Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 19

Search results for: Tsuyoshi Imai

19 High Efficient Biohydrogen Production from Cassava Starch Processing Wastewater by Two Stage Thermophilic Fermentation and Electrohydrogenesis

Authors: Peerawat Khongkliang, Prawit Kongjan, Tsuyoshi Imai, Poonsuk Prasertsan, Sompong O-Thong

Abstract:

A two-stage thermophilic fermentation and electrohydrogenesis process was used to convert cassava starch processing wastewater into hydrogen gas. Maximum hydrogen yield from fermentation stage by Thermoanaerobacterium thermosaccharolyticum PSU-2 was 248 mL H2/g-COD at optimal pH of 6.5. Optimum hydrogen production rate of 820 mL/L/d and yield of 200 mL/g COD was obtained at HRT of 2 days in fermentation stage. Cassava starch processing wastewater fermentation effluent consisted of acetic acid, butyric acid and propionic acid. The effluent from fermentation stage was used as feedstock to generate hydrogen production by microbial electrolysis cell (MECs) at an applied voltage of 0.6 V in second stage with additional 657 mL H2/g-COD was produced. Energy efficiencies based on electricity needed for the MEC were 330 % with COD removals of 95 %. The overall hydrogen yield was 800-900 mL H2/g-COD. Microbial community analysis of electrohydrogenesis by DGGE shows that exoelectrogens belong to Acidiphilium sp., Geobacter sulfurreducens and Thermincola sp. were dominated at anode. These results show two-stage thermophilic fermentation, and electrohydrogenesis process improved hydrogen production performance with high hydrogen yields, high gas production rates and high COD removal efficiency.

Keywords: cassava starch processing wastewater, biohydrogen, thermophilic fermentation, microbial electrolysis cell

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18 Metal Berthelot Tubes with Windows for Observing Cavitation under Static Negative Pressure

Authors: K. Hiro, Y. Imai, T. Sasayama

Abstract:

Cavitation under static negative pressure is not revealed well. The Berthelot method to generate such negative pressure can be a means to study cavitation inception. In this study, metal Berthelot tubes built in observation windows are newly developed and are checked whether high static negative pressure is generated or not. Negative pressure in the tube with a pair of a corundum plate and an aluminum gasket increased with temperature cycles. The trend was similar to that as reported before.

Keywords: Berthelot method, cavitation, negative pressure, observation

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17 Social Innovation, Change and the Future of Resilient Communities in Tokyo

Authors: Heide Imai

Abstract:

The paper will introduce and discuss specific examples of urban practices which take place within the dynamic urban landscape of contemporary Tokyo. The rising interest and importance of derelict places as resilient and creative clusters will be analysed, before relating this to the rediscovery of small urban niches and the emergence of different forms of social entrepreneurs. Secondly, two different case study areas will be introduced before discussing different forms of hybrid lifestyles, social micro scale enterprises and social innovations, understanding the concept of ‘small places of resilience’ as zones of human interaction, desire and care in which spontaneous practices take place.

Keywords: entrepreneurship, social innovation, Tokyo, urban regeneration

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16 Negative Pressures of Ca. -20 MPA for Water Enclosed into a Metal Berthelot Tube under a Vacuum Condition

Authors: K. Hiro, Y. Imai, M. Tanji, H. Deguchi, K. Hatari

Abstract:

Negative pressures of liquids have been expected to contribute many kinds of technology. Nevertheless, experiments for subjecting liquids which have not too small volumes to negative pressures are difficult even now. The reason of the difficulties is because the liquids tend to generate cavities easily. In order to remove cavitation nuclei, an apparatus for enclosing water into a metal Berthelot tube under vacuum conditions was developed. By using the apparatus, negative pressures for water rose to ca. -20 MPa. This is the highest value for water in metal Berthelot tubes. Results were explained by a traditional crevice model. Keywords

Keywords: Berthelot method, negative pressure, cavitation nuclei, water

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15 Utility Analysis of API Economy Based on Multi-Sided Platform Markets Model

