MINISTRY OF EDUCATIO N, RESEARCH, YOUTH AND SPORTS [621696]
MINISTRY OF EDUCATIO N, RESEARCH, YOUTH AND SPORTS
UNIVERSITY "OVIDIUS" OF CONSTANȚ A
FACULTY OF NATURAL SCIENCES AND AGRICULTURAL SCI ENCES
DOCTORAL SCHOOL – BIOLOGY
THE ABSTRACT OF DOCT ORAL THESIS
“Cyanobacteria in mesothermal sulphurous waters
(Obanul Mare – Mangalia)”
Scientific Coordinator,
Prof. Univ. Dr. IOAN ARDELEAN
Doctoral Candidate ,
BENLIAN (SARCHIZIAN) IRIS
CONSTANȚA
2012
TABLE OF CONTENTS
Thesis Abstract
INTRODUCTION ……………….. ……………….. ……………….. ……………….. ……………….. ……………..
PART I. THE STATE OF KNOWLEDGE OF CYANOBACTERIA RELATED TO
THE LATEST DATA FROM THE LITERATURE
CHAPTER 1. CYANOBACTERIA – GENERAL DATA
1.1. General characteristics of cyanobacteria ………………………………………………………………….
1.1.1 . Morphological diversity of cyanobacteria ……………………………………………………………..
1.1.2 . Physiological diversity of cyanobacteria………………………………………………………………..
1.1.2.1 . Criofile cyanobacteria……………………………………………………………………………………….
1.1.2.2 . Mesophilic cyanobacteria ………………………………………………… ………………………………
1.1.2.3 . Thermophilic cyanobacteria ………………………………………………………………………………
1.2. Classif ication of cyanobacteria ………………………………………………………………………………..
CHAPTER 2. THEORETIC AL AND PRACTICAL IM PORTANCE OF
CYANOBACTERIA
2.1. Cyanobacteria – the oxygen source of the planet in past and in presence………….. ………….
2.2. The importance of photosynthesis and respiration in cyanobacteria …………………………….
2.3. The Cyano bacteria and the Biotechnology………………………………………………………….. …..
2.3.1. Cyanobacteria – a model system for study of biological nanoparticles on prokaryotes…
2.3.2. Cyanobacteria –a model of biological system for the production of nanoparticles……..
PART II. PERSONAL CO NTRIBUTIONS
OBJECTIVES OF THESIS
CHAPT ER 3. MATERIALS AND METHODS
3.1. Sampling and description of the study area ……………………………………. ………………………..
3.2. Methods for fixation and preservation of water samples ……………. ……….. …………………….
3.3. Staining methods used to study cyanobacteria ……………….. …………………. ………………….. ..
3.3.1. Fuxin alkaline staining method ………………. ………………….. ……………………. …………………
3.3.2. Crystal violet staining method (Gram stain) …………………………… ………………………… …..
3.3.3. Negative staining method for capsule by India / China Ink. ……………………. ………………
3.3.4. Dou ble staining with aniline blue and China Ink……… ……………………………. ………………
3.3.5. Method of alkaline methylene blue staining Loeffler …………….. ……………………. …………
3.4. Staining and microscopy visual ization of cyanobacteria using epifluorescence
microscopy……………………………………………………………………………………………………. ……………
3.4.1. Method of visualisation natural fluorescence of chlorophyll a
3.4.2. Acridine orange s or DAPI staining method……………………… ………………….. …………… ….
3.4.3. Aniline blue staining method ………………………… ………………….. ………….. ……………………
3.4.4. Q uantum dots staining method …………………………………….. ……………………… ……………..
3.5. Methods for isolation of new strains of cyanobacteria from mesothermal sulphurous
water from Oban ul Mare – Mangalia ……………… …………. ………………………. …………………………
3.5.1. Methods for isolation and cultivation of unicellular cyanobacteria belong to genus
Synechocystis sp…………………………………………………………………… ……………………………………..
3.5.2. Methods for isolation and cultivation of oxygen ic filamentous diazotrofic
cyanobacteria belong to genus Nostoc sp……………………………… ………………………………………..
3.5.3. Methods for isolation and cultivation of oxygen ic filamentous nonheterecystous
cyanobacteria belong to genus Anabaena sp. ……………………………….. ……………………………. ….
