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

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 w

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

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

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

TABLE OF CONTENT

PART 1 INTRODUCTION 1

1.1.Background 2

1.1.1 Microalgae 2

1.1.2 General Arthrospira platensis 4

1.1.2.1 Morphology and taxonomy for Arthrospira platensis 5

1.1.2.2 Effect of temperatures 6

1.1.2.3 Effect of pH 6

1.1.3 Microfluidics devices 7

1.1.3.1 An introduction to soft lithography 8

1.1.3.2 Advantages of microfluidic for cell culture 9

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

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 3 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 4 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

LIST OF FIGURES

Figure 1.1 Various applications of microalgae products for human, animals

and industries 3

Figure 1.2 Helical trichomes and straight of Arthrospira platensis The scale

bar (a)=40 µm, (b)=20 µm, respectively (Source: C.Sili, 2012) 5

Figure 1.3 The microfluidic devices: (a) including 3 layers: positively charged glass slide, microwell layer, fluidic layer; (b) devices completed 7

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 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) 16

Figure 2.4 C005 str and Central Lab str were transferred new medium and

kept in the incubator from 1 to 5 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

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

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

LIST OF ABBREVIATIONS

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) 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) A 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

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‖

1.1 Background

1.1.1 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 1.1) 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)

Figure 1.1 Various applications of microalgae products for human, animals

reported as the main groups of microalgae to produce antimicrobial substances Those Antimicrobial substances are identified including of fatty acids, glycolipids, acrylic acid phenolics, cyclic peptides, N-glycosides, sulphate-polysaccharides, ß-diketone, isonitriles-containing indole, alkaloids

During the past decades, extensive collections of microalgae have been created by researchers in different countries An instance is the freshwater microalgae collection of the University of Coimbra (ACOI) in Portugal considered one of the world’s largest, having more than 4000 strains and 1000 species This collection attests to the large variety of different microalgae available to be selected for use in a broad diversity of applications, such as value

added products for pharmaceutical purposes, food crops for human consumption and an energy source (Singh el al, 2010)

1.1.2 General Arthrospira platensis

Arthrospira (Spirulina) is marketed widely under the name ―Spirulina” as

a food supplement for humans and animals They are filamentous, heterocystous cyanobacteria that are generally found in tropical and subtropical regions in warm bodies They are one the most cultivated commercial microalgae Cyanobacteria constitute one of the largest groups of prokaryotes They are some of the simplest life forms on earth and the cellular structure is simple prokaryote, which can perform photosynthesis like plants, but without the plant cell walls resembling primitive bacteria (Staniner et al, 1981) Like animals cell, they also have complex sugars, such as glycogen, on their cell membrane (Singh et al, 2005) However, they are truly prokaryotic lacking nuclear membranes, internal organelles and histone protein associated with chromosomes Cyanobacteria are able to live autotrophic utilizing CO2 as their sole carbon source using the reductive pentose phosphate pathway or Calvin cycle (Stal and Moezelaar, 1997) They are mainly aquatic and larger than other types of bacteria They are often mentioned as blue-green algae, which can be misleading since they are not algae (eukaryote) Also, they are all unicellular, although many grow in colonies or filaments (Singh et al, 2005)

non-The cyanobacteria biomass is rich in carotenoid, chlorophyll, phycocyanin, amino acid, minerals and many other bioactive components, which makes it ideal to use as a food additive along with many other applications within biology, biotechnology, food and medical industry (Sanchez et al, 2003) The composition of the nutrients in the biomass depends on the growth conditions such as light intensity, temperature, pH, salinity, etc., which can have a major affecton the lipid and pigment content and growth rate in the cells

The cyanobacterium A platensis is gaining more attention on key

biotechnology research of its economical, ecological and nutritional importance

A.platensis has a great potential to provide the industry with biological produced

ingredients that can be used for food production and related nutritional materials,

such as coloring agents, vitamins, γ-linolenic acid and enzymes (Borowitzka,

2013) There are many different proteins in A.platensis including phycobilin

proteins, which are a family of brilliantly colored, hydrophilic and stable fluorescent pigment proteins