Authors: Mami Sugiura, Shinichi Arakawa, Masayuki Murata, Satoshi Imai, Toru Katagiri, Motoyoshi Sekiya

Abstract:

API (Application Programming Interface) economy, where many participants join/interact and form the economy, is expected to increase collaboration between information services through API, and thereby, it is expected to increase market value from the service collaborations. In this paper, we introduce API evaluators, which are the activator of API economy by reviewing and/or evaluating APIs, and develop a multi-sided API economy model that formulates interactions among platform provider, API developers, consumers, and API evaluators. By obtaining the equilibrium that maximizes utility of all participants, the impact of API evaluators on the utility of participants in the API economy is revealed. Numerical results show that, with the existence of API evaluators, the number of developers and consumers increase by 1.5% and the utility of platformer increases by 2.3%. We also discuss the strategies of platform provider to maximize its utility under the existence of API evaluators.

Keywords: API economy, multi-sided markets, API evaluator, platform, platform provider

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14 Nitrogen-Doped Ultrananocrystalline Diamond/Hydrogenated Amorphous Carbon Composite Films Prepared by Coaxial Arc Plasma Deposition

Authors: Abdelrahman Zkria, Tsuyoshi Yoshitake

Abstract:

Diamond is one of the most interesting semiconducting carbon materials owing to its unique physical and chemical properties, yet its application in electronic devices is limited due to the difficulty of realizing n-type conduction by nitrogen doping. In contrast Ultrananocrystalline diamond with diamond grains of about 3–5 nm in diameter have attracted much attention for device-oriented applications because they may enable the realization of n-type doping with nitrogen. In this study, nitrogen-doped Ultra-Nanocrystalline diamond films were prepared by coaxial arc plasma deposition (CAPD) method, the nitrogen content was estimated by X-ray photoemission spectroscopy (XPS). The electrical conductivity increased with increasing nitrogen contents. Heterojunction diodes with p-type Si were fabricated and evaluated based on current–voltage (I–V) and capacitance–voltage (C–V) characteristics measured in dark at room temperature.

Keywords: heterojunction diodes, hopping conduction mechanism, nitrogen-doping, ultra-nanocrystalline diamond

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13 Aerodynamic Bicycle Torque Augmentation with a Wells Turbine in Wheels

Authors: Tsuyoshi Yamazaki, Etsuo Morishita

Abstract:

Cyclists often run through a crosswind and sometimes we experience the adverse pressure. We came to an idea that Wells turbine can be used as power augmentation device in the crosswind something like sails of a yacht. Wells turbine always rotates in the same direction irrespective of the incoming flow direction, and we use it in the small-scale power generation in the ocean where waves create an oscillating flow. We incorporate the turbine to the wheel of a bike. A commercial device integrates strain gauges in the crank of a bike and transmitted force and torque applied to the pedal of the bike as an e-mail to the driver’s mobile phone. We can analyze the unsteady data in a spreadsheet sent from the crank sensor. We run the bike with the crank sensor on the rollers at the exit of a low-speed wind tunnel and analyze the effect of the crosswind to the wheel with a Wells turbine. We also test the aerodynamic characteristics of the turbine separately. Although power gain depends on the flow direction, several Watts increase might be possible by the Wells turbine incorporated to a bike wheel.

Keywords: aerodynamics, Wells turbine, bicycle, wind engineering

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12 CO2 Gas Solubility and Foam Generation

Authors: Chanmoly Or, Kyuro Sasaki, Yuichi Sugai, Masanori Nakano, Motonao Imai

Abstract:

Cold drainage mechanism of oil production is a complicated process which involves with solubility and foaming processes. Laboratory experiments were carried out to investigate the CO2 gas solubility in hexadecane (as light oil) and the effect of depressurization processes on microbubble generation. The experimental study of sensitivity parameters of temperature and pressure on CO2 gas solubility in hexadecane was conducted at temperature of 20 °C and 50 °C and pressure ranged 2.0–7.0 MPa by using PVT (RUSKA Model 2370) apparatus. The experiments of foamy hexadecane were also prepared by depressurizing from saturated pressure of 6.4 MPa and temperature of 50 °C. The experimental results show the CO2 gas solubility in hexadecane linearly increases with increasing pressure. At pressure 4.5 MPa, CO2 gas dissolved in hexadecane 2.5 mmol.g-1 for temperature of 50 °C and 3.5 mmol.g-1 for temperature of 20 °C. The bubbles of foamy hexadecane were observed that most of large bubbles were coalesced shortly whereas the small one keeps presence. The experimental result of foamy hexadecane indicated large depressurization step (∆P) produces high quality of foam with high microbubble distribution.

