandigarh has reached a tipping point. There needs to be a more empirical and sustainable model of cars per household, which should be based on specific parameters of livable neighborhoods. Sector 22 in Chandigarh is one of the first residential sectors to be established in the city. There is scope to think, reflect, and work out a method to know how many cars we need to sell our citizens before we lose the argument to traffic problems, parking problems, and road rage. This is where the true challenge of a planner or a designer of the city lies. Currently, in Chandigarh city, there are no clear visible answers to this problem. The way forward is to look at spatial mapping, planning, and design of car parking units to address the problem, rather than suggesting extreme measures of banning cars (short-term) or promoting plans for citywide transport (very long-term). This is a chance to resolve the problem with a pragmatic approach from a citizen’s perspective, instead of an orthodox development planner’s methodology. Since citizens are at the center of how the problem is to be addressed, acceptable solutions are more likely to emerge from the car and traffic problem as defined by the citizens. Thus, the idea and its implementation would be interesting in comparison to the known academic methodologies. The novel and innovative process would lead to a more acceptable and sustainable approach to the issue of number of car parks in the neighborhood of Chandigarh city.

Keywords: cars, Chandigarh, neighborhood, sustainable living, walkability

Procedia PDF Downloads 148
447 Effects of Microbial Biofertilization on Nodulation, Nitrogen Fixation, and Yield of Lablab purpureus

Authors: Benselama Amel, Ounane S. Mohamed, Bekki Abdelkader

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A collection of 20 isolates from fresh Nodules of the legume plant Lablab purpureus was isolated. These isolates have been authenticated by seedling inoculation grown in jars containing sand. The results obtained after two months of culture have revealed that the 20 isolates (100% of the isolates) are able to nodulate their host plants. The results obtained were analyzed statistically by ANOVA using the software statistica and had shown that the effect of the inoculation has significantly improved all the growth parameters (the height of the plant and the dry weight of the aerial parts and roots, and the number of nodules). We have evaluated the tolerance of all strains of the collection to the major stress factors as the salinity, pH and extreme temperature. The osmotolerance reached a concentration up to 1710mm of NaCl. The strains were also able to grow on a wide range of pH, ranging from 4.5 to 9.5, and temperature, between 4°C and 40°C. Also, we tested the effect of the acidity, aluminum and ferric deficit on the Lablab-rhizobia symbiosis. Lablab purpureus has not been affected by the presence of high concentrations of aluminum. On the other hand, iron deficiency has caused a net decrease in the dry biomass of the aerial part. The results of all the phenotypic characters have been treated by the statistical Minitab software, the numerical analysis had shown that these bacterial strains are divided into two distinct groups at a level of similarity of 86 %. The SDS-PAGE was carried out to determine the profile of the total protein of the strains. The coefficients of similarity of polypeptide bands between the isolates and strains reference (Bradyrhizobium, Mesorizobium sp.) confirm that our strain belongs to the groups of rhizobia.

Keywords: SDS-PAGE, rhizobia, symbiosis, phenotypic characterization, Lablab purpureus

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446 Current Epizootic Situation of Q Fever in Polish Cattle

Authors: Monika Szymańska-Czerwińska, Agnieszka Jodełko, Krzysztof Niemczuk

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Q fever (coxiellosis) is an infectious disease of animals and humans causes by C. burnetii and widely distributed throughout the world. Cattle and small ruminants are commonly known as shedders of C. burnetii. The aims of this study were the evaluation of seroprevalence and shedding of C. burnetii in cattle. Genotypes of the pathogen present in the tested specimens were also identified using MLVA (Multiple Locus Variable-Number Tandem Repeat Analysis) and MST (multispacer sequence typing) methods. Sampling was conducted in different regions of Poland in 2018-2021. In total, 2180 bovine serum samples from 801 cattle herds were tested by ELISA (enzyme-linked immunosorbent assay). 489 specimens from 157 cattle herds such as: individual milk samples (n=407), bulk tank milk (n=58), vaginal swabs (n=20), placenta (n=3) and feces (n=1) were subjected to C. burnetii specific qPCR. The qPCR (IS1111 transposon-like repetitive region) was performed using Adiavet COX RealTime PCR kit. Genotypic characterization of the strains was conducted utilizing MLVA and MST methods. MLVA was performed using 6 variable loci. The overall herd-level seroprevalence of C. burnetii infection was 36.74% (801/2180). Shedders were detected in 29.3% (46/157) cattle herds in all tested regions. ST 61 sequence type was identified in 10 out of 18 genotyped strains. Interestingly one strain represents sequence type which has never been recorded previously. MLVA method identified three previously known genotypes: most common was J but also I and BE were recognized. Moreover, a one genotype has never been described previously. Seroprevalence and shedding of C. burnetii in cattle is common and strains are genetically diverse.

