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 3.2 Filaments in the Zarrouk’s medium from stationary cell culture We conducted to with total 10 single filaments, including filaments C005 str and filaments Central Lab str In C005 str, it has filaments that fragment (rate 40%) and filaments change the color (Fig 3.4) when we collect picture from our experiment In Central Lab str, it has filaments that fragment (rate 60%) and filaments change the color (Fig 3.4) So, based this rate, we can easily see, the strain Central Lab is still able to grow faster To prove in detail, we continually compare the growth length, fragmentation time, the growth rate of single filament of each strain Additionally, to find the cause that the phenomenon of color change of these two strains We think that the main reason which the environment lacked nutrients Because, the medium was filted when cell cultured in the station phase Figure 3.4 The phenomenon color- changed filaments of C005 str (a) and Central Lab str (b) 25 3.2.1 Comparison of growth length of single filaments before fragmenting The growth length of single filaments of each strain which they are different We realize that the length of C005 str is longer than Central Lab str It is demonstrated based on graph, average and SD of each strain (Fig 3.5) The both mother filaments of C005 str are longer than mother filaments of the Central Lab star The mother filament is 920.160 µm (date 3/3-C005) the longest and 476.117 µm the shortest (date 19/3-Central Lab) Following, we compared the growth length of offspring of each strain In C005 str, the length is 527.470 µm (date 13/3-off 2) the longest and 360.160 µm (date 13/3-off 1) the shortest In Central Lab str, the length is 546.214 µm (date 16/3-off 1) the longest and 385.715 µm (date 19/3-off 1) the shortest So, we found that the growth length of filaments of the Central Lab str is equivalent Figure 3.5 Compare growth length of single filaments when they were cultured in medium from stationary cell culture 3.2.2 Compare fragmentation time of single filament Fragmentation time of C005 str is shorter than Central Lab str It was demonstrated by average, SD, time that showed Fig 3.5 We can easily realize that fragmentation time of the Central Lab str is five times longer than C005 str In C005 str, maximum time is 390 minutes (date 3/3off 1&2) and minimum time is 120 minute (date 6/3-off 1) In Central Lab str, maximum time is 2070 minute (date 19/3-off 2) and minimum time is 150 minutes (date 16/3-off 1) Furthermore, growth length and fermentation time of the two strains are inverse proportion together 26 Figure 3.6 Comparison of fragmentation time of single filaments when they were cultured in medium from stationary cell culture 3.2.3 Comparison of growth rate of single filaments We observed the development of two filaments of C005 str and three filaments of the Central Lab str As mentioned the rate above, we realize that the growth rate of Central Lab is faster than C005 str, to make sure for this hypothesis We compared the growth rate of offspring of the Central Lab str and C005 str that showed Fig 3.6 Figure 3.7 Compare the growth rate of single filaments when they were cultured in medium from stationary cell culture 27 In C005 str, growth rate of filaments is 7.940 µm/min (date 3/3-off 2) the fastest and 3.441 µm/min (date 3/3-off 1) the slowest In Central Lab str, the growth rate of filaments is 150.080 µm/min (date 31/3-off 1) the fastest and 18.612 µm/min (date 16/3-off 2) the slowest Additionally, we can based on average and SD of each strain The growth rate of Central Lab str is nearly 20 times higher than C005 str The basic information of each strain in Zarrouk’s medium from stationary cell culture and showed table 3.2 Table 3.2 Basic information of each Arthrospira platensis single filaments in the modified Zarrouk’s medium from stationary cell culture 3.3 Compare growth rate of the same strain in the modified standard Zarrouk’s media and Zarrouk’s medium from stationary cell culture Through table 3.3, we realize that the growth rate of two strains in the standard Zarrouk’s medium had significantly faster than two strains in the Zarrouk’s medium from stationary cell culture So, to explain why this growth rate is different, we give a reason: When the cell was cultured in stationary phase, the cellular was able to the growth very bad The medium had been filtered from stationary Arthrospira platensis cell culture and this medium was used to kept filaments in the microwell Additionally, the nutrient was contented to reduce in this medium Therefore, the cell growth is normal very low Therefore, the cell in 28 this environment could dead, changed colors, slow movement, fragmentation time from mother filaments until offspring filaments were very long Table 3.3 Comparison of growth rate based on average and SD of each strain Standard Strains Average (µm/min) Stationary SD Average (µm/min) SD C005 148.116 52.83 5.990 1.65 Central Lab 194.670 52.59 81.24 44.37 3.4 Discussion: Life cycle of Arthrospira platensis From the experiment, we found that the growth manner of our Arthrospira platensis was not in the same old manner that was described by Ciferri and Tiboni, 1983 (Fig 3.7) Figure 3.8 Life cycle of Arthrospira by Ciferri and Tiboni, 1983 Our Arthrospira platensis didn’t separate into small fragment and grown to the hormogonia, necridia A.platensis fragmented into two long filaments and further elongated (Fig 3.8) They will continually elongate, fragment and didn’t stop They will only stop when the nutrients decrease in the environment 29 Figure 3.9 Life cycles of Arthrospira platensis from our experiment (C005 str and Central Lab str) 3.8.a: Mother filaments and continually elongate 3.8.b: Mother filaments fragment into two offspring (off 1&2) 3.8.c: One offspring fragment and create three filaments 3.8.d: Four filaments 30 PART CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions 4.1.1 Cells/filaments were cultured in the standard Zarrouk’s medium The average length of C005 str is about 540 µm; Central Lab str is about 815 µm Therefore, The length of Central Lab str is longer than C005 str The fragmentation time of C005 str is the same Central Lab str, around 1500 minutes Two strains is different growth rate even they were from the same batch So, this phenomena reflect the inhomogeneity among the Arthrospira platensis population The average growth rate of C005 str is about 148 µm/min and Central Lab str is 194 µm/min Thus, Central Lab str grew faster than C005 str 4.1.2 Cells/ filaments in the Zarrouk’s medium from stationary cell culture The length of the two strains is shorter than filaments was kept in the standard Zarrouk’s medium Furthermore, the average length of Central Lab str is about 508 µm and C005 str is 583 µm So, the length of C005 str is longer than Central Lab str The average for fragmentation time of C005 str is 285 minutes and Central Lab str is 1435 minutes Regarding, fragmentation time of Central Lab is the five times higher than C005 str Beside that, the average growth rate of C005 is µm/min and Central Lab str is 80 µm/min The growth rate of Central Lab str is approximately twenty times faster than C005 str The growth rate of Arthrospira platensis is not constant Two strains were cultured in standard Zarrouk’s medium, it had grown faster than two strains in Zarrouk’s medium from stationary cell culture Thus, Central Lab str has always grown faster than C005 str The maximum length at fragmentation time of Central Lab str is longer than C005 str 4.2 Suggestions - Continually experiment about 10 filaments of each strain for clearly and convince conclusion 31 - Continually study, 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