Keywords: CO2 gas solubility, depressurization process, foamy hexadecane, microbubble distribution

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11 Aerodynamic Devices Development for Model Aircraft Control and Wind-Driven Bicycle

Authors: Yuta Moriyama, Tsuyoshi Yamazaki, Etsuo Morishita

Abstract:

Several aerodynamic devices currently attract engineers and research students. The plasma actuator is one of them, and it is very effective to control the flow. The actuator recovers a separated flow to an attached one. The actuator is also inversely applied to a spoiler. The model aircraft might be controlled by this actuator. We develop a model aircraft with the plasma actuator. Another interesting device is the Wells turbine which rotates in one direction. The present authors propose a bicycle with the Wells turbine in the wheels. Power reduction is measured when the turbine is driven by an electric motor at the exit of a wind tunnel. Several Watts power reduction might be possible. This means that the torque of the bike can be augmented by the turbine in the cross wind. These devices are tested in the wind tunnel with a three-component balance and the aerodynamic forces and moment are obtained. In this paper, we introduce these devices and their aerodynamic characteristics. The control force and moment of the plasma actuator are clarified and the power reduction of the bicycle is quantified.

Keywords: aerodynamics, model aircraft, plasma actuator, Wells turbine

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10 Surface Characteristics of Bacillus megaterium and Its Adsorption Behavior onto Dolomite

Authors: Mohsen Farahat, Tsuyoshi Hirajima

Abstract:

Surface characteristics of Bacillus megaterium strain were investigated; zeta potential, FTIR and contact angle were measured. Surface energy components including Lifshitz-van der Waals, Hamaker constant, and acid/base components (Lewis acid/Lewis base) were calculated from the contact angle data. The results showed that the microbial cells were negatively charged over all pH regions with high values at alkaline region. A hydrophilic nature for the strain was confirmed by contact angle and free energy of adhesion between microbial cells. Adsorption affinity of the strain toward dolomite was studied at different pH values. The results showed that the cells had a high affinity to dolomite at acid pH comparing to neutral and alkaline pH. Extended DLVO theory was applied to calculate interaction energy between B. megaterium cells and dolomite particles. The adsorption results were in agreement with the results of Extended DLVO approach. Surface changes occurred on dolomite surface after the bio-treatment were monitored; contact angle decreased from 69° to 38° and the mineral’s floatability decreased from 95% to 25% after the treatment.

Keywords: Bacillus megaterium, surface modification, flotation, dolomite, adhesion energy

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9 Genetic Characteristics of Chicken Anemia Virus Circulating in Northern Vietnam

Authors: Hieu Van Dong, Giang Thi Huong Tran, Giap Van Nguyen, Tung Duy Dao, Vuong Nghia Bui, Le Thi My Huynh, Yohei Takeda, Haruko Ogawa, Kunitoshi Imai

Abstract:

Chicken anemia virus (CAV) has a ubiquitous and worldwide distribution in chicken production. Our group previously reported high seroprevalence of CAV in chickens in northern Vietnam. In the present study, 330 tissue samples collected from commercial and breeder chicken farms in eleven provinces in northern Vietnam were tested for the CAV infection. We found that 157 out of 330 (47.58%) chickens were positive with CAV genes by real-time PCR method. Nine CAV strains obtained from the different location and time were forwarded to the full-length sequence of CAV VP1 gene. Phylogenetic analysis of the Vietnamese CAV vp1 gene indicated that the CAVs circulating in northern Vietnam were divided into three distinct genotypes, II, III, and V, but not clustered with the vaccine strains. Among the three genotypes, genotype III was the major one widely spread in Vietnam, and that included three sub-genotypes, IIIa, IIIb, and IIIc. The Vietnamese CAV strains were closely related to the Chinese, Taiwanese, and USA strains. All the CAV isolates had glutamine at amino acid position 394 in the VP1 gene, suggesting that they might be highly pathogenic strains. One strain was defined to be genotype V, which had not been reported for Vietnamese CAVs. Additional studies are required to further evaluate the pathogenicity of CAV strains circulating in Vietnam.