Keywords: Coxiella burnetii, cattle, MST, MLVA, Q fever

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445 Resonant Auxetic Metamaterial for Automotive Applications in Vibration Isolation

Authors: Adrien Pyskir, Manuel Collet, Zoran Dimitrijevic, Claude-Henri Lamarque

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During the last decades, great efforts have been made to reduce acoustic and vibrational disturbances in transportations, as it has become a key feature for comfort. Today, isolation and design have neutralized most of the troublesome vibrations, so that cars are quieter and more comfortable than ever. However, some problems remain unsolved, in particular concerning low-frequency isolation and the frequency-dependent stiffening of materials like rubber. To sum it up, a balance has to be found between a high static stiffness to sustain the vibration source’s mass, and low dynamic stiffness, as wideband as possible. Systems meeting these criteria are yet to be designed. We thus investigated solutions inspired by metamaterials to control efficiently low-frequency wave propagation. Structures exhibiting a negative Poisson ratio, also called auxetic structures, are known to influence the propagation of waves through beaming or damping. However, their stiffness can be quite peculiar as well, as they can present regions of zero stiffness on the stress-strain curve for compression. In addition, auxetic materials can be easily adapted in many ways, inducing great tuning potential. Using finite element software COMSOL Multiphysics, a resonant design has been tested through statics and dynamics simulations. These results are compared to experimental results. In particular, the bandgaps featured by these structures are analyzed as a function of design parameters. Great stiffness properties can be observed, including low-frequency dynamic stiffness loss and broadband transmission loss. Such features are very promising for practical isolation purpose, and we hope to adopt this kind of metamaterial into an effective industrial damper.

Keywords: auxetics, metamaterials, structural dynamics, vibration isolation

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444 Clinical, Demographic and Molecular Characterization of Dengue, Chikungunya and Zika Viruses Causing Hemorrhagic Fever in North India

Authors: Suruchi Shukla, Shantanu Prakash, Amita Jain

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Introduction: Arboviral diseases are one of the most common causes of viral hemorrhagic fever (VHF). Of which, Dengue and Chikungunya pose a significant health problem in India. Arbovirus has a tendency to cross the territories and emerge in the new region. Considering the above issues, in the current study active surveillance was conducted among viral hemorrhagic fever (VHF) cases reported from Uttar Pradesh (UP), India. We studied the arboviral etiology of VHF; mainly Dengue, Chikungunya, and ZIKA. Methods: Clinical samples of 465 suspected VHF cases referred to tertiary care referral center of UP, India were enrolled in the study during a period from 15th May 2016 to 9th March 2018. Serum specimens were collected and analyzed for the presence of Dengue, Chikungunya, and ZIKA either by serology and/or by molecular assays. Results: Of all tested, 165 (35.4%) cases were positive for either Dengue or Chikungunya. Dengue (21.2%) was found to be the most prevalent, followed by Chikungunya, (6.6%). None of the cases tested positive for ZIKA virus. Serum samples of 35 (7.5%) cases were positive for both Dengue and Chikungunya. DEN-2 serotype was the most predominant serotype. Phylogenetic and sequence analysis of DEN-2 strains showed 100% clustering with the Cosmopolitan genotype strain. Bleeding from several sites, jaundice, abdominal pain, arthralgia, haemoconcentration, and thrombocytopenia were significantly higher in dengue hemorrhagic cases. However, the rash was significantly more common in Chikungunya patients. Most of the Dengue and Chikungunya positive cases (Age group 6-40 years) were seen in post monsoon season (September to November). Conclusion: Only one-third of total VHF cases are positive for either Dengue/Chikungunya or both. This necessitates the screening of other etiologies capable of causing hemorrhagic manifestations.

Keywords: viral hemorrhagic fever, dengue, chikungunya, zika, India

Procedia PDF Downloads 155
443 Development of Multifunctional Yarns and Fabrics for Interactive Textiles

Authors: Muhammad Bilal Qadir, Danish Umer, Amir Shahzad

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The use of conductive materials in smart and interactive textiles is gaining significant importance for creating value addition, innovation, and functional product development. These products find their potential applications in health monitoring, military, protection, communication, sensing, monitoring, actuation, fashion, and lifestyles. The materials which are most commonly employed in such type of interactive textile include intrinsically conducting polymers, conductive inks, and metallic coating on textile fabrics and inherently conducting metallic fibre yarns. In this study, silver coated polyester filament yarn is explored for the development of multifunctional interactive gloves. The composite yarn was developed by covering the silver coated polyester filament around the polyester spun yarn using hollow spindle technique. The electrical and tensile properties of the yarn were studied. This novel yarn was used to manufacture a smart glove to explore the antibacterial, functional, and interactive properties of the yarn. The change in electrical resistance due to finger movement at different bending positions and antimicrobial properties were studied. This glove was also found useful as an interactive tool to operate the commonly used touch screen devices due to its conductive nature. The yarn can also be used to develop the sensing elements like stretch, strain, and piezoresistive sensors. Such sensor can be effectively used in medical and sports textile for performance monitoring, vital signs monitoring and development of antibacterial textile for healthcare and hygiene.

Keywords: conductive yarn, interactive textiles, piezoresistive sensors, smart gloves

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442 Prophylactic Effects of Dairy Kluyveromyces marxianus YAS through Overexpression of BAX, CASP 3, CASP 8 and CASP 9 on Human Colon Cancer Cell Lines

Authors: Amir Saber Gharamaleki, Beitollah Alipour, Zeinab Faghfoori, Ahmad YariKhosroushahi

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Colorectal cancer (CRC) is one of the most prevalent cancers and intestinal microbial community plays an important role in colorectal tumorigenesis. Probiotics have recently been assessed as effective anti-proliferative agents and thus this study was performed to examine whether CRC undergo apoptosis by treating with isolated Iranian native dairy yeast, Kluyveromyces marxianus YAS, secretion metabolites. The cytotoxicity assessments on cells (HT-29, Caco-2) were accomplished through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as well as qualitative DAPI (4',6-diamidino-2-phenylindole staining) and quantitative (flow cytometry assessments) evaluations of apoptosis. To evaluate the main mechanism of apoptosis, Real time PCR method was applied. Kluyveromyces marxianus YAS secretions (IC50) showed significant cytotoxicity against HT-29 and Caco-2 cancer cell lines (66.57 % and 66.34 % apoptosis) similar to 5-Fluorouracil (5-FU) while apoptosis only was developed in 27.57 % of KDR normal cells. The prophylactic effects of Kluyveromyces marxianus (PTCC 5195), as a reference yeast, was not similar to Kluyveromyces marxianus YAS indicating strain dependency of bioactivities on CRC disease prevention. Based on real time PCR results, the main cytotoxicity is related to apoptosis phenomenon and the core related mechanism is depended on the overexpression of BAX, CASP 9, CASP 8 and CASP 3 inducing apoptosis genes. However, several investigations should be conducted to precisely determine the effective compounds to be used as anticancer therapeutics in the future.

