The 30th Special CU – af Seminar 2022

The 30th Special CU-af Seminar 2022 “Research to Excellence for Sustainability” September 2nd, 2022 Meeting Room 202, 2nd Floor, Chamchuri 4 Building Chulalongkorn University



The 30th Special CU-af Seminar 2022 September 2, 2022 MESSAGE from PRESIDENT, CHULALONGKORN UNIVERSITY Professor Bundhit Eua-Arporn, Ph.D. Mr. Hiroyuki Watanabe, Senior Executive Director of the Asahi Glass Foundation, President of AGC Flat Glass (Thailand) PCL., AGC Chemicals (Thailand) Co.,Ltd., AGC Automotive (Thailand) Co.,Ltd. Distinguished guests, ladies and gentlemen Thank you all for joining us today at Chulalongkorn University. After last year, when the ceremony and seminar were organized only online due to the Covid-19 pandemic. I’m very delighted that we could reunite with each other in this very warm face-to-face meeting. So, once again, I would like to extend a cordial welcome to all of you to this 30th Special Chulalongkorn University and the Asahi Glass Foundation Seminar, as well as my congratulations to ten researchers granted this year. As research and development is one of the major factors in the development of our country, we are gearing towards an emphasis on research in university that can both enhance and enrich our teaching to be more up-to-date and relevant to the contemporary and sustainable society. Moreover, the research for new knowledge and technological advancement is indispensable and must also be carried out in view of particular needs and practical uses on both national and global levels. However, Chulalongkorn University cannot achieve these goals without the supports from our precious partners, the Asahi Glass Foundation. As we are stepping into the 41st year of grant presentation ceremony, the university has been receiving continuous and generous support from the Asahi Glass Foundation of Japan, enabling us to promote various impactful research works. The annual CU–af seminar is one of the showcase that allows researchers to publicise their findings and to exchange ideas among distinguished audience. I am confident that this kind of academic exchange will greatly contribute to the existing body of knowledge and enhance the atmosphere of intellectual spirit. Therefore, I would like to express my warmest thanks to the Asahi Glass Foundation for the generous support for decades. Along with my best wishes for the success of this event and hope that you all enjoy the exciting speakers to come during the seminar. May I take this opportunity to declare the 30th Special Seminar open. Thank you. I

The 30th Special CU-af Seminar 2022 September 2, 2022 CONGRATULATORY ADDRESS from SENIOR EXECUTIVE DIRECTOR, THE ASAHI GLASS FOUNDATION Mr.Hiroyuki Watanabe Thank you very much for your kind introduction. Good afternoon, everyone. My name is Hiroyuki Watanabe, I am Senior Executive Director of the Asahi Glass Foundation. Unfortunately, our chairman Shimamura-san cannot attend this ceremony, I will make a congratulatory address on behalf of him.It is both honor and pleasure for me to be this ceremony, especially in person, as a representative of Asahi Glass Foundation in Japan. Firstly, please allow me to extend my sincere gratitude to Professor Bundhit Eua-arporn, President of the University, Professor Chakkaphan Sutthira, Vice President, our distinguished guests and the members hosting today’s ceremony.Thank you very much. Secondly, let me introduce Asahi Glass Foundation briefly. It was established in 1933 to celebrate the 25th anniversary of the founding of Asahi Glass Co., Ltd.Asahi Glass Co., Ltd. was renamed the AGC Inc. in 2018. Now we provide 3 major programs such as the Research Grant Program, Scholarship Program and Commendation Program named “Blue Planet Prize”. In addition, we have publicized the annual survey on the global environment named “Environment Doomsday Clock”.In terms of the relationship between AGC Inc. and Thailand, In 1964, AGC began to produce flat glass in Thailand, and expanding its business to automotive glass, chemicals and electric materials. Today total annual sales revenue of group is approximately 52 billion THB, 1.3 Billion USD in Thailand. And many alumni of the Chulalongkorn University have joined AGC Flat Glass, Thailand, AGC Chemicals, Thailand and so on. In 1982, Asahi Glass Foundation initiated our grant program with Chulalongkorn University. This is the 41st grant presentation ceremony. So AGC Inc, and Asahi Glass Foundation have a long history with Thailand and Chulalongkorn University as well. We have supported 315 grant themes and contributed approximately 2 M USD from the beginning. I believe these researches can contribute enough for the development and prosperity of Thailand. We wish our support can be helpful for your researchers. As one of other major activities of Foundation, We provide “Blue Planet Prize”, from 1992, which has become internationally recognized award successfully. The foundation has awarded the Blue Planet Prize to two people who have made outstanding contributes towards solving global environment issues with a certificate of merit, commemorative trophy and 50 million Japanese yen or 370 thousand USD per each in prize money. Prof. Veerabhadran Ramanathan from USA and Prof. Mohan Munasinghe from Sri Lanka were winners in 2021. And His Majesty Jigme Singye Wangchuck, the 4th King of Bhutan and Professor Stephen Carpenter from USA are winners this year. Please visit our website, which explains the winner’s achievement in detail. And you can also reach the names and research themes of this year’s new grantees and the research reports of the past year’s grantees. II

The 30th Special CU-af Seminar 2022 September 2, 2022 As you know, our environmental situation is getting worse every year. Humankind has sacrificed its environment in exchange for its prosperity. That is the reason why we established the “Blue Planet Prize”. I hope the winner of this prize from Thailand will be awarded someday. I sincerely hope all of researchers here can contribute to our precious Blue planet through your researches. Once again, I’d like to express my deepest appreciation to Professor Bundhit Eua-arporn, Professor Chakkaphan Sutthira, our distinguished guests, the members hosting today’s ceremony. and all members concerned kindly supported the seminar, and award ceremony as well. Thank you for your kind attention. Hiroyuki Watanabe Senior Executive Director of Asahi Glass Foundation September 2nd, 2022 III

The 30th Special CU-af Seminar 2022 September 2, 2022 REPORT ADDRESS by VICE PRESIDENT, CHULALONGKORN UNIVERSITY Professor Chakkaphan Sutthirat, Ph.D., DGemG. Mr. Hiroyuki Watanabe, Senior Executive Director of the Asahi Glass Foundation, Professor Dr. Bundhit Eua-arporn, President of Chulalongkorn University, Distinguished guests, ladies and gentlemen It is our great pleasure to welcome everyone back to Chulalongkorn University after two years of social distancing due to the Covid-19 pandemic. We are fortunate to have great cooperation with the Asahi Glass Foundation both in term of support and strong relationship. Back in 1982, Chulalongkorn University received the first support from the Asahi Glass Foundation for research. Initially, this support was focused on the research in the field of engineering. Subsequently, it has expanded to other vital areas, such as environmental science, medical science, pharmaceutical science, and information technology. While last year, the support has also opened a new door to the field of social science. Up to now, the findings of these research projects are compiled and published with the support of the Asahi Glass Foundation for dissemination to various agencies worldwide. The seminars that have been organized regularly, are based on the research findings that help providing venues for the exchange of knowledge and expertise among academics, external organizations and agencies in both the governmental and private sectors. It is expected that the seminar will promote the application of the research in the industrial sector as well. We are proud that this special seminar in the year 2022 marks the 30th of such gathering. While the grant presentation ceremony reached their 41st year. Today, we are also particularly honored by the presence of today keynote speaker, Professor Dr.Yuttanant Boonyongmaneerat, Deputy Director of Metallurgy and Materials Science Research Institute, Chulalongkorn University, on the topic of “Intermediary Role in Driving Impactful Materials Research: From Kawasaki Fontale’s Chanathip to a Research Group that builds Industrial Clusters and iBDS”. After his lecture, eight research papers will be presented. Two projects from Faculty of Engineering, a project from Faculty of Science, Faculty of Medicine, together with projects from Metallurgy and Materials Science Research Institute, Aquatic Resources Research Institute, Environmental Research Institute and Institute of Asian Studies. Please allow me to take this opportunity to express our sincere appreciation to the Asahi Glass Foundation, Japan, for the continuous support for the research projects of Chulalongkorn University. We also would like to thank all our researchers who have worked with strong commitment on their projects that will help enrich their teaching and their societies. Next, may I now invite Professor Dr. Bundhit Eua-arporn, President of Chulalongkorn University, to give an address on the opening ceremony. Thank you. IV

The 30th Special CU-af Seminar 2022 September 2, 2022 The 30th Special CU-af Seminar 2022 “Research to Excellence for Sustainability” September 2, 2022 Meeting Room 202, 2nd Floor, Chamchuri 4 Building Chulalongkorn University 13.00 – 13.30 Registration 13.30 – 14.00 Opening Ceremony • Welcome Remark and Report Prof. Chakkaphan Sutthirat, Ph.D., DGemG. Vice President, Chulalongkorn University • Opening Address Prof. Bundhit Eua-arporn, Ph.D. President, Chulalongkorn University • Greetings from The Asahi Glass Foundation, Japan Mr. Hiroyuki Watanabe Senior Executive Director, The Asahi Glass Foundation, Japan Grantees (1) Asst. Prof. Dr. Supareak Praserthdam Faculty of Engineering (2) Asst. Prof. Dr. Jittima Luckanagul Faculty of Pharmaceutical Sciences (3) Dr. Pichaya In-na Faculty of Science (4) Dr. Rueangwit Sawangkeaw The Institute of Biotechnology and Genetic Engineering (5) Dr. Chaiyaboot Ariyachet Faculty of Medicine (6) Dr. Theerayut Phengsaart Faculty of Engineering (7) Asst. Prof. Dr. Tanatorn Khotavivattana Faculty of Science (8) Dr. Junjuda Unruangsri Faculty of Science (9) Dr. Nuttapon Pombubpa Faculty of Science (10) Dr. Chanat Aonbangkhen Faculty of Science V