Keywords: chicken anemia virus, genotype, genetic characteristics, Vietnam

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8 Effect of Chromium Behavior on Mechanical and Electrical Properties Of P/M Copper-Chromium Alloy Dispersed with VGCF

Authors: Hisashi Imai, Kuan-Yu Chen, Katsuyoshi Kondoh, Hung-Yin Tsai, Junko Umeda

Abstract:

Microstructural and electrical properties of copper-chromium alloy (Cu-Cr) dispersed with vapor-grown carbon fiber (VGCF) prepared by powder metallurgy (P/M) process have been investigated. Cu-0.7 mass% Cr pre-alloyed powder (Cu-Cr) made by water atomization process was used as raw materials, which contained solid solute Cr elements in Cu matrix. The alloy powder coated with un-bundled VGCF by using oil coating process was consolidated at 1223 K in vacuum by spark plasma sintering, and then extruded at 1073 K. The extruded Cu-Cr alloy (monolithic alloy) had 209.3 MPa YS and 80.4 IACS% conductivity. The extruded Cu-Cr with 0.1 mass% VGCF composites revealed a small decrease of YS compared to the monolithic Cu-Cr alloy. On the other hand, the composite had a higher electrical conductivity than that of the monolithic alloy. For example, Cu-Cr with 0.1 mass% VGCF composite sintered for 5 h showed 182.7 MPa YS and 89.7 IACS% conductivity. In the case of Cu-Cr with VGCFs composites, the Cr concentration was observed around VGCF by SEM-EDS analysis, where Cr23C6 compounds were detected by TEM observation. The amount of Cr solid solution in the matrix of the Cu-Cr composites alloy was about 50% compared to the monolithic Cu-Cr sintered alloy, and resulted in the remarkable increment of the electrical conductivity.

Keywords: powder metallurgy Cu-Cr alloy powder, vapor-grown carbon fiber, electrical conductivity

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7 Phylogenetic Analyses of Newcastle Disease Virus Isolated from Unvaccinated Chicken Flocks in Kyrgyzstan from 2015 to 2016

Authors: Giang Tran Thi Huong, Hieu Dong Van, Tung Dao Duy, Saadanov Iskender, Isakeev Mairambek, Tsutomu Omatsu, Yukie Katayama, Tetsuya Mizutani, Yuki Ozeki, Yohei Takeda, Haruko Ogawa, Kunitoshi Imai

Abstract:

Newcastle disease virus (NDV) is a contagious viral disease of the poultry industry and other birds throughout the world. At present, very little is known about molecular epidemiological data regarding the causes of ND outbreak in commercial poultry farms in Kyrgyzstan. In the current study, the NDV isolated from the one out of three samples from the unvaccinated flock was confirmed as NDV. Phylogenetic analysis indicated that this NDV strain is clustered in the Class II subgenotype VIId, and closely related to the Chinese NDV isolate. Phylogenetic analyses revealed that the isolated NDV strain has an origin different from the 4 NDV strains previously identified in Kyrgyzstan. According to the mean death time (MDT: 61.1 h) and a multibasic amino acid (aa) sequence at the F0 proteolytic cleavage site (¹¹²R-R-Q-K-R-F¹¹⁷), the NDV isolate was determined as mesogenic strain. Several mutations in the neutralizing epitopes (notably, ³⁴⁷E→K) and the global head were observed in the hemagglutinin-neuraminidase (HN) protein of the current isolate. The present study represents the molecular characterization of the coding gene region of NDV in Kyrgyzstan. Additionally, further study will be investigated on the antigenic characterization using monoclonal antibody.