Keywords: anticancer, anti-proliferative, apoptosis, cytotoxicity, yeast

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441 Mycorrhizal Autochthonous Consortium Induced Defense-Related Mechanisms of Olive Trees against Verticillium dahliae

Authors: Hanane Boutaj, Abdelilah Meddich, Said Wahbi, Zainab El Alaoui-Talibi, Allal Douira, Abdelkarim Filali-Maltouf, Cherkaoui El Modafar

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The present work aims to investigate the effect of arbuscular mycorrhizal fungi (AMF) in improving the olive tree resistance to Verticillium wilt caused by Verticillium dahliae. Inoculated plants with a mycorrhizal autochthonous consortium 'Rhizolive consortium' and pure strain 'Glomus irregulare' were infected after three months with V. dahliae. The improving of olive tree resistance was determined through disease severity, incidence, and defoliation. On the other hand, the defense mechanisms of olive plants were evaluated through lignin content, phenylalanine ammonia lyase (PAL) activity, and polyphenol content. The results revealed that both AMF significantly (p < 0.05) reduced disease development and the rate of defoliation in infected olive plants. Moreover, the contents of lignin were boosted after mycorrhizal inoculation in both the roots and the stems of olive plants, which remained significantly (p < 0.001) higher after the 90th days of V. dahliae inoculation. PAL activity was increased after V. dahliae inoculation in the stems of 'Rhizolive consortium' treatment that were 17 times higher than those in the roots of olive plants. The polyphenol content in the stems was about twice higher than those in the roots. The reduction of disease severity was accompanied by increased levels of lignin content, PAL activity, and polyphenol content, particularly in the stems of olive plants, indicating the strengthening of the olive plant immune system against V. dahliae.

Keywords: olive tree, Mycorrhizal autochthonous consortium, Glomus irregulare, Verticillium dahliae, defense mechanisms

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440 In-Vitro Dextran Synthesis and Characterization of an Intracellular Glucosyltransferase from Leuconostoc Mesenteroides AA1

Authors: Afsheen Aman, Shah Ali Ul Qader

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Dextransucrase [EC 2.4.1.5] is a glucosyltransferase that catalysis the biosynthesis of a natural biopolymer called dextran. It can catalyze the transfer of D-glucopyranosyl residues from sucrose to the main chain of dextran. This unique biopolymer has multiple applications in several industries and the key utilization of dextran lies on its molecular weight and the type of branching. Extracellular dextransucrase from Leuconostoc mesenteroides is most extensively studied and characterized. Limited data is available regarding cell-bound or intracellular dextransucrase and on the characterization of dextran produced by in-vitro reaction of intracellular dextransucrase. L. mesenteroides AA1 is reported to produce extracellular dextransucrase that catalyzes biosynthesis of a high molecular weight dextran with only α-(1→6) linkage. Current study deals with the characterization of an intracellular dextransucrase and in vitro biosynthesis of low molecular weight dextran from L. mesenteroides AA1. Intracellular dextransucrase was extracted from cytoplasm and purified to homogeneity for characterization. Kinetic constants, molecular weight and N-terminal sequence analysis of intracellular dextransucrase reveal unique variation with previously reported extracellular dextransucrase from the same strain. In vitro synthesized biopolymer was characterized using NMR spectroscopic techniques. Intracellular dextransucrase exhibited Vmax and Km values of 130.8 DSU ml-1 hr-1 and 221.3 mM, respectively. Optimum catalytic activity was detected at 35°C in 0.15 M citrate phosphate buffer (pH-5.5) in 05 minutes. Molecular mass of purified intracellular dextransucrase is approximately 220.0 kDa on SDS-PAGE. N-terminal sequence of the intracellular enzyme is: GLPGYFGVN that showed no homology with previously reported sequence for the extracellular dextransucrase. This intracellular dextransucrase is capable of in vitro synthesis of dextran under specific conditions. This intracellular dextransucrase is capable of in vitro synthesis of dextran under specific conditions and this biopolymer can be hydrolyzed into different molecular weight fractions for various applications.

Keywords: characterization, dextran, dextransucrase, leuconostoc mesenteroides

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439 Crustal Deformation Study across the Chite Fault Using GPS Measurements in North East India along the Indo Burmese Arc

Authors: Malsawmtluanga, J. Malsawma, R. P. Tiwari, V. K. Gahalaut

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North East India is seismically one of the six most active regions of the world. It is placed in Zone V, the highest zone in the seismic zonation of India. It lies at the junction of Himalayan arc to the north and the Burmese arc to the east. The region has witnessed at least 18 large earthquakes including two great earthquakes Shillong (1987, M=8.7) and the Assam Tibet border (1950, M=8.7).The prominent Chite fault lies at the heart of Aizawl, the capital of Mizoram state and this hilly city is the home to about 2 million people. Geologically the area is a part of the Indo-Burmese Wedge and is prone to natural and man-made disasters. Unplanned constructions and urban dwellings on a rapid scale have lead to numerous unsafe structures adversely affecting the ongoing development and welfare projects of the government and they pose a huge threat for earthquakes. Crustal deformation measurements using campaign mode GPS were undertaken across this fault. Campaign mode GPS data were acquired and were processed with GAMIT-GLOBK software. The study presents the current velocity estimates at all the sites in ITRF 2008 and also in the fixed Indian reference frame. The site motion showed that there appears to be no differential motion anywhere across the fault area, thus confirming presently the fault is neither accumulating strain nor slipping aseismically. From the geological and geomorphological evidence, supported by geodetic measurements, lack of historic earthquakes, the Chite fault favours aseismic behaviour in this part of the Indo Burmese Arc (IBA).