3.5.4. Methods for isolation and cultivation of oxygen ic filamentous cyanobacteria belong
to genus Tychonema sp…………………………………… ………… ………………………………………………..
3.5.5. Methods for isolation and cultivation of anoxygenic unicellular and filamentous
cyanobacteria …………………………………………………………………………………………………………….. . 1
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3.5.6. Methods for isolation and cultivation of anoxygenic and oxygenic filamentous
thermotolerant cyanobacteria…. ……………………………………………………………………………………..
3.6.Methods for purification of cyanobacteria isolates ……………………………………………………..
3.6.1. Obtaining of axenic cultures of diazotrophic cyanobacteria using antibiotics tienam,
augmentin, nalidixic acid and cephalexin ……………………………………………………………………….
3.6.2. Obtaining axenic cultures of cyanobacteria using lysozyme methods…… …………………..
3.7. Methods for identifying new strains of cyanobacteria isolated …………………. ………….
3.7.1. Identifcation key for S ubsection III ……………………………………. …………………… ………… ..
3.7.2. Identification key for S ubsection IV …………………………………… …………………… …….. ……
3.7.3. Using digital image analysis to study the morphological and physiological
characteristics of the isolated cyanobacteria …………… ………………………………. …………………….
3.7.3.1. Vi sualisation and studying morphological characters of cyanobacteria using CellC
software …………………………………………………………………………… ………….. ……………………….
3.7.3.2. Visualisation and studying morphological characters of cyanobacteria using ImageJ
software …………………………………………………………. ……………………………………….. ……………
3.8. Methods for determin ation the growth rate of cyanobacteria in various conditions –
oxygenic photosynthesis on BG 0 and BG 11 media ………………………………………. …………………..
3.8.1. Spectrophotometric method for the determination of nitrate and amm onium content …..
3.8.2. Methods for determinin ation the growth rate of cyanobacteria by spectrophotometric
method…………………….. ………………………….. ……………………………………… ……………………….
3.8.3. Methods for determinination the rate growth of cyanobacteria by calculating the
frequency of dividing cells… ……………………………………………….. ………………………………….
3.8.3.1. Dete rmination method of growth rate for Anabaena sp ……………………….. ………………
3.8.3.2. Determination method of growth rate for Tychonema sp ……………….. ……………… ……
3.8.4. Direct viable count m ethod (Kogure et al ., 1979)…….. ……… ………………………………….
3.8.4.1. Direct viable count m ethod in Anabaena sp ………………………………………………………
3.8.4.2. Direct viable count m ethod in Synechocystis PCC 6803 …………………………… ………..
3.8.4.3. Direct viable count m ethod in Synechocystis sp………………………………………………….
3.8.4.4. Direct viable count methods in natural populations of cyanobacteria ………………… .
3.9. Methods for studying cellular redox properties of some isolated strain…………… ..
3.9.1. Spectrophotometric measurement of dehydrogenase activity in some populations of
cyanobacteria…………………………………………………… …………………………………………………….
3.9.2. Quantification of redox properties at individual le vel (filament of cyanobacteria)
Anabaena sp………………………………………………………………………………… ………………………….
3.9.3. Quanti fication of redox properties at individual cell level from cyanobacteria l
filament Anabaena sp ………………………………………………………………………………………………….
3.10. Methods for investigating the interaction between quantum dots (CdSe / ZnS) and
cyanobacterial populations ……………………………………………………………………………………….
3.10.1. Passive nonspecific labeling using quantum dots in natural samples and
cyanobacterial cultures ……….. ………………………………………………………………………. ………….
3.10.2. Highlighting the fluorescence of marked cyanobacteria with quantum dots…….
3.10.3. Methods for study the cytotoxic effect of quantum dots on cyanobacteria………………..
CHAPTER 4. RESULTS AND DISCUSSION ……………………………………………………………
4.1. Unicellular and filament ous cyanobacteria popula tions in natural samples from
mesothermal sulphurous spring Obanul Mare – Mangalia …………………………………. ……………..
4.2. Isolation of new strains of cyanobacteria ………………………….. ……………………………… ……..
4.3. Obtaining axenic cultures of diazotrophic cyanobacteria using antibiotics t ienam,
augmentin, nalidixic acid, cephalexin ……………. …………………………….. ……………………………….