Keywords: Kyrgyzstan, Newcastle disease, genotype, genome characterization

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6 Changes of pH and Pseudomonas Aeruginosa Growth in Liquid Media

Authors: Sayaka Ono, Ryutaro Imai, Tomoko Ehara, Tetsuya Matsumoto, Hajime Matsumura

Abstract:

Background: Wound pH affects a number of important factors in wound healing. We previously measured the pH value of the exudates collected from second-degree burns and found that the increase in pH was observed in the burn wounds in which colonized by Staphylococcus spp., and the increase in pH was evident prior to the clinical findings of local infection. To investigate the relationship between the changes of pH value and bacterial growth, we performed in vitro study using Pseudomonas aeruginosa and liquid medium as a locally infected wound equivalent model. Methods: Pseudomonas aeruginosa standard strain (ATCCR 10145TM) was cultured at 37 °C environment in Luria Broth Miller medium. The absorbance rate which means the amount of bacteria was measured by a microplate reader 2300EnSpireTM). The pH was measured using pH-indicator strips (MColorpHastTM). The statistical analysis was performed using the product-moment correlation coefficient of Pearson's. Results: The absorbance rate and pH value were increased along with culture period. There was a positive correlation between pH value and absorbance rate (n = 27, Pearson's r = 0.985). Moreover, there was a positive correlation between pH value and the culture period (n = 18, Pearson's r = 0.901). The bacteria was well growth in the media from pH 6.6 to pH 8.0 and the pH of culture media converged at 8 -9 along with the bacterial growth. Conclusion: From these results, we conclude that pH value of the wound is correlated with the number of viable bacteria and bacterial growth periods.

Keywords: colonization, potential of hydrogen, Pseudomonas aeruginosa, wound

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5 Situated Urban Rituals: Rethinking the Meaning and Practice of Micro Culture in Cities in East Asia

Authors: Heide Imai

Abstract:

Contemporary cities, especially in Japan, have reached an indescribable complexity and excessive, global investments blur formal, rooted structures. Modern urban agglomerations blindly trust a macro understanding, whereas everyday activities which portray the human degree of living space are being suppressed and erased. The paper will draw upon the approach ‘Micro-Urbanism’ which focus on the sensitive and indigenous side of contemporary cities, which in fact can hold the authentic qualities of a city. Related to this approach is the term ‘Micro-Culture’ which is used to clarify the inner realities of the everyday living space on the example of the Japanese urban backstreet. The paper identifies an example of a ‘micro-zone’ in terms of ‘street space’, originally embedded in the landscape of the Japanese city. And although the approach ‘Micro-Urbanism’ is more complex, the understanding of the term can be tackled by a social analysis of the street, as shown on the backstreet called roji and closely linked examples of ‘situated’ urban rituals like (1) urban festivities, (2) local markets/ street vendors and (3) artistic, intellectual tactics. Likewise, the paper offers insights in a ‘community of streets’ which boundaries are specially shaped by cultural activity and social networks.

Keywords: urban rituals, community, streets as micro-zone, everyday space

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4 Survey of Epidemiology and Mechanisms of Badminton Injury Using Medical Check-Up and Questionnaire of School Age Badminton Players

Authors: Xiao Zhou, Kazuhiro Imai, Xiaoxuan Liu

Abstract:

Badminton is one type of racket sports that requires repetitive overhead motion, with the shoulder in abduction/external rotation and requires players to perform jumps, lunges, and quick directional changes. These characteristics could be stressful for body regions that may cause badminton injuries. Regarding racket players including badminton players, there have not been any studies that have utilized medical check-up to evaluate epidemiology and mechanism of injuries. In addition, epidemiology of badminton injury in school age badminton players is unknown. The first purpose of this study was to investigate the badminton injuries, physical fitness parameters, and intensity of shoulder pain using medical check-up so that the mechanisms of shoulder injuries might be revealed. The second purpose of this study was to survey the distribution of badminton injuries in elementary school age players so that injury prevention can be implemented as early as possible. The results of this study revealed that shoulder pain occurred in all players, and present shoulder pain players had smaller weight, greater shoulder external rotation (ER) gain, significantly thinner circumference of upper limbs and greater trunk extension. Identifying players with specific of these factors may enhance the prevention of badminton injury. This study also shows that there are high incidences of knee, ankle, plantar, and shoulder injury or pain in elementary school age badminton players. Injury prevention program might be implemented for elementary school age players.