Keywords: Chite fault, crustal deformation, geodesy, GPS, IBA

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438 Effect of Built in Polarization on Thermal Properties of InGaN/GaN Heterostructures

Authors: Bijay Kumar Sahoo

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An important feature of InₓGa₁-ₓN/GaN heterostructures is strong built-in polarization (BIP) electric field at the hetero-interface due to spontaneous (sp) and piezoelectric (pz) polarizations. The intensity of this electric field reaches several MV/cm. This field has profound impact on optical, electrical and thermal properties. In this work, the effect of BIP field on thermal conductivity of InₓGa₁-ₓN/GaN heterostructure has been investigated theoretically. The interaction between the elastic strain and built in electric field induces additional electric polarization. This additional polarization contributes to the elastic constant of InₓGa₁-ₓN alloy. This in turn modifies material parameters of InₓGa₁-ₓN. The BIP mechanism enhances elastic constant, phonon velocity and Debye temperature and their bowing constants in InₓGa₁-ₓN alloy. These enhanced thermal parameters increase phonon mean free path which boost thermal conduction process. The thermal conductivity (k) of InxGa1-xN alloy has been estimated for x=0, 0.1, 0.3 and 0.9. Computation finds that irrespective of In content, the room temperature k of InₓGa₁-ₓN/GaN heterostructure is enhanced by BIP mechanism. Our analysis shows that at a certain temperature both k with and without BIP show crossover. Below this temperature k with BIP field is lower than k without BIP; however, above this temperature k with BIP field is significantly contributed by BIP mechanism leading to k with BIP field become higher than k without BIP field. The crossover temperature is primary pyroelectric transition temperature. The pyroelectric transition temperature of InₓGa₁-ₓN alloy has been predicted for different x. This signature of pyroelectric nature suggests that thermal conductivity can reveal pyroelectricity in InₓGa₁-ₓN alloy. The composition dependent room temperature k for x=0.1 and 0.3 are in line with prior experimental studies. The result can be used to minimize the self-heating effect in InₓGa₁-ₓN/GaN heterostructures.

Keywords: built-in polarization, phonon relaxation time, thermal properties of InₓGa₁-ₓN /GaN heterostructure, self-heating

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437 Species Profiling of Scarab Beetles with the Help of Light Trap in Western Himalayan Region of Uttarakhand

Authors: Ajay Kumar Pandey

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White grub (Coleoptera: Scarabaeidae), locally known as Kurmula, Pagra, Chinchu, is a major destructive pest in western Himalayan region of Uttarakhand state of India. Various crops like cereals (up land paddy, wheat, and barley), vegetables (capsicum, cabbage, tomato, cauliflower, carrot etc) and some pulse (like pigeon pea, green gram, black gram) are grown with limited availability of primary resources. Among the various limitations in successful cultivation of these crops, white grub has been proved a major constraint in for all crops grown in hilly area. The losses incurred due to white grubs are huge in case of commercial crops like sugarcane, groundnut, potato, maize and upland rice. Moreover, it has been proved major constraint in potato production in mid and higher hills of India. Adults emerge in May-June following the onset of monsoon and thereafter defoliate the apple, apricot, plum, and walnut during night while 2nd and 3rd instar grubs feed on live roots of cultivated as well as non cultivated crops from August to January. Survey was conducted in hilly (Pauri and Tehri) as well as plain area (Haridwar district) of Uttarakhand state. Collection of beetle was done from various locations from August to September of five consecutive years with the help of light trap and directly from host plant. The grub was also collected by excavating one square meter area from different locations and reared in laboratory to find out adult. During the collection, the diseased or dead cadaver were also collected and brought in the laboratory and identified the causal organisms. Total 25 species of white grub was identified out of which Holotrichia longipennis, Anomala dimidiata, Holotrichia lineatopennis, Maladera insanabilis, Brahmina sp. make complex problem in different area of Uttarakhand where they cause severe damage to various crops. During the survey, it was observed that white grubs beetles have variation in preference of host plant, even in choice of fruit and leaves of host plant. It was observed that, a white grub species, which identified as Lepidiota mansueta Burmeister., was causing severe havoc to sugarcane crop grown in major sugarcane growing belt of Haridwar district. The study also revealed that Bacillus cereus, Beauveria bassiana, Metarhizium anisopliae, Steinernema, Heterorhabditis are major disease causing agents in immature stage of white grub under rain-fed condition of Uttarakhand which caused 15.55 to 21.63 percent natural mortality of grubs with an average of 18.91 percent. However, among the microorganisms, B. cereus found to be significantly more efficient (7.03 percent mortality) then the entomopathogenic fungi (3.80 percent mortality) and nematodes (3.20 percent mortality).

Keywords: Lepidiota, profiling, Uttarakhand, whitegrub

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436 Perspectives of charitable organisations on the impact of the COVID-19 pandemic on family carers of people with profound and multiple intellectual disabilities.