The 30th Special CU-af Seminar 2022 September 2, 2022 14.10 – 14.30 Keynote Speech Intermediary Role in Driving Impactful Materials Research: From Kawasaki Fontale’s Chanathip to a Research Group that builds Industrial Clusters and iBDS Prof. Yuttanant Boonyongmaneerat, Ph.D. Metallurgy & Materials Science Research Institute (MMRI), 14.30 – 14.40 Group Photo Session 14.40 – 15.00 Coffee Break 15.00 – 17.00 Concurrent Sessions (Session 1, Session 2) Presentation: 10-15 minutes each Q&A: 5 minutes each Concurrent Sessions Session 1: Physical Science Chairperson: Assistant Professor Dr. Supareak Praserthdam Department of Chemical Engineering, Faculty of Engineering 15.00 – 15.30 The effect of ZrO2 addition on nickel aluminide developments on pure nickel substrate prepared by pack aluminization Assistant Professor Dr. Sirichai Leelachao Department of Metallurgical Engineering, Faculty of Engineering 15.30 – 16.00 Comparative Study of Silk Fibroin-Based Hydrogels and Their Potential as Material for 3-dimensional (3D) Printing Associate Professor Dr. Juthamas Ratanavaraporn Biomedical Engineering Program, Faculty of Engineering 16.00 – 16.30 Improvement of Aqueous Zinc-ion Battery Performance by combining Experiments and Molecular Dynamics Simulations Dr. Manaswee Suttipong Department of Chemical Technology, Faculty of Science 16.30 – 17.00 Development of polyacrylonitrile/bio-related polyurethane electrospun fiber mats as separator in Zn-ion battery Dr. Manunya Okhawilai Metallurgy and Materials Science Research Institute VI

The 30th Special CU-af Seminar 2022 September 2, 2022 Session 2: Life Science & Social Science Chairperson: Dr. Chanat Aonbangkhen Department of Chemistry, Faculty of Science 15.00 – 15.30 Who will Take Over the Farm? : Youth Farmers and Agrarian Transition in Timor-Leste Dr. Pyone Myat Thu Asian Research Center of Migration, Institute of Asian Studies 15.30 – 16.00 Enhancing Local Capability toward Sustainable Municipal Solid Waste Management: Case Study of Nan Municipality, Thailand Dr. Wilailuk Niyommaneerat Environmental Research Institute 16.00 – 16.30 Characterization and product distribution via thermal conversion of abandoned, lost or otherwise discarded fishing gear (ALDFG) waste Dr. Yotwadee Hawangchu Aquatic Resources Research Institute 16.30 – 17.00 Label-Free Identification and Classification of Circulating Tumor Cells using Deep Learning and High-Content Imaging Dr. Sira Sriswasdi Research Affairs, Faculty of Medicine VII



The 30th Special CU-af Seminar 2022 September 2, 2022 TABLE OF CONTENTS Page Message form President, Chulalongkorn University I Congratulatory Address form Chairman, The Asahi Glass Foundation II Report Address by Vice President, Chulalongkorn University IV Schedule V Keynote Lecture 1 Intermediary Role in Driving Impactful Materials Research: From Kawasaki Fontale’s Chanathip to a Research Group that builds Industrial Clusters and iBDS Prof. Yuttanant Boonyongmaneerat, Ph.D. Metallurgy & Materials Science Research Institute Invited Papers 3 Session 1: Physical Science 1. The effect of ZrO2 addition on nickel aluminide developments on pure nickel substrate prepared by pack aluminization Assistant Professor Dr. Sirichai Leelachao 2. Comparative Study of Silk Fibroin-Based Hydrogels and Their Potential 15 as Material for 3-dimensional (3D) Printing Associate Professor Dr. Juthamas Ratanavaraporn 3. Improvement of Aqueous Zinc-ion Battery Performance by combining 29 Experiments and Molecular Dynamics Simulations Dr. Manaswee Suttipong 4. Development of polyacrylonitrile/bio-related polyurethane electrospun 43 fiber mats as separator in Zn-ion battery Dr. Manunya Okhawilai Session 2: Life Science & Social Science 1. Label-Free Identification and Classification of Circulating Tumor Cells 57 using Deep Learning and High-Content Imaging Dr. Sira Sriswasdi 2. Enhancing Local Capability toward Sustainable Municipal Solid Waste 71 Management: Case Study of Nan Municipality, Thailand Dr. Wilailuk Niyommaneerat, Dr. Vacharaporn Soonsin, Dr. Puntita Tanwattana IX

The 30th Special CU-af Seminar 2022 85 99 September 2, 2022 111 3. Characterization and product distribution via thermal conversion of abandoned, lost or otherwise discarded fishing gear (ALDFG) waste Dr. Yotwadee Hawangchu, Prof. Dr. Viboon Sricharoenchaikul, Dr. Duangduen Atong 4. Who will Take Over the Farm? : Youth Farmers and Agrarian Transition in Timor-Leste Dr. Pyone Myat Thu Appendix X



Keynote Lecture Intermediary Role in Driving Impactful Materials Research: From Kawasaki Fontale’s Chanathip to a Research Group that builds Industrial Clusters and iBDS Prof. Yuttanant Boonyongmaneerat, Ph.D.

The 30th Special CU-af Seminar 2022 September 2, 2022 Keynote Lecture Intermediary Role in Driving Impactful Materials Research: From Kawasaki Fontale’s Chanathip to a Research Group that builds Industrial Clusters and iBDS Prof. Yuttanant Boonyongmaneerat, Ph.D.1* Abstract Through a framework that has been persistently orchestrated since 2012, this talk aims to exemplify a role of a so-called ‘intermediary’ (or what a football coach would call ‘attacking midfielder’) that researchers could adopt and exercise to promote impactful (materials) research from the initiation of meaningful projects to implementation in the real world. The Surface & Coating Technology for Metals and Materials Research Unit (SurfMet RU) at Metallurgy & Materials Science Research Institute (MMRI) of Chulalongkorn University represents a research group that are determined to develop fundamental and applied research for benefits of the industry and society. Challenges always arise when laboratory-scaled knowledge is to be translated to a larger scale and go across the chasm for implementation and commercialization. In fact, the challenge starts from the shaping up of the projects that are to be relevant and aligned to users’ needs. Working closely with the implementers, i.e. the surface finishing industry in this case, is the key. Back in the year 2012, the SurfMet group thus strategically develop a triple-helix consortium of surface finishing industry, which later was supported by the national innovation policy agency NXPO and strategic international partner Fraunhofer Institute IPA, and became the strong TEPNET association as of today. The consortium serves as the advisory board for initiation of meaningful projects, supporting manufacturing-scale research, and also organizes educational and technical-related activities to prepare and enable the industrial community to absorb the scientific and research know-hows. Assembling of the consortium was however challenging – after all many members are business competitors. So the keen acts of intermediary role that build trust and encourage collaboration are very critical. Next we would need an army of individuals that serve as a bridge between academic researchers and private sectors, providing innovation & business development service (iBDS) that aims for implementation of technology and ultimately nurturing the development of innovation-driven enterprises (IDEs) for SMEs. With the support by Chulalongkorn University through the C2F program, Nexurf a spinoff iBDS venture was thus established in 2021, and has been pioneering activities that effectively link the academics, markets and supply chains closer together, leading to expansive developments of spinoffs and impactful innovations.Nexurf, in many ways, serves as intermediary itself. From these building blocks, a stream of impactful research and innovation that directly connect with the market needs and are scalable have thus been developed and deployed. To name a few, these include highly-corrosion resistant coating for harsh environments, precious metal recovery system, energy-saving anodizing process, automatic brush plating system, metallizing of bio-polymers, anti-microbial alloys, and vaccine packages carried by autonomous drone. 1Metallurgy & Materials Science Research Institute, Chulalongkorn University *E-mail: [email protected] 1

The effect of ZrO2 addition on nickel aluminide developments on pure nickel substrate prepared by pack aluminization Sirichai LEELACHAO

The 30th Special CU-af Seminar 2022 September 2, 2022 The effect of ZrO2 addition on nickel aluminide developments on pure nickel substrate prepared by pack aluminization Sirichai LEELACHAO1* Abstract This study aims to investigate the effect of ZrO2 on phase evolution and oxidation behavior of the aluminized Ni substrate via pack aluminization at 1,000 °C for 6.25 h where the process contained different contents ZrO2 powder. The aluminized specimens were cyclically oxidized at 1,000 °C with rapid cooling and heating. A scanning electron microscope (SEM) equipped with energy-dispersive X-ray spectroscopy (EDS) and an X-ray diffractometer (XRD) were utilized for phase identification. The coatings consisted of β-NiAl, γ’-Ni3Al and Al-dissolved γ-Ni where thicknesses decreased with increasing ZrO2 contents. A reaction between Al and ZrO2 was suggested for an absence of δ-Ni2Al3. The reduced Zr was incorporated as intermetallics. While the specimen with moderate ZrO2 content showed parabolic oxidation kinetics, a multiple linear kinetics along with a high-exponent power-law oxidation were observed in other doping conditions. A modification of the thermodynamic Al activity can be achieved using less-stable oxides in pack aluminization. Zr could promote the stable α-Al2O3 scale even for a lower Al concentration of aluminides. 1Department of Metallurgical Engineering Faculty of Engineering, Chulalongkorn University Bangkok, Thailand 3