Keywords: badminton injury, epidemiology, medical check-up, school age players

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3 Effects of Forest Bathing on Cardiovascular and Metabolic Parameters in Middle-Aged Males

Authors: Qing Li, Maiko Kobayashi, Shigeyoshi Kumeda, Hiroko Ochiai, Toshiya Ochiai, Takashi Miura, Takahide Kagawa, Michiko Imai, Toshiaki Otsuka, Tomoyuki Kawada

Abstract:

In the present study, we investigated the effects of a forest bathing program on cardiovascular and metabolic parameters. Nineteen healthy male subjects (mean age: 51.3 ± 8.8 years) were selected after obtaining informed consent. These subjects took day trips to a forest park named Akasawa Shizen Kyuyourin, Agematsu, Nagano Prefecture (situated in central Japan), and to an urban area of Nagano Prefecture as a control in August 2015. On both trips, they walked 2.6 km for 80 min each in the morning and afternoon on Saturdays. Blood and urine were sampled in the morning before and after each trip. Cardiovascular and metabolic parameters were measured. Blood pressure and pulse rate were measured by an ambulatory automatic blood pressure monitor. The Japanese version of the profile of mood states (POMS) test was conducted before, during and after the trips. Ambient temperature and humidity were monitoring during the trips. The forest bathing program significantly reduced pulse rate, and significantly increased the score for vigor and decreased the scores for depression, fatigue, and confusion in the POMS test. The levels of urinary noradrenaline and dopamine after forest bathing were significantly lower than those after urban area walking, suggesting the relaxing effect of the forest bathing program. The level of adiponectin in serum after the forest bathing program was significantly greater than that after urban area walking. There was no significant difference in blood pressure between forest and urban area trips during the trips.

Keywords: ambient temperature, blood pressure, forest bathing, forest therapy, human health, POMS, pulse rate

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2 Visco-Hyperelastic Finite Element Analysis for Diagnosis of Knee Joint Injury Caused by Meniscal Tearing

Authors: Eiji Nakamachi, Tsuyoshi Eguchi, Sayo Yamamoto, Yusuke Morita, H. Sakamoto

Abstract:

In this study, we aim to reveal the relationship between the meniscal tearing and the articular cartilage injury of knee joint by using the dynamic explicit finite element (FE) method. Meniscal injuries reduce its functional ability and consequently increase the load on the articular cartilage of knee joint. In order to prevent the induction of osteoarthritis (OA) caused by meniscal injuries, many medical treatment techniques, such as artificial meniscus replacement and meniscal regeneration, have been developed. However, it is reported that these treatments are not the comprehensive methods. In order to reveal the fundamental mechanism of OA induction, the mechanical characterization of meniscus under the condition of normal and injured states is carried out by using FE analyses. At first, a FE model of the human knee joint in the case of normal state – ‘intact’ - was constructed by using the magnetron resonance (MR) tomography images and the image construction code, Materialize Mimics. Next, two types of meniscal injury models with the radial tears of medial and lateral menisci were constructed. In FE analyses, the linear elastic constitutive law was adopted for the femur and tibia bones, the visco-hyperelastic constitutive law for the articular cartilage, and the visco-anisotropic hyperelastic constitutive law for the meniscus, respectively. Material properties of articular cartilage and meniscus were identified using the stress-strain curves obtained by our compressive and the tensile tests. The numerical results under the normal walking condition revealed how and where the maximum compressive stress occurred on the articular cartilage. The maximum compressive stress and its occurrence point were varied in the intact and two meniscal tear models. These compressive stress values can be used to establish the threshold value to cause the pathological change for the diagnosis. In this study, FE analyses of knee joint were carried out to reveal the influence of meniscal injuries on the cartilage injury. The following conclusions are obtained. 1. 3D FE model, which consists femur, tibia, articular cartilage and meniscus was constructed based on MR images of human knee joint. The image processing code, Materialize Mimics was used by using the tetrahedral FE elements. 2. Visco-anisotropic hyperelastic constitutive equation was formulated by adopting the generalized Kelvin model. The material properties of meniscus and articular cartilage were determined by curve fitting with experimental results. 3. Stresses on the articular cartilage and menisci were obtained in cases of the intact and two radial tears of medial and lateral menisci. Through comparison with the case of intact knee joint, two tear models show almost same stress value and higher value than the intact one. It was shown that both meniscal tears induce the stress localization in both medial and lateral regions. It is confirmed that our newly developed FE analysis code has a potential to be a new diagnostic system to evaluate the meniscal damage on the articular cartilage through the mechanical functional assessment.