Authors: Mark Linden, Trisha Forbes, Michael Brown, Lynne Marsh, Maria Truesdale, Stuart Todd, Nathan Hughes

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Background The COVID-19 pandemic resulted in a reduction of health care services for many family carers of people with profound and multiple intellectual disabilities (PMID). Due to lack of services, family carers turned to charities for support during the pandemic. We explored the views of charity workers across the UK and Ireland who supported family carers during the COVID-19 pandemic and explored their views on effective online support programmes for family carers. Methods This was a qualitative study using online focus groups with participants (n = 24) from five charities across the UK and Ireland. Questions focused on challenges, supports, coping and resources which helped during lockdown restrictions. Focus groups were audio recorded, transcribed verbatim, and analysed through thematic analysis. Findings Four themes were identified (i) ‘mental and emotional health’, (ii) ‘they who shout the loudest’ (fighting for services), (iii) ‘lack of trust in statutory services’ and (iv) ‘creating an online support programme’. Mental and emotional health emerged as the most prominent theme and included three subthemes named as ‘isolation’, ‘fear of COVID-19’ and ‘the exhaustion of caring’. Conclusions The withdrawal of many services during the COVID-19 pandemic further isolated and placed strain on family carers. Even after the end of the pandemic family cares continue to report on the struggle to receive adequate support. There is a critical need to design services, including online support programmes, in partnership with family carers which adequately address their needs.

Keywords: intellectual disability, family carers, COVID-19, charities

Procedia PDF Downloads 74
435 Histopathological Examination of BALB/C Mice Receiving Strains of Acinetobacter baumannii Resistant to Colistin Antibiotic

Authors: Shahriar Sepahvand, Mohammad Ali Davarpanah

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Infections caused by Acinetobacter baumannii are among the common hospital-acquired infections that have seen an increase in antibiotic resistance in recent years. Colistin is the last treatment option against this pathogen. The aim of this study is to investigate the histopathology of BALB/C mice receiving sensitive and resistant strains of Acinetobacter baumannii to colistin. A total of 68 female laboratory mice weighing 30 to 40 grams of the BALB/C breed were studied in this research for three weeks under appropriate laboratory conditions in terms of food and environment. The experimental groups included: control group, second group, third group, fourth group. Lung, liver, spleen, and kidney tissues were removed from anesthetized mice and, after washing in physiological serum, were fixed in 10% formalin for 14 days. For dehydration, alcohol with ascending degrees of 70, 80, 90, and 100 was used. After clearing and soaking in paraffin, the samples were embedded in paraffin. Then, sections with a thickness of 5 microns were prepared and, after staining by hematoxylin-eosin, the samples were ready for study with a light microscope. In liver, spleen, lung, and kidney tissues of mice receiving the colistin-sensitive strain of Acinetobacter baumannii, infiltration of inflammatory cells and hyperemia were observed compared to control group mice. Liver and lung tissues of mice receiving strains of Acinetobacter baumannii resistant to colistin showed tissue destruction in addition to infiltration of inflammatory cells and hyperemia, with more destruction observed in lung tissue.

Keywords: acinetobacter baumannii, colistin antibiotic, histopathological examination, resistant

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434 Process Development of pVAX1/lacZ Plasmid DNA Purification Using Design of Experiment

Authors: Asavasereerat K., Teacharsripaitoon T., Tungyingyong P., Charupongrat S., Noppiboon S. Hochareon L., Kitsuban P.

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Third generation of vaccines is based on gene therapy where DNA is introduced into patients. The antigenic or therapeutic proteins encoded from transgenes DNA triggers an immune-response to counteract various diseases. Moreover, DNA vaccine offers the customization of its ability on protection and treatment with high stability. The production of DNA vaccines become of interest. According to USFDA guidance for industry, the recommended limits for impurities from host cell are lower than 1%, and the active conformation homogeneity supercoiled DNA, is more than 80%. Thus, the purification strategy using two-steps chromatography has been established and verified for its robustness. Herein, pVax1/lacZ, a pre-approved USFDA DNA vaccine backbone, was used and transformed into E. coli strain DH5α. Three purification process parameters including sample-loading flow rate, the salt concentration in washing and eluting buffer, were studied and the experiment was designed using response surface method with central composite face-centered (CCF) as a model. The designed range of selected parameters was 10% variation from the optimized set point as a safety factor. The purity in the percentage of supercoiled conformation obtained from each chromatography step, AIEX and HIC, were analyzed by HPLC. The response data were used to establish regression model and statistically analyzed followed by Monte Carlo simulation using SAS JMP. The results on the purity of the product obtained from AIEX and HIC are between 89.4 to 92.5% and 88.3 to 100.0%, respectively. Monte Carlo simulation showed that the pVAX1/lacZ purification process is robust with confidence intervals of 0.90 in range of 90.18-91.00% and 95.88-100.00%, for AIEX and HIC respectively.