4.4. The u se of lysozyme method to obtain axenic cultures of cyanobacteria ……………………
4.5. Identification of isolates from axenic cultures …………………… ………………………………………
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4.6. Studying some physiological aspects of isolated strains ……………….. …….. ……………………
4.6.1. Determination the growth rate of cyanobacteria by s pectrophotometric methods…………
4.6.2. Determination the growth rate of cyanobacteria by the frequency of dividing cells……..
4.6.2.1. Determ ination the growth rate in Anabaena sp …………………. ………………………………..
4.6.2.2. Determination the growth rate in Tychonema sp ………………….. ……………………………..
4.6.3. Determining the direct viable count cells in dif ferent strains of cyanobacteria……………
4.6.3.1. Determining the direct viable count cells in Anabaena sp……………………………………..
4.6.3.2. Determining the direct viable count cells in strain Synechocystis PCC 6803… ………….
4.6.3.3. Determining the direct viable count cells in unicellular strain Synechocystis sp……….
3.6.3.4. Determining the direct viable count cells in natur al populations of cyanobacteria…….
4.6.4. Quantification of redox properties of some strains of cyanobacteria ………………………….
4.6.4.1. Spectrophotometric measurement of dehydrogenase activity in some populations of
cyanobacteria ……………………………………………………………………………….. ……………………………
4.6.4.2. Quantification of redox properties at the individual level (c yanobacteria filament of
Anabaena sp.)……………………………………………………………………………………………………. …..
4.6.4.3. Quant ification of redox properties at individual cell level from Anabaena sp…………..
4.6.5. Using quantum dots to study the cytotoxicity of cyanobacteria ……………… …………
4.6.5.1. Passive nonspecific labeling using quantum dots of cyanobacteria from natural
samples and enriched cultures …………………………………………………………………………………
4.6.5.2. Study the cytotoxic effect of quantum dots ……………………………………. ………………
CONCLUSIONS ……………………………………………………………………………………………………..
REFERENCES ……………………………………………………………………………………………………..
ANNEX …………………………………………………………………………………………………………….. ….
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Keywords: cyanobacteria ; identification ; growth rate ; determination of cell’s number
capable of growth and multiplication ; redox properties at population level and
individual cell level ; cytotoxicity of quantum dots ( CdSe / ZnS); digital image analysis .
INTRODUCTION
Cyanobacteria have been always in the top of scientific world's attention, the interest
from this group of microorganisms is determined because cyanobacteria has a vital role in the
evolution of specific biosphere. Thus, I just reminding only metabolic diversity of
cyanobact eria in a wide range of environmental factors, including extreme conditions, such as
sulfurous mesothermal water, we can affirm that the thesis topic is taking place within the
actual international research .
The choice of research topic titled "Cyanobacter ia in mesother mal sulphurous
waters (Obanul Mare – Mangalia)" was made in consultation with the existing literature, but
insufficient data on cyanobacteria from aquatic sulfurous environments in Romania leded me
to approach this new and fascinating topic .
Doctoral research lies in the fact that scientific studies raise only questions and
problems, so approach their own research go in was quite difficult, highlighting the originality
of methods combining theoretical and practical research, the classical wit h modern methods ,
leading to gen eral conclusions of the thesis.
Aim of the thesis is to isolate, purify and identify to genus level on the principles of
bacterial taxonomy the cyanobacteria strains isolated from natural samples collected from
mesothermal sulphurous spring at Oban ul Mare – Mangalia, using classical methods improved
by adding carbon source before antibiotic , the study of physiological aspects of isolated, such
as spectrophotometric methods to determine the growth rate of cyanobacteria cultu red
aerobically on different media: BG 0 and BG 11 or by calculating cell division rate, determine
number of cells capable of growth and multiplication method of Kogure et al. for
heterotrophic bacteria (1979), studying the redox properties using spetrophoto metric methods
at the population level in some strains of cyanobacteria isolated and the automatic analysis of
digital images obtained from microscope, marking cyanobacteria with quantum dots (CdSe /
ZnS) and study the cytotoxic effect of quantum dots on c yanobacteria.
The doctoral thesis is divided into five chapters, contained in two parts: the current
state of knowledge cyanobacteria reported in the latest data of literature (two chapters) and
experimental research (including two chapters) and a chapter of general conclusions.