The 30th Special CU-af Seminar 2022 September 2, 2022 Introduction and Objectives Nickel-based superalloys are commonly used as materials for petrochemical pipelines, turbine blades in power plants or jet engines due to its high melting point and excellent machinability among other heat-resistant alloys. High-temperature oxidation, as a significant degradation of materials, can be mitigated by increasing Al concentration of the alloys via a surface modification called as an aluminization where intermetallic compounds of aluminium and metal substrates are served as elevated Al sources during oxidation which provide a protective Al2O3 layer. A pack-cementation is cost-effective and enables us to adjust a chemical reactivity of diffusing species, so-called thermodynamic activity, through the contents in pack mixtures. Therefore, an alteration on phase existence or chemical composition of the coatings can be promptly achieved. δ-Ni2Al3 is generally produced in conditions where Al concentration is high[1]. However, its brittleness, as compared with other intermetallics,[2] makes it undesirable in mechanical aspects. To avoid a formation of δ-Ni2Al3, a high-temperature aluminization is proposed which based on a principle of Al-activity reduction[3,4]. Unexpectedly, a polymorphism of Al2O3 can lower oxidation resistance of alumina-forming alloys by inducing cracks in the protective film during phase transformation[5]. Hence, an incorporation with Zr in which accelerates the formation of stable α-Al2O3 may overcome the problem[3, .6-8] Due to a negative Gibb’s energy difference of the following reaction, i.e. 4Al+ 3ZrO2 = 3Zr + 2Al2O3, at temperatures ≤ 700 °C[9], the thermodynamic activity of diffusible Al-rich species can be modified. Therefore, it may allow a simultaneous incorporation of Zr into aluminide coatings prepared by a pack aluminization using Al and ZrO2 powder as ingredients where a change in phase evolution of such coatings are expectable. Methods All chemicals used were commercial-grade except ZrO2 powder which was analytical-grade (Riedel-de Haën, Germany). Compositions in pack mixture containing Al, NH4Cl, Al2O3, and ZrO2 powders were listed in Table 1 where a nominal atomic ratio presented is calculated from the remaining amount of Al in the packs and obtained Zr atoms after the reduction reaction fully completed. Commercially pure nickel strips (Kenis, Japan) were cut with dimensions 15×15×0.3 mm3. After being ultrasonically cleaned in acetone, substrates were vertically embedded into the mixture in separate alumina crucibles. Supplementary Al2O3 powder was added on top of the pack to fill up the crucible. An alumina lid was sealed using an air-setting refractory mortar. All crucibles were simultaneously placed in a horizontal furnace to perform aluminization at 1,000 °C for 6.25 h with a constant heating rate of 5 °C·min-1. A quartz reactor was flushed with industrial Ar gas (99.5%-purity, Linde, Thailand) at a rate of 3 L·min-1 from a beginning to a cool-down stage at a temperature of about 600 °C; the crucible was then furnace-cooled to room temperature. Prior to aluminizing step, a baking at 100 °C for 10 min is intentionally performed to remove any adsorbed moisture. Aluminized samples were dissected using a Struers Accutom-5 precision cutting machine. Microstructure analysis and phase identification of diffusion layers were examined using a JOEL JSM-IT100 scanning electron microscope equipped with an energy dispersive spectroscope (SEM-EDS) and a Bruker D8 Advance X-ray diffractometer (XRD) using Cu Kα source, respectively. By excluding Ni from quantitative EDS mapping scans, an average atomic fraction of Al and Zr in three different locations of the coatings was used to determine the incorporated Zr concentration. 4

The 30th Special CU-af Seminar 2022 September 2, 2022 Cyclic oxidation was performed in a box furnace at 1,000 °C for 8 h per cycle under an ambient atmosphere. Alumina boats containing each specimen at room temperature were inserted into a preheated furnace to reduce an oxidation during heating periods. After 8 h of oxidation, the specimens were air-quenched to aggravate a failure of oxides. An OHAUS PA214 analytical balance was used to measure a weight change without removing the specimen from the boat. Phase identification of the oxidized layers were analyzed using a Rigaku D/Max 2200P/C glancing incident angle X-ray diffractometer (GIXRD) at an incident angle of 3° using a parallel Cu Kα beam whereas their topography was examined using a Hitachi S4800 scanning electron microscope. ICSD numbers used for phase identification are listed as follow: Pure Ni (#004-0850), β-NiAl (#044-1188), γ’-Ni3Al (#009-0097), α-Al2O3 (#046-1212), θ-Al2O3 (#023-1009), γ-Al2O3 (#050-0741), δ-Al2O3 (#046-1215), NiAl2O4 (#010-0339) and NiO (#044-1159). Results and Discussion Phase evolution of the aluminized specimens The coatings with Kirkendall voids from the Zr10 (Figure.1a) composed of chemical compositions in each sublayer as listed in Table 2; it corresponds to the following sequence: (1) Stoichiometric β-NiAl, (2) Ni-rich β-NiAl, (3) γ’-Ni3Al and (4) Al-dissolved γ-Ni, respectively. Faded interface between the stoichiometric and the Ni-rich β-NiAl layer is likely to be caused by a competitive diffusion[10]. Figure 1b shows diffraction patterns of the Zr10 specimen in which exhibits a predominate β-NiAl phase as shown in Figure.1b. According to SEM and XRD results, there is no Zr incorporated into the specimen. This is suggested to be due to the following reasons: (i) multiple species of zirconium chlorides (ZrCl4, ZrCl3, ZrCl2, ZrCl) than those of aluminium (AlCl3, AlCl2, AlCl) are possible so that disproportionation reactions are more complicated, and (ii) an efficiency of redox reactions is not 100%. As shown later, it can be seen that an overall Zr concentration in the coatings was lower than the nominal values for all doping conditions. Figure 1: (a) Cross- sections and (b) X-ray diffraction patterns of as-prepared Zr10 specimen. 5

The 30th Special CU-af Seminar 2022 September 2, 2022 The coatings were changed into two layered structure with bright precipitates where Kirkendall voids and the thickness of the coatings were significantly reduced with higher content of ZrO2 in the pack mixture. Phase fraction of the precipitates was remarkably higher for the Zr30 specimen, as being compared in Figure.2a and 2b. EDS results of the Zr20 and the Zr30 are listed in Table 3 and 4, respectively. Ni-rich β-NiAl intermetallic became a predominant layer with a thin outer layer of stoichiometric β-NiAl. Highly-contrast precipitates were found to contain high amount of Zr which being assigned as Ni7Zr2. A ternary intermetallic AlNi2Zr were observed in fainted regions of the Zr30 specimen, as represented in Spectrum 4 in Figure.2b, along with Ni7Zr2 and β-NiAl. X-ray diffractograms of both conditions (Figure.2c and 2d) were in accordance with the suggested phases. Noticeable Al2O3 peaks might indicate the outward layer growth[12]. The total thickness of the coatings was decreased with increasing the ZrO2 content in the pack whereas the incorporated Zr concentration was lower than the theoretical values as shown in Figure 3. Figure 2: Microstructures of the aluminized layers of (a) Zr20 and (b) Zr30 along with corresponding X-ray diffractograms of (c) Zr20 and (d) Zr30, respectively. 6

The 30th Special CU-af Seminar 2022 September 2, 2022 7

The 30th Special CU-af Seminar 2022 September 2, 2022 Figure 3: Correlation of the expected Zr concentration on thicknesses of the coatings (▲) and the obtained Zr/(Zr+Al) measured by EDS mappings analyses which excluded Ni (●). Above-mentioned results are clear evidences for a redox reaction between ZrO2 and Al powders which took place during aluminization with limited conversion efficiency. While a reduction in thermodynamic activity of Al can attribute to either a thickness reduction or a predomination of Ni-rich β-NiAl layer, an incorporation of Zr can be simultaneously achieved in forms of intermetallics which might be due to either a limited solubility[13-14] or a slow diffusivity of Zr in nickel aluminides[15]. Oxidation behavior of the aluminized specimens Specific mass gain of the different ZrO2 content aluminized specimens is illustrated in Figure 4. Detrimental effect of excessive Zr additions is evidently demonstrated. It is noteworthy mentioning that the Zr20 specimen exhibited the lowest oxidation rate with parabolic kinetics. While the Zr10 specimen showed a two-step linear oxidation at a transition time of 24 h, the Zr30 strangely displayed three steps: two linear oxidations with a transition time at 16 h, followed by power-law oxidation behavior. An equation of Δm=kw(x-τ)n + b was used to determine an exponent (n) and a rate constant (kw) of the power-law oxidation where Δm is a specific mass gain, b is a constant and τ is a transition time of 40 h. Calculated rate constants are listed in Table 5. Investigation of oxides evolution can be achieved by comparing XRD patterns of the oxidized specimens at different times. As shown in Figure 5a-5c, transient θ- and γ-Al2O3 was observed for the oxidized Zr10 specimen at the early stage of oxidation. It is clear- ly seen that Al in aluminides was gradually consumed as a transformation of Ni-rich β-NiAl into γ’ -Ni3Al. As the oxidation proceeded, the effect of Zr incorporation in the coatings took place. While transient aluminas were still major constituents for the oxidized Zr10 (Figure 5d), the stable α-Al2O3 was found to be predominant with remaining γ’-Ni3Al for the Zr20 and the Zr30 specimens (Figure 5e-5f). Figure 4: Mass change of Zr10 (+), Zr20 (▲) and Zr30 (●) subjected to a cyclic oxidation at 1,000 °C with rapid heating and cooling.Arrows indicate the XRD measurements at 24 h and 80 h oxidation. 8

The 30th Special CU-af Seminar 2022 September 2, 2022 Comparing between high oxidation rate specimens, oxide scale of the Zr10 specimen which subjected to 96 h of oxidation (Figure 6a) exhibited cracks and needle-like structures (suggested to be θ-Al2O3) on the rugged surface while the oxidized Zr30 (Figure 6c) showed cracks and delaminated patches composing of finer θ-Al2O3 needles; the patch-like structure observed in the study is comparably similar to ridges or cellular oxide scale caused by Zr-rich precipitates reported in[16]. Observed cracks are evidently clear for their linear oxidation behaviors[17-18]. Rapid and slow linear oxidation kinetics in the Zr10 specimen are attributed to a formation of θ-Al2O3 and α-Al2O3, respectively, as the transient shows a higher growth rate[19]. For the oxidized Zr30, two first linear oxidations might also involve alumina formations and cracks because the corresponding rate constants are in the same order of magnitude as those of the oxidized Zr10. As shown in Figure 6d, a smooth topography underneath the spiked patches in the Zr30 suggested that there are, at least, two different Al2O3 were involved. It might be therefore relevant to a power-law oxidation with greater exponent than 0.5, implying on the simultaneous oxidation behaviors. The scale from the oxidized Zr20 specimen (Figure 6b) consisted of nodular α-Al2O3, coarser needles of θ-Al2O3 with a smoother surface without observable cracks. The structure highly corresponds to a continuous protective layer whose oxidation kinetic is parabolic .[17-18] The parabolic rate constant kp at 1.94×10-2 mg2·cm-4·h-1 is in good agreement with the reported rate constant of Zr-doped NiAl alloys[20], the upper bound α-Al2O3 formation from a stoichiometric β-NiAl[14] or the reported value for ε-Ni3Al[21]. In this study, a formation of α-Al2O3 is likely to originate from Ni-rich β-NiAl layer. Detrimental effect of excessive Zr on oxidation behavior of nickel aluminides were also reported[8] as the result of internal oxidation[22]. The results strongly indicate that higher Al concentration does not always be beneficial for oxidation resistance of aluminides whereas a formation of stable α-Al2O3 can be promoted by an incorporation of Zr where a continuous Zr-rich precipitates is suggested to behave as a constant supply of Zr during oxidation of aluminides. 9