Keywords: AIEX, DNA vaccine, HIC, puification, response surface method, robustness

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433 Influence of BaTiO₃ on the Biological Behaviour of Hydroxyapatite: Collagen Composites

Authors: Cristina Busuioc, Georgeta Voicu, Sorin-Ion Jinga

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The human bone presents in its dry form piezoelectric properties, which means that a mechanical stress results in electric polarization and an applied electric field causes strain. The immediate consequence was the revealing of piezoelectricity role in bone remodelling, as well as the integration of ceramic materials with piezoelectric behaviour in the composition of unitary or composite biomaterials. Thus, we prepared hydroxyapatite - collagen hybrid materials with barium titanate addition in order to achieve a better osseointegration. Barium titanate powder synthesized by a combined sol-gel-hydrothermal method, commercial hydroxyapatite and laboratory extracted collagen gel were employed as starting materials. Before the composites, fabrication, the powder with piezoelectric features was characterized in detail from the compositional, structural, morphological and electrical point of view. The next step was to elucidate the influence of barium titanate presence especially on the biological properties of the final materials. The biocompatibility of the hybrid supports without or with piezoelectric addition was investigated on mouse osteoblast cells through LDH cytotoxicity assay, LIVE/DEAD cell viability assay, and MTT cell proliferation assay. All results indicated that the analysed materials do not exert cytotoxic effects and present the ability to sustain cell survival and to promote their proliferation. In conclusion, barium titanate nanoparticles exhibit a good biocompatibility and osteoinductive properties, while the derived composite materials based on hydroxyapatite as oxide phase and collagen as polymeric phase can be successfully used for tissue engineering applications.

Keywords: barium titanate, hybrid composites, piezoelectricity, tissue engineering

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432 Expression of Tissue Plasminogen Activator in Transgenic Tobacco Plants by Signal Peptides Targeting for Delivery to Apoplast, Endoplasmic Reticulum and Cytosol Spaces

Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

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Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA4404. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.

Keywords: tPA, recombinant, transgenic, tobacco

Procedia PDF Downloads 145
431 The Effect of Shredded Polyurethane Foams on Shear Modulus and Damping Ratio of Sand

Authors: Javad Saeidaskari, Nader Khalafian

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The undesirable impact of vibrations induced by road and railway traffic is an important concern in modern world. These vibrations are transmitted through soil and cause disturbances to the residence area and high-tech production facilities alongside the train/traffic lines. In this paper for the first time a new method of soil improvement with vibration absorber material, is used to increase the damping factor, in other word, to reduce the ability of wave transitions in sand. In this study standard Firoozkooh No. 161 sand is used as the host sand. The semi rigid polyurethane (PU) foam which used in this research is one of the common materials for vibration absorbing purposes. Series of cyclic triaxial tests were conducted on remolded samples with identical relative density of 70% of maximum dry density for different volume percentage of shredded PU foam. The frequency of tests was 0.1 Htz with shear strain of 0.37% and 0.75% and also the effective confining pressures during the tests were 100 kPa and 350 kPa. In order to find out the best soil-PU foam mixture, different volume percent of PU foam varying from 10% to 30% were examined. The results show that adding PU foam up to 20%, as its optimum content, causes notable enhancement in damping ratio for both shear strains of 0.37% (52.19% and 69% increase for effective confining pressures of 100 kPa and 350 kPa, respectively) and 0.75% (59.56% and 59.11% increase for effective confining pressures of 100 kPa and 350 kPa, respectively). The results related to shear modulus present significant reduction for both shear strains of 0.37% (82.22% and 56.03% decrease for effective confining pressures of 100 kPa and 350 kPa, respectively) and 0.75% (89.32% and 39.9% decrease for effective confining pressures of 100 kPa and 350 kPa, respectively). In conclusion, shredded PU foams effectively affect the dynamic properties of sand and act as vibration absorber in soil.

Keywords: polyurethane foam, sand, damping ratio, shear modulus

Procedia PDF Downloads 449
430 Fatigue Life Prediction under Variable Loading Based a Non-Linear Energy Model

Authors: Aid Abdelkrim

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A method of fatigue damage accumulation based upon application of energy parameters of the fatigue process is proposed in the paper. Using this model is simple, it has no parameter to be determined, it requires only the knowledge of the curve W–N (W: strain energy density N: number of cycles at failure) determined from the experimental Wöhler curve. To examine the performance of nonlinear models proposed in the estimation of fatigue damage and fatigue life of components under random loading, a batch of specimens made of 6082 T 6 aluminium alloy has been studied and some of the results are reported in the present paper. The paper describes an algorithm and suggests a fatigue cumulative damage model, especially when random loading is considered. This work contains the results of uni-axial random load fatigue tests with different mean and amplitude values performed on 6082T6 aluminium alloy specimens. The proposed model has been formulated to take into account the damage evolution at different load levels and it allows the effect of the loading sequence to be included by means of a recurrence formula derived for multilevel loading, considering complex load sequences. It is concluded that a ‘damaged stress interaction damage rule’ proposed here allows a better fatigue damage prediction than the widely used Palmgren–Miner rule, and a formula derived in random fatigue could be used to predict the fatigue damage and fatigue lifetime very easily. The results obtained by the model are compared with the experimental results and those calculated by the most fatigue damage model used in fatigue (Miner’s model). The comparison shows that the proposed model, presents a good estimation of the experimental results. Moreover, the error is minimized in comparison to the Miner’s model.

Keywords: damage accumulation, energy model, damage indicator, variable loading, random loading

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429 Assessing Storage of Stability and Mercury Reduction of Freeze-Dried Pseudomonas putida within Different Types of Lyoprotectant

Authors: A. A. M. Azoddein, Y. Nuratri, A. B. Bustary, F. A. M. Azli, S. C. Sayuti