Keywords: finite element analysis, hyperelastic constitutive law, knee joint injury, meniscal tear, stress concentration

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1 A High-Throughput Enzyme Screening Method Using Broadband Coherent Anti-stokes Raman Spectroscopy

Authors: Ruolan Zhang, Ryo Imai, Naoko Senda, Tomoyuki Sakai

Abstract:

Enzymes have attracted increasing attentions in industrial manufacturing for their applicability in catalyzing complex chemical reactions under mild conditions. Directed evolution has become a powerful approach to optimize enzymes and exploit their full potentials under the circumstance of insufficient structure-function knowledge. With the incorporation of cell-free synthetic biotechnology, rapid enzyme synthesis can be realized because no cloning procedure such as transfection is needed. Its open environment also enables direct enzyme measurement. These properties of cell-free biotechnology lead to excellent throughput of enzymes generation. However, the capabilities of current screening methods have limitations. Fluorescence-based assay needs applicable fluorescent label, and the reliability of acquired enzymatic activity is influenced by fluorescent label’s binding affinity and photostability. To acquire the natural activity of an enzyme, another method is to combine pre-screening step and high-performance liquid chromatography (HPLC) measurement. But its throughput is limited by necessary time investment. Hundreds of variants are selected from libraries, and their enzymatic activities are then identified one by one by HPLC. The turn-around-time is 30 minutes for one sample by HPLC, which limits the acquirable enzyme improvement within reasonable time. To achieve the real high-throughput enzyme screening, i.e., obtain reliable enzyme improvement within reasonable time, a widely applicable high-throughput measurement of enzymatic reactions is highly demanded. Here, a high-throughput screening method using broadband coherent anti-Stokes Raman spectroscopy (CARS) was proposed. CARS is one of coherent Raman spectroscopy, which can identify label-free chemical components specifically from their inherent molecular vibration. These characteristic vibrational signals are generated from different vibrational modes of chemical bonds. With the broadband CARS, chemicals in one sample can be identified from their signals in one broadband CARS spectrum. Moreover, it can magnify the signal levels to several orders of magnitude greater than spontaneous Raman systems, and therefore has the potential to evaluate chemical's concentration rapidly. As a demonstration of screening with CARS, alcohol dehydrogenase, which converts ethanol and nicotinamide adenine dinucleotide oxidized form (NAD+) to acetaldehyde and nicotinamide adenine dinucleotide reduced form (NADH), was used. The signal of NADH at 1660 cm⁻¹, which is generated from nicotinamide in NADH, was utilized to measure the concentration of it. The evaluation time for CARS signal of NADH was determined to be as short as 0.33 seconds while having a system sensitivity of 2.5 mM. The time course of alcohol dehydrogenase reaction was successfully measured from increasing signal intensity of NADH. This measurement result of CARS was consistent with the result of a conventional method, UV-Vis. CARS is expected to have application in high-throughput enzyme screening and realize more reliable enzyme improvement within reasonable time.

Keywords: Coherent Anti-Stokes Raman Spectroscopy, CARS, directed evolution, enzyme screening, Raman spectroscopy

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