The 30th Special CU-af Seminar 2022 September 2, 2022 Figure 5: XRD spectra after 24 h of cyclic oxidation of (a) Zr10, (b) Zr20 and (c) Zr30; 80 h of cyclic oxidation of (d) Zr10, (e) Zr20 and (f) Zr30. Each symbol is assigned as follows: α = α-Al2O3, β = NiAl, γ’ = Ni3Al, θ = θ-Al2O3, γ = γ-Al2O3, δ = δ-Al2O3, * = NiAl2O4 and ◊ = NiO. (Online color) 10

The 30th Special CU-af Seminar 2022 September 2, 2022 Figure 6: Oxide scale topography of the 96h-oxidized (a) Zr10, (b) Zr20, (c) Zr30 and (d) higher magnification of the selected area in the oxidized Zr30. Cracks are addressed by arrows. Conclusion Adding of ZrO2 powder into pack mixtures are confirmed to a potential approach for reducing the thermodynamic Al activity in aluminization process. This facilitates a formation of β-NiAl phase so that such intermetallic can be achieved without any post-process heat treatment. In addition, it also envisions a possibility in a simultaneous doping by using less-stable metal oxides in which are beneficial for alumina phase transformation. Conversion of redox reaction is found to be limited so that a sufficient ZrO2 content in the pack is essential for incorporating Zr. The recommended ratio of ZrO2 and Al powder in the pack should be greater than 1:1, according to this study. Zr is rather found as intermetallic forms, suggesting on either low solubility or sluggish diffusion of Zr in NixAly. Higher Al concentration of aluminides does not attribute to an α-Al2O3 formation. The lowest oxidation rate with parabolic kinetics is achieved when a weight ratio of ZrO2 to Al at 1:1 where the excessive Zr incorporating concentration tends to have an adverse effect on alumina phase formation. References 1. Huang, W. and Chang, Y.A., Intermetallics, 6(6): 487-498. 2. Donachie, M.J., and Donachie, S.J. (2002), Superalloys: a Technical Guide (2nd Edition), United States of America: ASM International. 3. Bose, S. (2018), High temperature coatings, United Kingdom: Butterworth-Heinemann. 4. Erdeniz, D., and Dunand, D.C., Intermetallics, 50: 43-53. 5. Lamouri, S., Hamidouche, M., Bouaouadja, N., Belhouchet, H., Garnier, V., Fantozzi, G., and Trelkate, J.F., Boletín de la Sociedad Española de Cerámica y Vidrio, 56(2): 47-54. 6. Kim, D., Shang, S., Li, Z., Gleeson, B., and Liu, Z.-K., Oxid. Met., 92(3): 303-313. 7. White, R., and Weaver M., Oxid. Met., 92(3): 227-242. 8. Zhou, Y., Zhao, X., Zhao, C., Hao, W., Wang, X., and Xiao, P., Corros. Sci., 123: 103-115. 9. Zare Mohazabie, M.S., and Shahriari Nogorani, F., Surf. Coat. Technol., 378(25), 125066-1-8. 10. Bozza, F., Bolelli, G., Giolli, C., Giorgetti, A., Lusvarghi, L., Sassatelli, P., Scrivani, A., Candeli, A., and Thoma, M., Surf. Coat. Technol., 239: 147-159. 11. Shankar, S., and Seigle, L.L., Metall. Trans. A, 9(10): 1467-1476. 12. Mohseni Bababdani, S. and Shahriari Nogorani, F., Metall. Mater. Trans. A, 45: 2116–2122. 13. Chen, Q., Huang, L.H., Liu, H.S., Zheng, F., and Jin, Z.P., J. Phase Equilibria Diffus., 34(5): 390-402. 11

The 30th Special CU-af Seminar 2022 September 2, 2022 14. Noebe, R.D., Bowman, R.R., and Nathal, M.V. (1996). The Physical and Mechanical Metallurgy of NiAl. In N.S. Stoloff & V.K. Sikka (Eds.), Physical Metallurgy and processing of Intermetallic Compounds (pp. 212-296), Boston: Springer US. 15. Wierzba, B., Romanowska, J., Zagula-Yavorska, M., Markowski, J., and Sieniawski, J., High Temp. Mater. Process., 34(5): 495-502. 16. Doychak, J., Smialek, J.L., and Barrett, C.A. (1988). The oxidation of Ni-rich Ni-Al intermetallics (NASA TM-101455). 17. Davis, J.R. (1997), ASM Specialty Handbook: Heat-Resistant Materials, United States of America: ASM International. 18. Khanna, A.S. (2013). High-Temperature Oxidation. In M. Kutz (Eds.), Handbook of Environmental Degradation of Materials (2nd Edition) (pp. 127-194) Elsevier. 19. Brumm, M.W. and Grabke, H.J., Corros. Sci., 33(11): 1677-1690. 20. An, T.F., Guan, H.R., Sun, X.F. and Hu, Z.Q., Oxid. Met., 54(3/4): 301–316. 21. Lee, D.B., and Santella, M.L., Mater. Sci. Eng. A, 374(1): 217-223. 22. Kaplin, C., and Brochu, M., Surf. Coat. Technol., 205(17–18): 4221-4227. 12



Comparative Study of Silk Fibroin-Based Hydrogels and Their Potential As Material for 3-dimensional (3D) Printing Juthamas RATANAVARAPORN

The 30th Special CU-af Seminar 2022 September 2, 2022 Comparative Study of Silk Fibroin-Based Hydrogels and Their Potential As Material for 3-dimensional (3D) Printing Juthamas RATANAVARAPORN1* Abstract Three-dimensional (3D) printing is regarded as a critical technology in material engineering for biomedical applications. From a previous report, silk fibroin (SF) has been used as a biomaterial for tissue engineering due to its biocompatibility, biodegradability, non-toxicity and robust me-chanical properties which provide a potential as material for 3D-printing. In this study, SF-based hydrogels with different formulations and SF concentrations (1–3%wt) were prepared by natural gelation (SF/self-gelled) and sodium tetradecyl sulfate-induced (SF/STS). From the results, 2%wt SF-based (2SF) hydrogels showed suitable properties for extrusion, such as storage modulus, shear-thinning behavior and degree of structure recovery. The 4-layer box structure of all 2SF-based hydrogel formulations could be printed without structural collapse. In addition, the mechanical stability of printed structures after three-step post-treatment was investigated. The printed structure of 2SF/STS hydrogel exhibited high stability with high degree of structure recovery as 70.4% compared to 2SF/self-gelled construct as 38.9%. The 2SF/STS hydrogel showed a great potential to use as material for 3D-printing due to its rheological properties, printability and structure stability. 1Biomedical Engineering Program, Faculty of Engineering, Chulalongkorn University, Bangkok 15

The 30th Special CU-af Seminar 2022 September 2, 2022 Introduction and Objectives Three-dimensional-printing technology has attracted considerable attention as a promising tool for tissue engineering and regenerative medicine[1]. Structural-complex scaffolds can be precisely designed using software and fabricated in a high resolution using a layer manufacture. The development of printable material is one of the critical parts in 3D-printing researches[2]. The critical features required for printa-ble materials are biocompatibility, bioactivity, biodegradation and mechanical stability[3,4]. In addition, the material should be able to encapsulate cells and maintain cell viability for long-term tissue culture. Natural-derived polymers are attractive candidates to be applied as bioink. Silk fibroin (SF) is a naturally derived fibrous protein produced by domesticated Bombyx mori silkworms. Due to its characteristics, including biocompatibility, robust mechanical properties, biodegradability, sterilizability, high thermal stability and microbial resistance, SF has been widely studied in biomedical fields[5,6]. The primary structure of SF contains a large number of glycine–alanine repetitive sequences which are accounted for the formation of thermodynamically stable β-sheet structure. There-fore, comparing to other natural polymers, SF possesses a slower degradation rate as well as higher mechanical robustness[7]. From our previous studies, SF-based materials were developed and applied for various applications such as bone tissue regenera-tion, wound healing and drug-controlled release systems[8–10]. Moreover, the cytotoxicity of SF-based bone scaffold has been proved according to ISO 10993[11]. Different techniques have been reported for fabrication of various Thai SF formats such as film[12], scaffold[13], tube[14], sponge[15], microsphere[16], fiber[17] and hydrogel[18–20]. For 3D-printing, SF-based hydrogels showed an interesting feature which could be appropriate to serve as the material for 3D-printing, e.g., high water content, shape plasticity, suitable rheological properties, low surface tension and the ability of crosslinking[21]. Generally, SF hydrogels can be spontaneously formed from the regenerated SF so-lution within a couple of weeks to months by self-assembly processes[22]. The long gelation time of self-gelled SF may limit its application. Different strategies were in-troduced to induce the rapid gelation of SF. The gelation of SF through enzymatic crosslinking can be induced using enzymes, such as horseradish peroxidase or transglutaminase, in which the gelation can occur under physiological conditions, allowing the cell encapsulation during the gelation[23]. Moreover, photopolymerization, and the chemical crosslinkers, such as carbodiimides, glutaraldehyde, or genipin were used. Our previous studies demonstrated various formulations of SF-based hydrogels that can be induced by different agents, such as alcohols[24], an anionic surfactant, sodium tetradecyl sulfate (STS)[25], and a phospholipid 1, 2-dimyristoyl-sn-glycerol-3-phospho-(1’-rac-glycerol) (DMPG)[26]. STS is as anionic surfactant that have been shown to accelerate the gelation of SF rather than the cationic and nonionic surfactant. Moreover, STS has been used in medical products approved by the U.S. Food and Drug Administration (US-FDA). DMPG is a phosphorylglycerol with negative charged am-phipathic lipids. It has the ability to induce conformational changes in several water-soluble proteins due to electrostatic and hydrophobic interactions. For STS and DMPG, the gel formation time ranged from 20 minutes to less than an hour depending on the amount of the additivesand the gelation mechanisms. The electrostatic and hydrophobic interactions between SF and either STS or DMPG induced the structural transition from the amorphous random coil to the stable β-sheet structure, leading to the gel formation. In addition, the cytocompatibility and in vitro degradation of SF/STS and SF/DMPG hydrogels were also 16

