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 c 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 c 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 1.
Nguyen Xuan Vu 3.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.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 c 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 do 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 c TABLE OF CONTENT PART 1. General Arthrospira platensis. Morphology and taxonomy for Arthrospira platensis. Effect of temperatures.
Effect of pH. An introduction to soft lithography. Advantages of microfluidic for cell culture. Microfluidic devices for cell biology.
Microfluidic devices for single cell analysis. Scope of study. Equipments and materials. Microfluidic devices design: an electrostatic using microwell based microfluidic devices.
Algal strains cultivation. Make microfluidic devices. Cell loading and cultivation in the microwell. Imaging of cells and analysis methods.
RESULTS AND DISCUSSION. Cells/filaments in the standard Zarrouk’s medium. Comparison of growth length of single filament before fragmenting. Compare fragmentation time of single filaments.
Comparison of growth rate of single filaments. Filaments in the Zarrouk’s medium from stationary cell culture. Comparison of growth length of single filaments before fragmenting. Compare fragmentation time of single filament.
Comparison of growth rate of single filaments. Compare growth rate of the same strain in the modified standard Zarrouk’s media and Zarrouk’s medium from stationary cell culture. Discussion: Life cycle of Arthrospira platensis. CONCLUSIONS AND SUGGESTIONS.
Cells/filaments were cultured in the standard Zarrouk’s medium. Cells/ filaments in the Zarrouk’s medium from stationary cell culture. 33 iv c LIST OF FIGURES Figure 1. Various applications of microalgae products for human, animals and industries.
Helical trichomes and straight of Arthrospira platensis. The scale bar (a)=40 µm, (b)=20 µm, respectively (Source: C. The microfluidic devices: (a) including 3 layers: positively charged glass slide, microwell layer, fluidic layer; (b) devices completed. Overview of advantages of both macroscopic and microfluidic cell culture (Halldorsson et al, 2015).
Medium culture: (a) standard Zarrouk’s medium, (b) Zarrouk’s medium from stationary cell culture. Morphology of Arthrospira platensis in the microwell. The scale bar represents 100 µm, respectively. The fabricated device.
The device is composed 3 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). C005 str and Central Lab str were transferred new medium and kept in the incubator from 1 to 5 days. The process of making microfluidic devices.
The process of cell loading and cultivation in the microwell-based microfluidic devices. 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). Measure the length of filaments of C005 strain (a) and Central Lab strain (b). Growth length of single filaments of C005 str and Central Lab str in modified Zarrouk’s medium.
Fragmentation time of single filaments of C005 str and Central Lab str in modified Zarrouk’s medium. Compare the growth rate of single filament of C005 and Central Lab str in modified Zarrouk’s media. The phenomenon color- changed filaments of C005 str (a) and Central Lab str (b). Compare growth length of single filaments when they were cultured in medium from stationary cell culture.
Comparison of fragmentation time of single filaments when they were cultured in medium from stationary cell culture. Compare the growth rate of single filaments when they were cultured in medium from stationary cell culture. Life cycle of Arthrospira by Ciferri and Tiboni, 1983. Life cycles of Arthrospira platensis from our experiment (C005 str and Central Lab str).
30 vi c LIST OF TABLES Table 2. Equipments for studies. Constituents of Zarrouk’s medium. Basic information of each Arthrospira platensis single filaments in modified Zarrouk’s medium.
Basic information of each Arthrospira platensis single filaments in the modified Zarrouk’s medium from stationary cell culture. Comparison of growth rate based on average and SD of each strain. 29 vii c 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 c PART 1. INTRODUCTION At the present, we are standing the challenge of energy, food crisis due to the population explosion of the world.
One of factors to solve this difficult is phytoplantonic. That is algae-Arthrospira platensis (A.platensis is a filamentous cyanobacterium that has been studied and produced by large factories in large-sized countries such as China, India and the United States (Pulz and Gross, 2004). platensis is an ideal food and dietary supplement for the 21st century by Food and Agriculture Organization (FAO) of the United Nation (Pelizer, 2003). The successful commercial exploitation of A.platensis because of its high nutritional value, chemical composition such as protein, pigments, antioxidants, fatty acids (γ-linoleic acid) (Pulz and Gross, 2004), pharmaceutical compounds (Kuntanawat, 2014).
They are safety of the biomass has made it one of the most important industrially cultivated microalgae. Knowledge of its biology, chemistry and physiology, which is essential for understanding the growth kinetic, morphology, have been used in the different conditioned medium. Microfluidics, the study of fluid flow at microscale and its application in biological, biomedical, biotechnology and chemical analyses, has been large progress over the last two decades (Squires and Quake, 2005). Microfluidics systems have many advantages over traditional technicals such as low cost, low area, low reagent consumption, fast response time, flexibility of device design, experimental flexibility and control, single cell handling, real-time on a chip analysis.
Some microfluidic systems created new functions based on the combine physical, chemical and biological characteristics at microscale which are not available for macro-systems (flask, petri dish, etc). One important class of microfluidic systems are those for cell culture and the ability to control parameters of the cell microenvironment at growth length, fragmentation time, growth rate, cellular behaviors, growth kinetics in the specified physiological microenvironment. Studies of single-cell microalgae are grown interest, because these organisms are being used as model systems for the studies of many fundamental biological 1 c processes (Hoek et al, 1995), as well as in many commercial, industrial and biological applications. Gaining a understanding of single-cell in Arthrospira platensis that it will also be a great value to optimize the biotechnological applications.
As the reasons above, it inspires for us to study the properties of Arthrospira platensis that nobody had ever known before. Fundamental understanding of the cellular phenomena requires detailed investigations of the growth, observe the activities of a single cell. The kinetic parameters have measured the length, fragmentation time of each filament. Therefore, I was chosen the topic:―Algal cell culture in microfluidic devices and microenvironment‖.1 Microalgae Microalgae are sunlight-driven cell factories that convert dioxide to potential biofuels, foods, feeds and high-value bioactives, agricultural, chemical and pharmaceutical sectors (Yusuf Chisti, 2007) (Fig 1.
Microalgae reproduce themselves using photosynthesis to convert sun energy into chemical energy, completing entire growth cycles every few days (Sheehan J et al, 1998). Microalgae are prokaryotic or eukaryotic photosynthetic microorganisms that can grow rapidly and live in harsh conditions due to their unicellular or simple multicellular structure (Mata et al, 2010). Examples of prokaryotic microorganisms are Cyanobacteria (Cyanophyceae) and eukaryotic microalgae are for example green algae (Chlorophyta) and diatoms (Bacillariophyta) (Mata et al, 2010). Moreover, they can grow almost anywhere, requiring sunlight and some simple nutrients, although the growth rates can be accelerated by the addition of specific nutrients and sufficient aeration (Muhling et al, 2005).
Different microalgae species can be adapted to live in a variety of environmental conditions. Microalgae are presented in all existing earth ecosystems, not just aquatic but also terrestrial, representing a big variety of species living in a wide range of environmental conditions. It is estimated that more than 50.000 species exist, but only a limited number, of around 30.000, has been studied and analyzed (Mata et al, 2010). Various applications of microalgae products for human, animals and industries (http://ecomerge.com/2012/02/algae-as-sustainable- protein.html) As studied before, some active organic compounds can be extracted from algae.
The antioxidant agents such as carotenoid and phycobiliprotein can be extracted from green algae (Chlorella sp.) and blue-green algae (Spirulina sp. In addition, the Chlorella spp.