1.1.2.1 Morphology and taxonomy for Arthrospira platensis

Arthrospira platensis genus are multicellular filamentous cyanobacteria

recognizable by the main morphological feature of the genus: the arrangement of the multicellular cylindrical trichomes in an open left-hand helix along the entire

length (Fig 1.2) The blue color comes from phycocyanin and the green color is

from chlorophyll, however the two pigments cover a third group of pigment, the carotenoids, which normally appears as a red, orange or yellow color (Richmon and Soeder, 1986) Under light microscopy, the blue-green non-heterocystous filaments composed of vegetative cells that undergo binary fission in a single plane, show easily-visible transverse cross-walls Filaments are solitary and free floating and display gliding motility (Andersen, 2005)

Figure 1.2 Helical trichomes and straight of Arthrospira platensis The scale

bar (a)=40 µm, (b)=20 µm, respectively (Source: C.Sili, 2012)

Environmental factors, mainly temperature, physical and chemical conditions, may affect the helix geometry (Jeeji Bai and Seshadri, 1980) One drastic alteration of this geometry is the reversible transition from helix to spiral shape, after transferring the filaments from liquid to solid media Although the

helical shape of the trichome is considered a stable and constant property maintained in culture, there may be considerable variation in the degree of helicity between different strains of the same species and within the same strain

or different strains Variations in the trichome geometry of each strain may be observed Once a strain has converted to the straight form, both naturally or after physical or chemical treatments, such as UV radiation or chemicals, it does not revert back to the helical form (Pelosi et al, 1971) Follow Jeeji Bai, 1985 suggested that this is due to a mutation affecting some trichomes during certain growth conditions When, in a culture of a helically coiled strain, a few filaments happen to become straight (Central Lab strain) They tend to become predominant

As mentioned above, cell division occurs by binary fission on one plane at right angles to the long axis of the trichome Trichome elongation occurs through multiple intercalary cell division all along the filament Multiplication occurs only by fragmentation: the trichome breakage is transcellular by the destruction

of an intercalary cell, sacrificial cell (Balloni et al, 1980)

1.1.2.2 Effect of temperatures

A.platensis is a mesophile microorganism that can tolerate and still grow

in temperatures ranging from up to around 40˚C and down to around 15˚C, although this way deviates between strains (Richmond and Soeder, 1986) The

optimal temperature for A platensis growth is 30˚C, which has been shown by

Ogbonda who investigated the effect of temperature and pH on biomass

production for A.platensis species (Ogbonda et al, 2007)

1.1.2.3 Effect of pH

Cyanobacteria can be found in many different environments and

conditions For example, A.platensis thrives in extreme alkaline environments However, even among cyanobacteria, A.platensis’s ability to adapt pH values is

very unique because even though most cyanobacteria can tolerate alkaline conditions, they still have optimal growth in a neutral medium (pH=7) (Ciferri,

1983) This is not the case for A.platensis, which fails to grow at pH=7 or lower

and the optimal growth is obtained around pH 9 to 10 (Ogbonda, 2007) Even at

pH=11,5 the growth rate for A.platensis has been approximately 80% of the

optimal growth rate and it can therefore clearly be defined as an obligate alkaliphile microorganism (Schlessinger, 1996)

The application of microfluidics to biology and medicine has lead to a diversity of new research directions (Melin and Quake, 2007) Cell culture refers

to the maintenance and growth of cells in a controlled laboratory environment Such as invitro cell culture models are the mainstay of experimental cell biological research Microfluidic cell culture attempt to develop devices and techniques for culturing, maintaining, analyzing and experimenting with cells in microscale volumes (Meyvantsoon et al, 2008) Some important aspects of microfluidic cell culture systems, including the effect of surface modification on cellular behavior, cellular analysis, cellular microenvironment This technique was created by standard PDMS soft lithography