The 30th Special CU-af Seminar 2022 September 2, 2022 demonstrated. Our preliminary study on the application of these SF-based hydrogels in 3D-printing have been performed. Interestingly, we found that SF-based hydrogels induced by STS and DMPG tended to have printability potential. Therefore, it is worth exploring the properties and printability of these SF-based hydrogels and introducing them as material for 3D-printing applications. In this study, the gelation time, rheological properties, printability and post-treatment processes, and chemical structure of SF-based hydrogels induced by STS was systematically investigated in comparison to those of the self-gelled SF hydrogel. The information from this study would be useful for the selection of suitable SF-based hydrogels as material for 3D-printing application. Methods Preparation of Regenerated Silk Fibroin Solution Thai Bombyx mori silk cocoons (Nangnoi Srisaket 1) were kindly supplied from Queen Sirikit Sericulture Center, Nakhon Ratchasima, Thailand. The regenerated aqueous SF solution was prepared following an established protocol[27]. Firstly, silk cocoons were boiled in 0.02 M sodium carbonate (Na2CO3) for 20 min to remove sericin and then washed with deionized water before leaving to dry. Subsequently, the degummed silk fibers were dissolved in 9.3 M lithium bromide (LiBr) at 60 °C for 4 h. The SF solution was then dialyzed against deionized water for 3 days using dialysis membrane (Molecular weight cut-off = 12000–16000 Da, Vikase Company Inc., Osaka, Japan) to remove salt ions[28]. Then, the dialyzed SF solution was centrifuged to remove impurities. The final concentration of the obtained aqueous SF solution was approximately 6–7%wt. The regenerated SF solution was stored at 4 °C until used. Preparation of SF-based Hydrogels Three formulations of SF hydrogels including natural gelation (SF/self-gelled) and STS-induced (SF/STS) were prepared according to the methods reported in our previous researches with slight modifications (Table 1)[25]. The SF solution was loaded in the syringe and the hydrogel was formed within the syringe used for 3D-printing. The final concentrations of SF hydrogels were fixed at 1%, 2% and 3%wt while the optimal concentrations STS and DMPG were selected from previous studies. For SF/STS hydrogel, the final concentration of STS was fixed at 0.09%w/v with a ratio of SF to glycerol at 3:1 (w/w). As a control, the SF/self-gelled hydrogel was prepared by incubation of SF solution at 60 °C until complete gelation was occurred. After the complete gelation, all hydrogels were further incubated at 60 °C for 24 h before 3D-printing process. 17

The 30th Special CU-af Seminar 2022 September 2, 2022 Gelation Time Determination The mixed solutions of SF and STS were prepared as previously described at different concentrations of SF (1%, 2% and 3%). The gelation time was determined from the turbidity change by a measure of optical density[28]. The measurement was monitored at 550 nm using a Microplate Reader (FLUOstar Omega, Thermo Fisher Scientific, Massachusetts, USA) at 37 °C. Then, the gelation time was defined as the point where the average optical density reached as half-maximum value. Measurement of Rheological Properties The Haake Mars Rheometer (Thermo Fisher Scientific, Massachusetts, USA) with temperature maintained at 37 °C was used for all experiments. A 35 mm parallel plate geometry was used with a gap value of 1 mm. At the first step, a time-sweep experiment was conducted by applying 0.5% strain and a frequency of 1 Hz. This test measures complex modulus change after SF solution has been loaded as time advances. Secondly, a frequency- sweep experiment was carried out with an applied strain of 0.5% and the frequency ranged from 0.5 to 100 Hz to determine the mechanical stability of the hydrogels. For the third step, the shear-thinning behavior and structure recovery of SF hydrogel were measured using a thixotropic analysis. These properties were analyzed by obtaining cycle through a 2-step experiment including up curve by increasing shear rate as 100 s−1 for 2 min and down the curve by decreasing shear rate to 0.1 s−1 for 1 min and repeated for more 2 cycles. From the experiment data, the degree of structure recovery (% recovery) was calculated according to the following equation (1) %recovery=ƞ2/ƞ1 ×100 (1) where ƞ1 represents the average viscosity of hydrogel at the first cycle before applied shear rate, and ƞ2 represents the average viscosity of hydrogel at the second cycle after applied shear rate as 100 s−1. 3D-printing and Post-treatment Processes Before 3D-printing, all SF-based hydrogel formulations were prepared and gelled in a syringe and then connected to 3D Bioplotter (3D-Bioplotter® Manufacturer Series, EnvisionTEC, Gladbeck, Germany). To fabricate the printed construct, the printing parameters as extruded pressure and movement speed were optimized for each SF-based hydrogel formulations (Table 2). The SF hydrogels were extruded through the nozzle with diameter as 0.41 mm. The construct design was made using the Perfactory RP software (EnvisionTEC) by converting designed part file (STL) file to printing code (G-code). The 3D-structure was printed as a 30 × 30 ×5 mm3 box with a 4-layer thickness. The inner pattern was printed in an alternating pattern, in which each one was aligned 90° from the layer below it. The printed hydrogels obtained were placed on the Petri dish for further post-treatment processes. 18

The 30th Special CU-af Seminar 2022 September 2, 2022 For post-treatment processes, the printed hydrogels were crosslinked by UV irradiation for 20 min at room temperature, then dried at room temperature for 4 h and followed by 70%wt ethanol immersion for 2 h. After that, the printed hydrogels were washed and stored in DI water until further tests. Secondary Structure Analysis Secondary structures of SF-based hydrogels were determined through Fourier Transform Infrared Spectroscopy (FTIR, using IRPrestige 21, Shimadzu, Kyoto, Japan). in an attenuated total reflection (ATR) mode. The lyophilized samples were finely ground and casted onto the ZnSe cell, before collecting the spectrum from 4000 to 800 cm−1 with 2.0 cm−1 resolution and 1 cm−1 interval. The chemical structure of the SF solution, SF/STS and SF/DMPG mixtures, the printed hydrogel before and after post-treatment were evaluated. To quantify the secondary structure, Fourier self-deconvolution and curve fitting of the infrared spectra were performed. The deconvolution of the amide I region (1575–1725 cm−1) were conducted using Omnic 8.0 software. The Voigt line shape with a half bandwidth of 10 cm−1 and an enhancement factor of 3.0 were applied. The curve fitting of the deconvoluted spectrum was performed using Origin Pro 9.0 software. The amount of β-sheet conformation was calculated from the sum of the percentage of area under the peaks in the 1616 to 1637 cm−1 and 1696 to 1703 cm−1 regions. The amount of random coil (1638–1655 cm−1), alpha helix (1656–1662 cm−1), and β-turn (1663–1696 cm−1) structures were also calculated from the area under the defined peaks. Results Gelation Time of SF-based Hydrogels Gelation times of SF-based hydrogels were shown in Table 1. The addition of STS in the SF solution significantly accelerated the gelation. It can be observed that the gelation time of SF/STS hydrogels decreased with an increasing SF concentration. For SF/STS formulation, the 3SF/STS and 2SF/STS hydrogels were formed within 19 and 36 min, respectively, while the gelation of 1SF/STS was not completed within 2 h. On the other hand, the spontaneous gelation of regenerated SF solution took over two weeks; therefore, the gelation time of SF/self-gelled cannot be measured. Rheological Properties of SF-based Sol-gel The time-sweep measurement of SF-based hydrogels (Figure 1 a and b) reported the storage modulus (G’) and loss modulus (G”) over time. For SF/STS, the solution possessed a fluid-like behavior in the beginning as G’ and G” were not clearly distinguishable. Then, both G’ and G” of the hydrogels increased before maintain a plateau at their respective gelation time. The G’ was higher than the G” for all formulations that was the characteristic of formation from sol-state to gel-state. However, in case of SF/self-gelled, the completed hydrogel was used to measure due to its long gelation time, therefore, the G’ was higher than G” with constant value from an initial time. 19

The 30th Special CU-af Seminar 2022 September 2, 2022 Figure 1: Time-sweep (a and b) and frequency-sweep (d and e) experiments showing storage (G’) and loss (G”) modulus of SF-based hydrogels with different SF concentrations. The frequency-dependent behavior of SF-based hydrogels was assessed using oscillatory frequency sweeps as shown in Figure1 d and e. For all SF-based hydrogels, the storage modulus was constant within the frequency range of 0.1–10 Hz, before becoming fluctuated when applied the frequency was over 10 Hz. The storage modulus of 1SF/self-gelled, 2SF/self-gelled and 3SF/self-gelled were 332.5, 2739.9 and 10,614.2 Pa, respectively. For SF/STS formulation, the storage modulus showed 52.2, 790.7 and 2294.4 Pa for 1SF/STS, 2SF/STS and 3SF/STS, respectively. The storage modulus increased with increasing SF concentration for all SF-based hydrogel formulations. The shear-thinning behavior and structure recovery of SF-based hydrogels measured by thixotropic analysis were shown in Figure 2. All SF-based hydrogels showed shear thinning behavior with different degrees of structure recovery. An initial viscosity of SF-based hydrogel increased with increasing SF concentration for all formulations. When a shear rate of 100 s−1 was applied, the viscosity of all SF-based hydrogels decreased rapidly. After the shear rate was decreased to 0.1 s−1, the viscosity of SF-based hydrogels was subsequently recovered closely to their initial value. The structural recovery could be observed at every cycle of shear rate changed. Table 2 demonstrated the calculated recovery percentage of SF-based hydrogels in the resting stage after the high shear rate was applied. The degree of structure recovery of SF/self-gelled increased with an increasing of SF concentrations. For SF/STS formulation, the 2SF-based hydrogels showed the highest degree of structure recovery. The highest 20