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Pseudomonas putida is a potential strain in biological treatment to remove mercury contained in the effluent of petrochemical industry due to its mercury reductase enzyme that able to reduce ionic mercury to elementary mercury. Freeze-dried P. putida allows easy, inexpensive shipping, handling and high stability of the product. This study was aimed to freeze dry P. putida cells with addition of lyoprotectant. Lyoprotectant was added into the cells suspension prior to freezing. Dried P. putida obtained was then mixed with synthetic mercury. Viability of recovery P. putida after freeze dry was significantly influenced by the type of lyoprotectant. Among the lyoprotectants, tween 80/ sucrose was found to be the best lyoprotectant. Sucrose able to recover more than 78% (6.2E+09 CFU/ml) of the original cells (7.90E+09CFU/ml) after freeze dry and able to retain 5.40E+05 viable cells after 4 weeks storage in 4oC without vacuum. Polyethylene glycol (PEG) pre-treated freeze dry cells and broth pre-treated freeze dry cells after freeze-dry recovered more than 64% (5.0 E+09CFU/ml) and >0.1% (5.60E+07CFU/ml). Freeze-dried P. putida cells in PEG and broth cannot survive after 4 weeks storage. Freeze dry also does not really change the pattern of growth P. putida but extension of lag time was found 1 hour after 3 weeks of storage. Additional time was required for freeze-dried P. putida cells to recover before introduce freeze-dried cells to more complicated condition such as mercury solution. The maximum mercury reduction of PEG pre-treated freeze-dried cells after freeze dry and after storage 3 weeks was 56.78% and 17.91%. The maximum of mercury reduction of tween 80/sucrose pre-treated freeze-dried cells after freeze dry and after storage 3 weeks were 26.35% and 25.03%. Freeze dried P. putida was found to have lower mercury reduction compare to the fresh P. putida that has been growth in agar. Result from this study may be beneficial and useful as initial reference before commercialize freeze-dried P. putida.

Keywords: Pseudomonas putida, freeze-dry, PEG, tween80/Sucrose, mercury, cell viability

Procedia PDF Downloads 355
428 Variant Selection and Pre-transformation Phase Reconstruction for Deformation-Induced Transformation in AISI 304 Austenitic Stainless Steel

Authors: Manendra Singh Parihar, Sandip Ghosh Chowdhury

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Austenitic stainless steels are widely used and give a good combination of properties. When this steel is plastically deformed, a phase transformation of the metastable Face Centred Cubic Austenite to the stable Body Centred Cubic (α’) or to the Hexagonal close packed (ԑ) martensite may occur, leading to the enhancement in the mechanical properties like strength. The work was based on variant selection and corresponding texture analysis for the strain induced martensitic transformation during deformation of the parent austenite FCC phase to form the product HCP and the BCC martensite phases separately, obeying their respective orientation relationships. The automated method for reconstruction of the parent phase orientation using the EBSD data of the product phase orientation is done using the MATLAB and TSL-OIM software. The method of triplets was used which involves the formation of a triplet of neighboring product grains having a common variant and linking them using a misorientation-based criterion. This led to the proper reconstruction of the pre-transformation phase orientation data and thus to its micro structure and texture. The computational speed of current method is better compared to the previously used methods of reconstruction. The reconstruction of austenite from ԑ and α’ martensite was carried out for multiple samples and their IPF images, pole figures, inverse pole figures and ODFs were compared. Similar type of results was observed for all samples. The comparison gives the idea for estimating the correct sequence of the transformation i.e. γ → ε → α’ or γ → α’, during deformation of AISI 304 austenitic stainless steel.

Keywords: variant selection, reconstruction, EBSD, austenitic stainless steel, martensitic transformation

Procedia PDF Downloads 489
427 Analysis of Taxonomic Compositions, Metabolic Pathways and Antibiotic Resistance Genes in Fish Gut Microbiome by Shotgun Metagenomics

Authors: Anuj Tyagi, Balwinder Singh, Naveen Kumar B. T., Niraj K. Singh

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Characterization of diverse microbial communities in specific environment plays a crucial role in the better understanding of their functional relationship with the ecosystem. It is now well established that gut microbiome of fish is not the simple replication of microbiota of surrounding local habitat, and extensive species, dietary, physiological and metabolic variations in fishes may have a significant impact on its composition. Moreover, overuse of antibiotics in human, veterinary and aquaculture medicine has led to rapid emergence and propagation of antibiotic resistance genes (ARGs) in the aquatic environment. Microbial communities harboring specific ARGs not only get a preferential edge during selective antibiotic exposure but also possess the significant risk of ARGs transfer to other non-resistance bacteria within the confined environments. This phenomenon may lead to the emergence of habitat-specific microbial resistomes and subsequent emergence of virulent antibiotic-resistant pathogens with severe fish and consumer health consequences. In this study, gut microbiota of freshwater carp (Labeo rohita) was investigated by shotgun metagenomics to understand its taxonomic composition and functional capabilities. Metagenomic DNA, extracted from the fish gut, was subjected to sequencing on Illumina NextSeq to generate paired-end (PE) 2 x 150 bp sequencing reads. After the QC of raw sequencing data by Trimmomatic, taxonomic analysis by Kraken2 taxonomic sequence classification system revealed the presence of 36 phyla, 326 families and 985 genera in the fish gut microbiome. At phylum level, Proteobacteria accounted for more than three-fourths of total bacterial populations followed by Actinobacteria (14%) and Cyanobacteria (3%). Commonly used probiotic bacteria (Bacillus, Lactobacillus, Streptococcus, and Lactococcus) were found to be very less prevalent in fish gut. After sequencing data assembly by MEGAHIT v1.1.2 assembler and PROKKA automated analysis pipeline, pathway analysis revealed the presence of 1,608 Metacyc pathways in the fish gut microbiome. Biosynthesis pathways were found to be the most dominant (51%) followed by degradation (39%), energy-metabolism (4%) and fermentation (2%). Almost one-third (33%) of biosynthesis pathways were involved in the synthesis of secondary metabolites. Metabolic pathways for the biosynthesis of 35 antibiotic types were also present, and these accounted for 5% of overall metabolic pathways in the fish gut microbiome. Fifty-one different types of antibiotic resistance genes (ARGs) belonging to 15 antimicrobial resistance (AMR) gene families and conferring resistance against 24 antibiotic types were detected in fish gut. More than 90% ARGs in fish gut microbiome were against beta-lactams (penicillins, cephalosporins, penems, and monobactams). Resistance against tetracycline, macrolides, fluoroquinolones, and phenicols ranged from 0.7% to 1.3%. Some of the ARGs for multi-drug resistance were also found to be located on sequences of plasmid origin. The presence of pathogenic bacteria and ARGs on plasmid sequences suggested the potential risk due to horizontal gene transfer in the confined gut environment.