Figure 1.3 The microfluidic devices: (a) including 3 layers: positively charged

glass slide, microwell layer, fluidic layer; (b) devices completed

1.1.3.1 An introduction to soft lithography

Soft lithography sets of techniques that makes microstructures by printing, molding and embossing using a patterned, elastomeric stamp or mold or a

popularity since 1995 because it does not require complex equipments and has a short fabrication time The techniques were developed as an alternative to photolithography and electron-beam lithography, and share the name ―soft lithography‖ because they are all based on using a patterned elastomeric polymer

as a mask, stamp or mold as polymers, gels The tools of soft lithography are being used with increasing frequency in cell biology because of their simplicity, low cost and compatibility with cells (Whitesides et al, 2007)

polymer that is commercially available and has properties that make it well suited to applications in microbiology PDMS will cross-link, with the addition

of a curing agent containing a catalyst (Hassler et al, 2011) PDMS has been widely used to produce microfluidic devices It can be easily molded to create complex fluidic circuits, using soft lithography techniques, making, prototyping relatively simple and cost effective (Nag and Banerjee, 2012) Some characteristics of PDMS, such as gas permeability, optical transparency, and flexibility als make it appealing for cell culture In addition, it is generally regarded as inert, non-toxic PDMS prepolymer generally consists of two components of polymer base and curing agent PDMS generally consists of polymer base and curing agent mixed at a ratio of 10:1 Next, the mixture created many bubbles and eliminate them by pump machine PDMS can be cured in 80ºC oven in 1 hour After curing, the PDMS piece is removed from the mold Access holes are punched through the PDMS with a blunt-end syringe needle The PDMS piece is then bonded to another substrate such as glass or another piece of PDMS To bonded PDMS to glass, the two pieces can be treated with oxygen plasma and bonded together via hydrophilic interaction (Bhattacharya et al, 2005) PDMS can also be bonded to another piece of PDMS to create monolithic structures This can be achieved by treating both PDMS surfaces with oxygen

plasma and bonding them together The two pieces are then brought into contact and cured together This method has been useful in constructing PDMS valves and pumps where two layers of PDMS channels sandwich a thin PDMS layer in between them (Bertheier et al, 2012)

1.1.3.2 Advantages of microfluidic for cell culture

Microfluidic cell culture has significant advantages over macroscopic culture The example, that is culturally in flasks, petri dishes (Fig 2.1) to compare the most significant advantages when using macroscopic and microfluidic cell culture There is great flexibility in the design of microfluidic devices, which can

be tailored to study single cell in the microwell (Yeo et al, 2011) The advantages

of microfluidic cell culture include the ability to more closely mimic a cell’s natural microenvironment (Meyvantsson and Beebe, 2008) At the same time, microfluidic cell culture offers reduced consumption of reagents, reduced contamination risk and efficient high throughput experimentation (Rubakhin et

al, 2011)

Microfluidic cell culture, offers an alternative to macroscopic methods to study cell migration processes and their mechanisms at single cell resolution Taking into consideration the advantages of design flexibility, the ability to handle single cells for experimentation, real-time on chip analysis via time-lapse microscopy and low reagent consumption (Huang et al, 2011) developed a compartmentalized microfluidic cell culture device which resembles the physiological environment of migrating cells

Microfluidic cell culture devices also make it feasible to study complex cellular behavior, like the relationship between single cell movements and collective cell migration Follow Vidal at al, 2013, studied the role played by collective cellular interactions on cell motility at different cellular densities within a microfluidic device Single cell locomotive behavior (straight lines, curved and short distances with no directional) By capturing sufficient data on the locomotion of individual cells within culture chambers with independently varied conditions, these authors developed a mathematical model to predict the role of social interaction in motility (Halldorsson et al, 2015)