The 30th Special CU-af Seminar 2022 September 2, 2022 percentage of structural recovery of each formulation can be observed as 44.2% and 70.4% for 3SF/self-gelled and 2SF/STS, respectively. As the result, the SF-based hydrogels induced with STS exhibited higher degree of structure recovery than SF/self-gelled formulation. Figure 2: Viscosity of (a–c) SF/self-gelled and (d–f) SF/STS hydrogels when applying different shear rate, indicating shear thinning behavior and structural recovery of the hydrogels. Printability Printability of SF-based hydrogels was assessed by printing a 4-layer construct of box model. From the preliminary study, a clogging of hydrogel at the nozzle was observed when 3SF-based hydrogels were printed. Consequently, the 2SF-based hydrogels were chosen for extrusion from 3D-bioprinter. The 2SF-based hydrogels were printed and crosslinked to stabilize the printed constructs as shown in Figure 3. During printing, the temperature of the syringe was maintained at 37 °C. The SF-based hydrogels were extruded on the Petri dish at the optimized printing conditions. The deposition temperature was set at 25 °C. Low deposition temperature helped to stabilize the structure of printed constructs by preventing the spread or deformation of printed hydrogel. All 2SF-based hydrogels could be printed into a 4-layer box model without collapse. Subsequently, the printed constructs were crosslinked under UV-irradiation for 20 min, air dried at room temperature for 4 h, and immersion in 70%wt ethanol for 2 h. After post-treatment processes, it can be observed that the structures of printed constructs from 2SF/STS hydrogels were maintained, while those of 2SF/self-gelled hydrogel were partially deformed. 21

The 30th Special CU-af Seminar 2022 September 2, 2022 Figure 3: Structure of 4-layer box model constructs printed from 2SF-based hydrogels before and after post-treatment at each condition. Chemical Structure and Secondary Conformation of SF-based Hydrogels The chemical structures of the 2SF/self-gelled and 2SF/STS constructs after printed and post-treatment were characterized through ATR-FTIR technique using 2SF solution as a control (Figure 4). The spectra of 2SF-based hydrogels showed the characteristic peaks of protein including amide I, II and III at 1650, 1550 and 1300 cm−1, respectively. The characteristic peaks of CH3 and CH2 stretching at 2955, 2873 and 2849 cm−1 were clearly observed on the spectra of 2SF/STS formulation due to the structure of inducing agents. The FTIR spectra were deconvoluted of each secondary conformation of the hydrogels after printed and after post-treatment were shown in Figure 4d. All SF-based hydrogels exhibited higher content of β-sheet compared to SF solutions. To compare the β-sheet content between after printed and after post-treatment, the β-sheet content increased while the random coil content seemed to decrease. Figure 4: ATR-FTIR spectra of SF-based hydrogels (a) 2SF/self-gelled, (b) 2SF/STS and (c) Percentage of secondary conformation of the constructs after printed (P) and post-treatment (T), deconvoluted from the amide I region of spectra using SF solution (S) as a control. 22

The 30th Special CU-af Seminar 2022 September 2, 2022 Discussion In general, the regenerated SF solution can turn gel through a self-assembly process by a chain rearrangement and hydrogen bonding that produce the transformation of random coil to a stable β-sheet form[29]. This gelation processes takes over two weeks, which is not practicable for the applications as cell-encapsulated bioink or injectable hydrogels. To reduce the gelation time, some amphipathic chemicals, such as surfactants and phospholipids were added into SF solution[25,26]. It can enhance the formation of β-sheet structure due to electrostatic and hydrophobic interactions. In this study, the mixtures of SF with STS showed the complete gelation within about 8-96 min while the SF/self-gelled formulations took approximately more than 2 weeks. Moreover, the gelation time of SF solution depends on SF concentration that a higher SF concentration resulted in a shorter gelation time. In addition, the mixture of glycerol also reduced the gelation time of SF/STS hydrogels due to the polarity and hydrophobicity[24]. Moreover, glycerol can act as a physically stabilized agent that beneficial for the extrusion of SF/STS hydrogels[30]. When SF mixed with glycerol, the homogeneity of SF/STS hydrogel was enhanced, and the printable hydrogel was obtained. The shear-thinning properties as well as the structure recovery of the printing materials are required to confirm the applicability for material which the SF-based hydrogels should be extruded from the nozzle, encounter the high shear stress, and able to recover the initial structure immediately after finishing the printing process[31]. From Figure 1, the storage modulus of the hydrogels increased with an increasing of the SF concentration. These could imply that SF hydrogels with lower SF concentration required a lower pressure for extrusion and would be easier to be printed comparing to the stiffer hydrogels with a higher SF concentration. It is noted that the storage modulus of SF/self-gelled hydrogel was higher than SF/STS hydrogels. In general, the regenerated SF solution can turn gel through a self-assembly process by a chain rearrangement and hydrogen bonding to transform random coil to a stable beta sheet structure. With the long incubation time, the SF structure can crystallize and form more stable structure, resulting in improved strength of SF hydrogel. When the additive as STS is mixed with the SF solution, it can enhance the formation of beta sheet structure due to the electrostatic and hydrophobic interactions. Adding gel enhancing chemicals also interferes with the hydrogen bond between the protein and water due to the formation of hydrogen bond in their structures and the steric hindrance effect[22]. Thus, mixture of STS might lead to a decrease in the hydrogen bonds, resulting in a lower storage modulus of SF/STS compared to SF/self-gelled hydrogel. Furthermore, the elastic behavior was predominated over the viscous nature in the frequency range of 0.1-10 Hz. It could be explained that all SF-based hydrogel formulations had the gel-state in this frequency range. For this study, it found that 1SF-based hydrogels would not be mechanically sufficient to maintain their architectural structure upon extrusion due to the lowest storage modulus and the lowest degree of structure recovery. On the other hand, there was a clogging of hydrogel at the nozzle when using 3SF-based hydrogels even printed at high pressure. In the case of 2SF-based hydrogel, it showed suitable storage modulus for extrusion without clogging behavior. Consequently, 2SF-based hydrogel was chosen for a single-syringe injectable material for 3D-printing. From the previous report, the highest cell viability upon printing is observed when a low polymer content hydrogel is used in combination with low pressure .[32] Therefore, 23

The 30th Special CU-af Seminar 2022 September 2, 2022 the SF concentration and extruded pressure for 2SF-based hydrogels could be beneficial for cell-laden and cell viability during printing process. Moreover, these hydrogels exhibited high degree of structure recovery and shear-thinning behavior which can allow ready flow of the fluid through confined nozzle diameters of the bioprinter. At the same time, immediately after extrusion, the fluid can demonstrate instant shape stability to assemble a 3D-structural assembly without collapse. The printing parameters were optimized for each 2SF-based hydrogel formulations. The 2SF/self-gelled formulation required higher extruded pressure than 2SF/STS due to the higher storage modulus. During the printing process, 2SF-based hydrogel would be attained because of a high maintaining the shape of the as-printed and a less pressure required. After printed, there was no evidence of structural collapse of the 4-layer box structure. This supports flow behavior of 2SF-based hydrogel that can extrude in a filament rather than a drop-let during 3D-printing. Then, the printed structure was treated by three-step post-treatment. Initially, the printed hydrogels were irradiated by UV light which was proposed to enhance the crosslinking of SF molecules. Generally, the water molecules presented in the SF hydrogel could be intrigued by UV and could produce a large number of free radicals. The aromatic side chains of tyrosine and phenylalanine in the SF would be attacked by the free radicals, leading to the bond formation[33]. However, only the UV treatment was not enough to crosslink and stabilize the printed structures, then another treatment step was required. Therefore, the post-treatment with ethanol immersion was conducted. The ethanol immersion resulted in an immediate effect on the secondary structures by diffusion into the printed hydrogels and induced β-sheet formation[34,35]. However, the deformation of the printed 2SF/self-gelled constructs was observed after ethanol immersion for 2 h. It can be explained that the degree of structure recovery for 2SF/self-gelled was lower than 2SF/STS. Consequently, the printed structure was easily broken from diffusion of ethanol into the printed structure. Additionally, the induced β-sheet content after three-step post-treatment was investigated by ATR-FTIR technique by compared between after printed and after post-treatment processes using 2SF solution as control. There are three structures in which silk fibroin can exist: silk I, II and III, where silk I is a structural form consisting of more water and less β-sheet content, silk II is a structural form consisting of β-sheet and silk III is a three-fold helical structure that is observed at the air-water interface[36]. Furthermore, the β-sheet content of 2F-based hydrogel increased after post-treatment processes compared to after printed. It can be seen that the structure transformation to stable form with enhanced structure stability was occurred through the three-step post-treatment. From our results, the 2SF/STS hydrogel showed better flow behavior, higher structure recovery and higher structure stability than 2SF/self-gelled h y d r o g e l d u r i n g p r i n t i n g a n d a f t e r p o st - t r e a t m e n t p r o c e s s e s . I n a d d i t i o n , the non-cytotoxicity of SF/STS hydrogels were shown in previous studies[26]. Therefore, these SF-based hydrogel formulations demonstrated the potential as material for 3D-printing and applied for several biomedical applications, such as tissue engineering. Conclusion In summary, SF-based hydrogel with different formulations were studied their properties to use as material for 3D-printing. The SF hydrogel with inducing agent as STS can reduce the gelation time due to the electrostatic and hydrophobic interactions. 24

