Phân lập và tuyển chọn một số chủng nấm trichoderma có hoạt tính kháng nấm từ đất trồng cây cây ăn quả và cây công nghiệp tại tỉnh thái nguyên

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY HOANG THI MAI Topic title: ALGAL CELL CULTURE IN MICROFLUIDIC DEVICES AND MICROENVIRONMENT BACHELOR THESIS Study Mode : Full-time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2013 – 2017 Thai Nguyen, 12/6/2017 THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY HOANG THI MAI Topic title: ALGAL CELL CULTURE IN MICROFLUIDIC DEVICES AND MICROENVIRONMENT BACHELOR THESIS Study Mode : Full-time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2013 – 2017 Supervisors : Dr Panwong Kuntanawat Dr Nguyen Xuan Vu Mr Phongsakorn Kunhorm Thai Nguyen, 12/6/2017 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Major Biotechnology Student name Hoang Thi Mai Student ID DTN1353150021 Thesis title Algal cell culture in microfluidic devices and microenvironment Supervisors Dr Panwong Kuntanawat Dr Nguyen Xuan Vu Mr.Phongsakorn Kunhorm Abstract: Arthrospira platensis is a filamentous multicellular cyanobacterium that has two distinct shapes: helical and straight filaments They have high nutritional value, chemical composition such as protein, pigments, antioxidant, fatty acids Microfluidics devices that were applied in various fields such as biological, biomedical, biotechnology and chemical analyses A.platensis was captured in the microfluidics devices in order to observed activation, fragmentation time, change color, life cycles It was performed with total 20 filaments (10 filaments of C005 str and 10 filaments of Central Lab str) in two different conditioned medium The result was based on measure length to comparison growth length, fragmentation time, growth rate of filament and strain In the standard Zarrouk’s medium, length and growth rate of Central Lab str is faster than C005 str, fragmentation time is the same In the stationary from cell culture: Fragmentation was expressed with two filaments of C005 str (rate 40%) and three filaments of Central Lab str (rate 60%) Moreover, the growth rate of Central Lab str was faster than C005 str The both strains of standard Zarrouk’s medium were grew faster than Zarrouk’s stationary from cell culture Key words C005 str, Central Lab str, microfluidic devices, growth length, fragmentation time, growth rate Number of pages 38 i ACKNOWLEDGEMENT Foremost, I would like to express my deep and sincere gratitude to my supervisor Dr Panwong Kuntanawat from the School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (KMUTT), Thailand, for providing me the opportunity to conduct research in his lab and giving me endless support in the past six months His insights, wisdoms, advices and enthusiasm for research have greatly influenced me and made the completion of my dissertation possible I would also like to thank Dr Nguyen Xuan Vu from the Faculty of Biotechnology and Food of Thai Nguyen University of Agriculture and Forestry (TUAF) who used to help, support and give me encouragements during this thesis implementation I would also like to extend my heartfelt thanks to my lectures of Biotechnology and Food Department, TUAF who imparted me a lot of knowledge through four years of university The knowledge not only helped me with my research, but also created a basic and soul foundation for me to start the job in the future Further, I would also like to express my sincere gratitude to Ms Trinh Thi Chung for providing me the opportunity to develop my skills by doing an internship abroad I sincerely thank to the teachers, the laboratory staffs and students at the laboratory for their regards and giving me an opportunity to research in the laboratory I would also especially thank Mr Phongsakorn Kunhorm who always helped, cared, instructed and taught me during my practicing in Thailand Finally, I would like to thank my family and my friends for their love and support I could not have done this without you Many thank and best regards Student Hoang Thi Mai ii TABLE OF CONTENT PART INTRODUCTION 1.1 Background 1.1.1 Microalgae 1.1.2 General Arthrospira platensis 1.1.2.1 Morphology and taxonomy for Arthrospira platensis 1.1.2.2 Effect of temperatures 1.1.2.3 Effect of pH 1.1.3 Microfluidics devices 1.1.3.1 An introduction to soft lithography 1.1.3.2 Advantages of microfluidic for cell culture 1.1.3.3 Microfluidic devices for cell biology 10 1.1.3.4 Microfluidic devices for single cell analysis 11 1.2 Objectives 11 1.3 Scope of study 12 PART 2: METHODS 13 2.1 Equipments and materials 13 2.1.1 Equipments 13 2.1.2 Materials 13 2.1.2.1 Medium culture 13 2.1.2.2 Algal