Figure 1.4 Overview of advantages of both macroscopic and microfluidic cell

culture (Halldorsson et al, 2015)

Microfluidic devices offer the advantages of precise control over experimental conditions via custom designed chip architectures, parallelization, automation, and direct coupling to miniaturized downstream analysis platforms Although microfluidic cell culture provides great flexibility with respect to experimental design, moving cells from a macroscopic culture environment of dishes, flasks and well-plates to microfluidic cell culture requires revision of cultural protocols Several unique factors of microfluidic and macroscopic cell culture, such as different culture surfaces, reduced media volumes and thrive different rates, methods, medium exchange

1.1.3.3 Microfluidic devices for cell biology

In recent years, the applications of microfluidic devices in cell biology have grown, and many believe the development in microfluidics will greatly expedite advancements in cell biology Microfluidic devices will enable cell biology experimentation to be done effectively in a high-throughput manner,

with both multicellular and single cell studies Reagents requirements for microfluidics devices are a couple of orders magnitude smaller than macroscale devices Many microfluidic devices also offer functionalities that are unachievable by traditional cell biology instrumentations Such functionalities are based on the devices ability to control the microenvironment precisely in both spatial and time These capabilities have allowed microfluidics devices to make physiological conditions possible for experimentation The this section, various microfluidic devices application to cell biology will be introduced

1.1.3.4 Microfluidic devices for single cell analysis

Due to the small scale of microfluidic channel, automation capability, and ease of parallelization, microfluidics have been applied to various aspects of single cell analysis Beside from droplet generation, which will be discussed separately, microfluidic devices have been designed to capture or otherwise isolate single cells for further analysis The main approaches consist of cell traps, where single cells can be arrayed into easily-imaged patterns, and compartments, which randomly or determinstically segregate individual cells and avoid cross talk during assay reactions

Microfluidic devices consist of devices that have channel geometries on the scale of a few to several hundred micrometers in cross section, or approximately that of single cells or small tissues Reducing the scale of the fluidic channels provides a number of advantages for single cell analysis

Cell trapping has been demonstrated using both passive sedimentation of single cells into microwells as well as active, hydronamic trapping In passive trapping, microwells of the dimensions of single cells act only as physical restraints against cell motion due to device or cell movement and allow researchers to rapidly image large fields of single cells They have been used in various forms to analyze single cells, as well as improving image based electrostatic and analyze by freeware

1.2 Objectives

- To study the A.platensis’s cellular response of growth kinetics,

morphology alternation, fragmentation, variation among population of microalgae using microwell based on microfluidic devices

Figure 1.3 The microfluidic devices: (a) including 3 layers: positively charged

glass slide, microwell layer, fluidic layer; (b) devices completed

1.3 Scope of study

- Cell loading and cultivation, capturing in the microwell

- Collect information including pictures, fragmentation time and time lapse video of each filament (from mother to offspring filament)

- Measure length of single filament of each strain

- Compare growth length, fragmentation time, growth rate between two different strains (C005 str and Central Lab str) in the same condition

- Compare growth rate of filaments in the same strain of different condition the culture medium

PART II METHODS 2.1 Equipments and materials

2.1.1 Equipments

Table 2.1 Equipments for studies

1 Standard Zarrouk’s medium

2 Zarrouk’s medium from stationary cell culture (cell culture in Zarrouk’s medium about from 10-14 days, filtered to reject algae and receive medium)

Table 2.2 Constituents of Zarrouk’s medium

Figure 2.1 Medium culture: (a) standard Zarrouk’s medium, (b) Zarrouk’s

medium from stationary cell culture

2.1.2.2 Algal strains

Algal strains were used Arthrospira platensis obtained from the Applied

Algal Research Unit, Department of Biology, Faculty of Science, ChiangMai

University, Thailand A.platensis has two major morphology and their’s name