The 30th Special CU-af Seminar 2022 September 2, 2022 Each SF-based hydrogel formulations were optimized by SF concentration to generate materials optimal under printing conditions. We found that the hydrogel with 2%wt SF was optimal for extrusion into the 4-layer box construct in all formulations without structural collapse. The three-step post-treatment can enhance the structure stability via induced β-sheet content of printed structure. The 2SF/STS hydrogel exhibited a good flow behavior without the nozzle clogging during the printing process. In addition, these hydrogels showed a high degree of structure recovery after printing and a high structure stability after post-treatment processes. Therefore, our results showed that SF-based hydrogels have the potential proper-ties to be used as material for 3D-printing. References 1. Wang, Q.; Han, G.; Yan, S.; Zhang, Q., Materials 2019, 12, 504. 2. Gungor-Ozkerim, P.S.; Inci, I.; Zhang, Y.S.; Khademhosseini, A.; Dokmeri, M.R., Biomater. Sci. 2018, 6, 915–946. 3. Lee, H.J.; Kim, Y.B.; Ahn, S.H.; Lee, J.S.; Jang, C.H.; Yoon, H., Adv. Healthc. Mater. 2015, 4, 1359–1368. 4. Loo, Y.; Lakshmanan, A.; Ni, M.; Toh, L.L.; Wang, S.; Hauser, C.A.E., Nano Lett. 2015, 15, 6919–6925. 5. Altman, G.H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R.L.; Chen, J., Biomaterials 2003, 24, 401–416. 6. Vepari, C.; Kaplan D.L., Prog. Polym. Sci. 2007, 32, 991–1007. 7. Correia, C.; Bhumiratana, S.; Yan, L.P.; Oliveira, A.L.; Gimble, J.M.; Rockwood D., Acta Biomater. 2012, 8, 2483–2492. 8. Ratanavaraporn, J.; Damrongsakkul, S.; Kanokpanont, S.; Yamamoto, M; Tabaya Y., J. Biomater. Sci. 2011, 22, 1083–1098. 9. Wongputtaraksa, T.; Ratanavaraporn, J.; Pichyangkura, R.; Damrongsakkul, S., J. Biomed. Mater. Res. Part. B: Appl. Biomater. 2012, 100B, 2307–2315. 10. Lerdchai, K.; Kitsongsermthon, J.; Ratanavaraporn, J.; Kanokpanont, S.; Damrongsakkul, S., J. Pharm. Sci. 2016, 105, 221–230. 11. Tungtasana, H.; Shuangshoti, S.; Shuangshoti, S.; Kanokpanont, S.; Kaplan, D.L.; Bunaprasert, T., J. Mater. Sci.: Mater. Med. 2010, 21, 3151–3162. 12. Zhang, X.-S.; Brugger, J.;Kim, B., Nano Energy 2016, 20, 37–47. 13. Ghosh, S.; Parker, S.T.; Wang, X.; Kaplan, D.L.; Lewis, J.A., Adv. Funct. Mater. 2008, 18, 1883–1889. 14. Rockwood, D.N.; Preda, R.C.; Yucel, T.; Wang, X.; Lovett, M.L.; Kaplan, D.L., Nat. Protoc. 2011, 6, 1612–1631. 15. Sionkowska, A., Płanecka, A., J. Mol. Liq. 2013, 178, 5–14. 16. Cao, Z.; Chen, X.; Yao, J.; Huang, L.; Shao, Z., Soft Matter 2007, 3, 910–915. 17. Yi, B.; Zhang, H.; Yu, Z.; Yuan, H.; Wang, X.; Zhang, Y., J. Mater. Chem. B 2018, 6, 3934–3945. 18. Tabatabai, A.P.; Kaplan, D.L. and Blair, D.L., Soft Matter 2015, 11, 756–761. 19. Piyanuch Thitiwuthikiat, M.l.; Saito, T.; Asahi, M.; Kanokpanont, S.; Tabata, Y. A., Tissue Eng. Part A 2015, 21, 1309–1319. 20. Laomeephol, C.; Ferreira, H.; Kanokpanont, S.; Neves, N.M.; Kobayashi, H.; Damrongsakkul, S., Int. J. Pharm. 2020, 589, 119844. 25

The 30th Special CU-af Seminar 2022 September 2, 2022 21. Rodriguez, M.J.; Dixon, T.A.; Cohen, E.; Huang, W.; Omenetto, F.G.; Kaplan, D.L., Acta Biomater. 2018, 71, 379–387. 22. Agostinacchio, F.; Mu, X.; Dire, S.; Motta, A.; Kaplan, D.L., Trends in Biotechnology 2021, 39(7): p. 719-730. 23. Yin, Z.; Wu, F.; Xing, T.; Yadavalli, V.K.; Kunda, S.C.; Lu, S., Rsc Adv. 2017, 7, 24085–24096. 24. Kaewprasit, K.; Kobayashi, T.; Damrongsakkul, S., Int. J. Biol. Macromol. 2018, 118, 1726–1735. 25. Chantong, N.; Damrongsakkul, S.; Ratanavaraporn, J., J. Surfactants Deterg. 2019, 22, 1395–1407. 26. Laomeephol, C.; Guedes, M.; Ferreira, H.; Reis, R.L.; Kanokpanont, S.; Damrongsakkul, S.; Neves, Nuno M., J. Tissue Eng. Regen. Med. 2020, 14, 160–172. 27. Vachiraroj, N.; Ratanavaraporn, J.; Damrongsakkul, S.; Pichyangkura, R.; Banapresert, T.; Kanokpanont, S., Int. J. Biol. Macro-mol. 2009, 45, 470–477. 28. Jetbumpenkul, P.; Amornsudthiwat, P.; Kanokpanont, S; Damrongsakkul, S., Int. J. Biol. Macromol. 2012, 50, 7–13. 29. Matsumoto, A.; Chen, J.; Collette, A.L.; Kim, U.J.; Altman, G.H.; Cebe, P., J. Phys. Chem. B 2006, 110, 21630–21638. 30. Jose, R.R.; Brown, J.E.; Polodo, K.E.; Omenetto, F.G.; Kaplan, D.L., Acs Biomater. Sci. Eng. 2015, 1, 780–788. 31. Hospodiuk, M.; Dey, M.; Sosnoski, D.; Ozbolat, I.T., Biotechnol. Adv. 2017, 35, 217–239. 32. Cidonio, G.; Glinka, M.; Dawson, J.I.; Oreffo, R.O.C., Biomater. 2019, 209, 10–24. 33. Zheng, H.; B. Zuo, J. Mater. Chemistry B 2021, 9(5): p. 1238-1258. 34. Rodriguez, M.J.; Brown, J.; Giordano, J.; Lin, S.J.; Omenetto, F.G.; Kaplan, D.L., Biomater. 2017, 117, 105–115. 35. Hölzl, K.; Lin, S.; Tytgat, L.; Vlierberghe, S. Van; Gu, L.; Ovsianikov, A., Biofabrication 2016, 8, 032002(1)–023002(19). 36. Wray, L.S.; Hu, X.; Gallego, J.; Georgakoudi, I.; Omenetto, F.G.; Schmidt D., J. Biomed. Mater. Research. Part. Bapplied Biomater. 2011, 99, 89–101. 37. Chantong, N., Engineering program in Biomedical Engineering. 2017, Chulalongkorn University. 26



Improvement of Aqueous Zinc-ion Battery Performance by combining Experiments and Molecular Dynamics Simulations Manaswee SUTTIPONG

The 30th Special CU-af Seminar 2022 September 2, 2022 Improvement of Aqueous Zinc-ion Battery Performance by combining Experiments and Molecular Dynamics Simulations Manaswee SUTTIPONG1* Abstract Molecular dynamics (MD) simulations were performed to investigate structural and dynamical properties of ions in aqueous zinc sulfate (ZnSO4) electrolyte at various concentrations, with and without 0.1 M manganese sulfate (MnSO4) additive. The simulation results showed the effects of ZnSO4 concentration and addition of MnSO4 additive on solvation structure of Zn2+ ions. At low concentrations of ZnSO4, Zn2+ ions were coordinated by six water molecules. The addition of MnSO4 resulted in a strong interaction between Zn2+ ions and water molecules, particularly at 2.0 M ZnSO4. The self-diffusion coefficients and ionic conductivities of ions obtained for the systems of ZnSO4 (at 0.1, 0.5, and 1.0 M) with adding MnSO4 were higher than those without MnSO4. However, at 2.0 M ZnSO4, the addition of MnSO4 resulted in decreasing the self-diffusion coefficients and ionic conductivities of ions. As compared with experiments, the trend of ionic conductivity was in good agreement with MD simulations. 1Department of Chemical Technology Faculty of Science, Chulalongkorn University Bangkok, Thailand 29