strains 14 2.1.2.3 Microfluidic devices design: an electrostatic using microwell based microfluidic devices 15 2.2 Methods 16 2.2.1 Algal strains cultivation 16 iii 2.2.2 Make microfluidic devices 17 2.2.3 Cell loading and cultivation in the microwell 18 2.2.4 Imaging of cells and analysis methods 19 PART RESULTS AND DISCUSSION 21 3.1 Cells/filaments in the standard Zarrouk’s medium .21 3.1.1 Comparison of growth length of single filament before fragmenting 21 3.1.2 Compare fragmentation time of single filaments 22 3.1.3 Comparison of growth rate of single filaments 23 3.2 Filaments in the Zarrouk’s medium from stationary cell culture 25 3.2.1 Comparison of growth length of single filaments before fragmenting 26 3.2.2 Compare fragmentation time of single filament 26 3.2.3 Comparison of growth rate of single filaments 27 3.3 Compare growth rate of the same strain in the modified standard Zarrouk’s media and Zarrouk’s medium from stationary cell culture .28 3.4 Discussion: Life cycle of Arthrospira platensis .29 PART CONCLUSIONS AND SUGGESTIONS 31 4.1 Conclusions .31 4.1.1 Cells/filaments were cultured in the standard Zarrouk’s medium 31 4.1.2 Cells/ filaments in the Zarrouk’s medium from stationary cell culture 31 4.2 Suggestions 31 REFERENCE 33 iv LIST OF FIGURES Figure 1.1 Various applications of microalgae products for human, animals and industries Figure 1.2 Helical trichomes and straight of Arthrospira platensis The scale bar (a)=40 µm, (b)=20 µm, respectively (Source: C.Sili, 2012) Figure 1.3 The microfluidic devices: (a) including layers: positively charged glass slide, microwell layer, fluidic layer; (b) devices completed Figure 1.4 Overview of advantages of both macroscopic and microfluidic cell culture (Halldorsson et al, 2015) 10 Figure 2.1 Medium culture: (a) standard Zarrouk’s medium, (b) Zarrouk’s medium from stationary cell culture 14 Figure 2.2 Morphology of Arthrospira platensis in the microwell The scale bar represents 100 µm, respectively 15 Figure 2.3 The fabricated device The device is composed layers: microwell layer (b), the positive charged glass slide (c) and fluidic layer (e); PDMS was poured in the mold (a), glass slide and microwell were bonded by plasma machine (d); then microfluidic were created by bonding between (d) and e Microfluidic devices were displayed in (f) 16 Figure 2.4 C005 str and Central Lab str were transferred new medium and kept in the incubator from to days 17 Figure 2.5 The process of making microfluidic devices 18 Figure 2.6 The process of cell loading and cultivation in the microwell-based microfluidic devices 18 Figure 2.7 Process set experiment: (a) sample was kept in a petri dish; (b) A.platensis cell was checked; (c) keep the sample and microscopy inside the incubator (connect with computer) 19 Figure 2.8 Measure the length of filaments of C005 strain (a) and Central Lab strain (b) 19 Figure 3.1 Growth length of single filaments of C005 str and Central Lab str in modified Zarrouk’s medium 22 v Figure 3.2 Fragmentation time of single filaments of C005 str and Central Lab str in modified Zarrouk’s medium 22 Figure 3.3 Compare the growth rate of single filament of C005 and Central Lab str in modified Zarrouk’s media 23 Figure 3.4 The phenomenon color- changed filaments of C005 str (a) and Central Lab str (b) 25 Figure 3.5 Compare growth length of single filaments when they were cultured in medium from stationary cell culture 26 Figure 3.6 Comparison of fragmentation time of single filaments when they were cultured in medium from stationary cell culture 27 Figure 3.7 Compare the growth rate of single filaments when they were cultured in medium from stationary cell culture 27 Figure 3.8 Life cycle of Arthrospira by Ciferri and Tiboni, 1983 29 Figure 3.9 Life cycles of Arthrospira platensis from our experiment (C005 str and Central Lab str) 30 vi LIST OF TABLES Table 2.1 Equipments for studies 13 Table 2.2 Constituents of Zarrouk’s medium 14 Table 3.1 Basic information of each Arthrospira platensis single filaments in modified Zarrouk’s medium 24 Table 3.2 Basic information of each Arthrospira platensis single filaments in the modified Zarrouk’s medium from stationary cell culture 28 Table 3.3 Comparison of growth rate based on average and SD of each strain 29 vii LIST OF ABBREVIATIONS % Percentage µl Microliter µm Micrometer A.platensis Arthrospira platensis ACOI Coimbra Collection of Algae CL str Central Lab strain FAO Food and Agriculture Organization G Gram Min Minutes ºC Degree centigrade or Celcius Off Offspring PDMS Polydimethysiloxane Rpm Revolutions per minute SD Standard Deviation Str Strain DSLR Digital single-lens reflexs viii