Keywords: antibiotic resistance, fish gut, metabolic pathways, microbial diversity

Procedia PDF Downloads 144
426 Inclusion Body Refolding at High Concentration for Large-Scale Applications

Authors: J. Gabrielczyk, J. Kluitmann, T. Dammeyer, H. J. Jördening

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High-level expression of proteins in bacteria often causes production of insoluble protein aggregates, called inclusion bodies (IB). They contain mainly one type of protein and offer an easy and efficient way to get purified protein. On the other hand, proteins in IB are normally devoid of function and therefore need a special treatment to become active. Most refolding techniques aim at diluting the solubilizing chaotropic agents. Unfortunately, optimal refolding conditions have to be found empirically for every protein. For large-scale applications, a simple refolding process with high yields and high final enzyme concentrations is still missing. The constructed plasmid pASK-IBA63b containing the sequence of fructosyltransferase (FTF, EC 2.4.1.162) from Bacillus subtilis NCIMB 11871 was transformed into E. coli BL21 (DE3) Rosetta. The bacterium was cultivated in a fed-batch bioreactor. The produced FTF was obtained mainly as IB. For refolding experiments, five different amounts of IBs were solubilized in urea buffer with protein concentration of 0.2-8.5 g/L. Solubilizates were refolded with batch or continuous dialysis. The refolding yield was determined by measuring the protein concentration of the clear supernatant before and after the dialysis. Particle size was measured by dynamic light scattering. We tested the solubilization properties of fructosyltransferase IBs. The particle size measurements revealed that the solubilization of the aggregates is achieved at urea concentration of 5M or higher and confirmed by absorption spectroscopy. All results confirm previous investigations that refolding yields are dependent upon initial protein concentration. In batch dialysis, the yields dropped from 67% to 12% and 72% to 19% for continuous dialysis, in relation to initial concentrations from 0.2 to 8.5 g/L. Often used additives such as sucrose and glycerol had no effect on refolding yields. Buffer screening indicated a significant increase in activity but also temperature stability of FTF with citrate/phosphate buffer. By adding citrate to the dialysis buffer, we were able to increase the refolding yields to 82-47% in batch and 90-74% in the continuous process. Further experiments showed that in general, higher ionic strength of buffers had major impact on refolding yields; doubling the buffer concentration increased the yields up to threefold. Finally, we achieved corresponding high refolding yields by reducing the chamber volume by 75% and the amount of buffer needed. The refolded enzyme had an optimal activity of 12.5±0.3 x104 units/g. However, detailed experiments with native FTF revealed a reaggregation of the molecules and loss in specific activity depending on the enzyme concentration and particle size. For that reason, we actually focus on developing a process of simultaneous enzyme refolding and immobilization. The results of this study show a new approach in finding optimal refolding conditions for inclusion bodies at high concentrations. Straightforward buffer screening and increase of the ionic strength can optimize the refolding yield of the target protein by 400%. Gentle removal of chaotrope with continuous dialysis increases the yields by an additional 65%, independent of the refolding buffer applied. In general time is the crucial parameter for successful refolding of solubilized proteins.

Keywords: dialysis, inclusion body, refolding, solubilization

Procedia PDF Downloads 294
425 Levansucrase from Zymomonas Mobilis KIBGE-IB14: Production Optimization and Characterization for High Enzyme Yield

Authors: Sidra Shaheen, Nadir Naveed Siddiqui, Shah Ali Ul Qader

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In recent years, significant progress has been made in discovering and developing new bacterial polysaccharides producing organisms possessing extremely functional properties. Levan is a natural biopolymer of fructose which is produced by transfructosylation reaction in the presence of levansucrase. It is one of the industrially promising enzymes that offer a variety of industrial applications in the field of cosmetics, foods and pharmaceuticals. Although levan has significant applications but the yield of levan produced is not equal to other biopolymers due to the inefficiency of producer microorganism. Among wide range of levansucrase producing microorganisms, Zymomonas mobilis is considered as a potential candidate for large scale production of this natural polysaccharide. The present investigation is concerned with the isolation of levansucrase producing natural isolate having maximum enzyme production. Furthermore, production parameters were optimized to get higher enzyme yield. Levansucrase was partially purified and characterized to study its applicability on industrial scale. The results of this study revealed that the bacterial strain Z. mobilis KIBGE-IB14 was the best producer of levansucrase. Bacterial growth and enzyme production was greatly influenced by physical and chemical parameters. Maximum levansucrase production was achieved after 24 hours of fermentation at 30°C using modified medium of pH-6.5. Contrary to other levansucrases, the one presented in the current study is able to produce high amount of products in relatively short period of time with optimum temperature at 35°C. Due to these advantages, this enzyme can be used on large scale for commercial production of levan and other important metabolites.

Keywords: levansucrase, metabolites, polysaccharides, transfructosylation

Procedia PDF Downloads 497