The 30th Special CU-af Seminar 2022 September 2, 2022 Introduction and Objectives The growing demand for green and sustainable energy storage for various applications, such as portable and flexible electronics and grid-scale energy storage systems, has stimulated the development of advanced energy storage technologies. Among various electrochemical energy storage devices, battery is a kind of viable technology due to their ability to conserve the intermittent energy converted from renewable resources, such as solar, wind, and mechanical energy, which can be quickly released into the power grid when needed. Currently, lithium ion batteries (LIBs) have dominated the secondary battery applications ranging from portable electronic gadgets to automotive vehicles due to their high energy density, power density and mature manufacturing technology[1,2]. However, the long-term concerns about safety issues, high cost, and limited reserves of lithium for the continuous manufacturing of LIBs have prompted the search for alternative energy storage technologies, such as sodium ion batteries (SIBs), potassium ion batteries (PIBs), aluminum ion batteries (AIBs), zinc ion batteries (ZIBs) and so on[3,4,5,6]. Aqueous batteries have attracted special attention in recent years because of the advantages of non-flammability, low cost and good rate of performance .[7,8] In this regard, aqueous ZIBs have attracted considerable attention due to their high electrical conductivity, easy processability, high stability in water, non-flammability, low toxicity, and low price (high natural abundance and mass production). More importantly, Zn anode possesses a high theoretical capacity (820 mAh/g or 5854 mAh/cm3), suitable redox potential (-0.76 V vs. standard hydrogen electrode) and two-electron transfer during redox reaction, leading to a high energy density. Electrolytes are one of the most important parts of aqueous ZIBs system since they are responsible for the conduction of ions between the electrodes. An eligible electrolyte should maintain good zinc deposition/stripping reversibility and release a broad electrochemical window. In addition, the role of electrolyte additives in aqueous ZIBs is prominent. It was found that the additives can improve ionic conductivity by augmenting the type and number of conductive ions[9], adjust ion dissolution balance[10], improve capacity and stability[11], and enhance the reversibility and capacity retention[12]. Understanding fundamental electrolyte interactions and transport properties of ions are critical aspects controlling bulk electrolyte parameters, and thus the battery performance. However, detail regarding these properties is still rare. In this work, we employed molecular dynamics (MD) simulation, to investigate the solvation structure and the dynamical properties of aqueous zinc sulfate (ZnSO4) electrolyte with and without manganese sulfate (MnSO4) additive. The ionic conductivity obtained from the simulations was compared to the experimental data. Methods Computational methods The simulation systems, composed of Zn2+‒SO42- in aqueous solution with and without the addition of Mn2+‒SO42- additive, were studied using MD simulations. We considered four different concentrations of ZnSO4, including 0.1, 0.5, 1.0, 2.0 M in which the concentration of MnSO4 additive was 0.1 M. The OPLS-AA force field was applied to describe both zinc salt molecules[13,14]. The SPC/E was the water model employed[15]. The number of water molecules was sufficient to obtain a molecular density (the bulk density was approximately 1000 g/L for all systems), comparable to that of liquid water at ambient conditions. The size of simulation box was 6×6×6 nm3. The structures used in this study are given in Figure 1. 30

The 30th Special CU-af Seminar 2022 September 2, 2022 The details of all systems and corresponding number of ions/molecules are summarized in Table 1. Figure 1: Structures of all chemical entities in this study. MD simulations were carried out in a canonical ensemble at 298 K using the GROMACS, version 5.0.4. All systems were simulated for a time step of 0.001 ps. We first performed NPT (isothermal-isobaric) ensemble using Parrinello-Rahman[16,17] at a constant pressure of 1 bar for 1 ns, followed by the NVT (canonical) ensemble using Nose-Hoover thermostat[18] for 10 ns. Periodic boundary conditions were applied in all three Cartesian directions. The long-range electrostatic interactions were calculated with the particle-mesh Ewald method [19,20] in which the Lennard-Jones interactions were treated with a cutoff equal to 8 Å. We used the following equation to derive the radial distribution plots from pair of atoms x-y correlation functions. Here, n(r) represents the number of y particles at a radial distance r from the position of x. 4πr^2dr is the volume of a shell of thickness dr at the radial distance r. ρ is the bulk density of y particles. The radial distribution function enables quantification of molecular configurations in aqueous electrolytes of various ions and molecules. In addition to radial distribution function, we calculated the number integral or coordination number, which illustrates the number density of y particles around the reference x at the radial distance r and are mathematically expressed as 31

The 30th Special CU-af Seminar 2022 September 2, 2022 where r_min is corresponding to the minimum of first peak in the g_(x,y) (r). The transport properties of the systems, involving self-diffusion coefficient (D)[21] and ionic conductivity (σ), are related to the movement of particles and were calculated using the MD trajectories. The self-diffusion coefficient indicates the pace at which the ions/molecules in the system are transported. Einstein relation was used for calculating the self-diffusion coefficient and is expressed as where MSD(t) = [r(t)- r(0)]2 is mean square displacement of a molecular center of mass during time.r(t) and r(0) denotes the position of the particle at t = 0 and t, respectively. t is time and < > denotes an ensemble average. The Nernst-Einstein equation can be used for computing the ionic conductivity[22,23] of solution in MD simulations from the summation of cationic and anionic self-diffusion coefficients, and it is mathematically represented as where e is the electric charge. V is the volume of simulation box.kB is the Boltzmann constant. T is the temperature. n_+ and n_- are the number of cations and anions, respectively. The q_+ and q_- are point charges of ions in the model.D+ and D_- are self-diffusion coefficients of cations and anions, respectively. Experimental methods The aqueous ZnSO4 electrolyte of 0.1, 0.5, 1.0, and 2.0 M, as shown in Figure 2, was used to study the conductivity by using electrochemical impedance spectroscopy (EIS). We prepared a half-cell with two stainless steels grade 304 material of 19.17 mm in diameter. We punched a polypropylene membrane into discs of 25 mm in diameter and used them as a separator. The ZnSO4 electrolyte of 0.4 ml at concentrations of 0.1, 0.5, 1.0 and 2.0 M was added into the separator. The half-cell was covered with a vacuum bag using the vacuum sealer. Figure 3 illustrates the half-cell with two stainless steels, covered with the vacuum bag. The test cell used for impedance measurements is similar to that described elsewhere[24-25]. The membrane was tightly clamped between two stainless steel using half-cells by using fold black clip. Electrochemical impedance measurements, as shown in Figure 4, were carried out by using an Impedance Analyzer (Gamry Interface 1010E potentiostat) controlled by a computer. The experimental data were corrected by both software and the influence of connecting cables as well as by other parasite capacitances. The measurements were carried out using the frequency range from 1000 kHz to 0.5 mHz. The DC current was zero and the AC current was set to 10 mV at room temperature. 32

The 30th Special CU-af Seminar 2022 September 2, 2022 Figure 2: ZnSO4 in aqueous solution. Figure 3: Half-cell with two stainless steels, covered with vacuum bag. Figure 4: Electrochemical impedance measurements The ionic conductivity of electrolyte solutions was obtained by the calculation of bulk resistance (R) through impedance spectroscopy, and it is represented as where σ is the ionic conductivity of the solution. The units of σ are Siemens per meter (S/m). The Siemen is the reciprocal of the ohm, so 1 S = 1/ohm.l is the length carrying a uniform current, A is area and R is the resistance (from impedance measurement). 33

The 30th Special CU-af Seminar 2022 September 2, 2022 Results and Discussion Solvation structure Figure 5: Radial distribution function (solid lines, left y-axis), and coordination number (dashed lines, right y-axis) for Zn-Ow (left) and O(SO42-)-Ow (right) obtained from simulations of ZnSO4 and ZnSO4 + 0.1 M MnSO4 at (a,e) 0.1 M, (b,f) 0.5 M, (c,g) 1.0 M and (d,h) 2.0 M. To understand the solvation structure of Zn2+ ions and SO42- ions, we investigated the radial distribution function and the coordination number of Zn2+ ions and SO42- ions with Ow (oxygen atom of water) Left and right panels of Figure 5 show the Zn2+-Ow and Zn2+-(SO42-) radial distribution function (solid lines, left y-axis) and coordination number (dashed lines, right y-axis) for the systems of ZnSO4 and ZnSO4 + 0.1 M MnSO4 at various concentrations of ZnSO4. The first solvation shell of Zn2+ ions for both systems was found to be around 2 Å at all concentrations, consistent with the previous work[26,27]. When the system concentration increased, the amplitude of the first peak of radial distribution function decreased, 34

The 30th Special CU-af Seminar 2022 September 2, 2022 suggesting less interaction between Zn2+ ions and water molecules. Figure 5 (a) shows the Zn2+-Ow radial distribution function at 0.1 M ZnSO4, with and without 0.1 M MnSO4, the Zn2+ ions were coordinated by approximately six water molecules. The coordination number was found to decrease as the ZnSO4 concentration increased, as shown Figure 5 (b) to (d). This could be due to not enough water molecules being present in the systems to form a solvation shell identical to that obtained in the dilute systems, leading to the formation of different water-salt complexes at different concentrations. For Figure 5 (d), at 2.0 M ZnSO4 concentration, it demonstrates that the addition of MnSO4 resulted in strong interaction of between Zn2+ ions and water molecules, which indicates good solvation structure of Zn2+ ions. From Figure 5, right panels, the first solvation shell around SO42- ions is composed of oxygen from water at 2.7 Å in ZnSO4 with and without 0.1 M MnSO4 for all concentrations. The coordination number was found to not change as the ZnSO4 concentration increased. When adding MnSO4, the coordination number is slightly decreased for all concentrations. Association of salt Figure 6: Radial distribution function (solid lines, left y-axis), and coordination number (dashed lines, right y-axis) for Zn-O(SO42-) obtained from simulations of ZnSO4 and ZnSO4 + 0.1 M MnSO4 at (a) 0.1 M, (b) 0.5 M, (c) 1.0 M and (d) 2.0 M. Association of ions can be understood by studying the radial distribution function of Zn2+-Oanion pair. It involves the computation of oxygen atom of SO42- and their distribution around Zn2+ ions. Figure 6 shows the Zn2+-Oanion radial distribution function (solid lines, left y-axis) and coordination number (dashed lines, right y-axis) for the systems of ZnSO4 and ZnSO4 + 0.1 M MnSO4 at various ZnSO4 concentrations. The Zn2+-Oanion radial distribution function for both systems was observed at around 2 Å, which is in good agreement with the literature of Sun et al.[2]. In Figure 6 (a), the position of first peak represents the position that Zn2+ ions interact closely with SO42- anions. This is due to strong electrostatic force between cations and anions. For ZnSO4 systems, the comparatively smaller double peaks were observed at approximately 3.5 and 4.2 Å, respectively. For ZnSO4 + 0.1 M MnSO4 systems, RDF reveals a small peak at the second solvation shell, which was observed at approximately 35