First, the thesis is characterized by the actuality of the topic discussed since its
introduc tion in the sphere of concerns r omanian and foreign specialists occurred relatively
late, not possible in our country develop advance d techniques in M icrobiology at the single
cell level, combined with digital image analysis.
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In order to accomplish this thesis documentation and I found about 380 titles and
suggestive references, of which about 60 were published in the last five years, w hich allowed
me to obtain new experimental results correlated with those obtained internationally. Among
them are those who open researches revealed microbiology, general biology, to automatic
analysis of digital images, a course of topical international a nd national, which was made
possible through a permanent collaboration with international experts, which allowed
development within this thesis.
On my own contributions can say that the original information processing capacity of
synthesis and the interpr etation of the data , also the transdisciplinarity, which allowed
personal opinions strained argument throughout the paper, to emphasize their vis ion of the
phenomena analyzed. Finally, a private contributors is present in the form of proposals
submitted a t the end of chapters on modern methods used to facilitate their work effort by the
researcher to process a large set of precise and reproducible data at a time short.
CHAPTER 1. CYANOBACT ERIA – GENERAL DATA
Chapter 1 sets out the general characteristics of cyanobacteria, the morphological and
physiological diversity, as well as the main characteristics of criofile mesophilic and
thermophilic cyanobacteria .
Cyanobacteria are the largest and most diverse group of photosinthetic bacteria.
Originally, cyanobacteria were considered the following characteristics: large, are bodies
oxygen ic phototrophs (H2O used as electron donor with the production of O 2), contains PS I
and PSII responsible for decompo sition by light energy absorbed H 2O, contain chlorophyll a
and β-carotene, ficobiliproiens as accessory pigments: photosynthesis is similar to plants.
Cyanobacteria can be found in all aquatic ecosystems, ranging from hydrothermal
vents, to arctic ar eas (Carmichael et al., 1990). Being the oldest oxygen -producing organisms
(Schöpf, 2000), cyanobacteria have played a key role in the evolution of the Earth since their
first appearance now 2.15 billion years ago (Hoffmann, 1975; Knopf 2006; Ramussen 2008 ) .
Long history of cyanobacteria is responsible for their ability to be better adapted to
environmental stress, including rare and abundant nutrients (Paerl, 2006), exposure to UV
radiation, high solar radiation and above all at high temperatures ( Paerl et al 1985; Robarts &
Zohary 1987; Briand, 2004). These special conditions may favor the dominance of
cyanobacteria in many aquatic habitats. Ability to be very tolerant cyanobacteria when
subjected to various stress factors suggest that cyanobacteria are likely to benefit from
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environmental changes associated with global warming (Paerl and Huisman, 2008, Paerl ,
2009).
Recently, Whitton and Potts (2000) have demonstrated morphological diversity of
cyanobacteria – filamentous and unicellular forms – which c an aggregate into colonies, cells in
colonies can be arranged in different ways (radial, flat or irregular). Some have specialized
cells for nitrogen fixation (heterocists ) cells that can survive unde r conditions of stress
(akinrets) and dispersion (hormog onia).
The chapter concludes with the presentation of cyanobact eria classified as prokaryotes,
concerning to Bergey's Manual of illustrated determinative Bacteriology (2001), employing
Cyanobacteria group, comprising five orders with 34 genera (Castenholz, 2001).
CHAPTER 2. THEORETIC AL AND PRACTICAL IM PORTANCE OF
CYANOBACTERIA
To better understand the structure of cyanobacteria, we studied the importance of
theoretical and applied them as cyanobacteria is one of the few groups of organisms can
perform photosynthesis and oxygen respiration simultaneously in the same compartment,
some species are able to fix nitrogen. This unusual combination of metabolic pathways and
metabolic flexibility may be responsible for the development of cyanobacteria and th eir
ability to thrive in extreme conditions. Cyanobacteria are the oldest organisms in terms of
evolution: microfosilele found to have 3.5 billion years old were assigned as belonging to
cyanobacteria (Schopf, 1993). An important question is the successful combination of
cyanobacterial e volution of metabolic pathways.
Characteristic of all species of cyanobacteria is operating photosystem I and II, and
the use of water as a source of electrons for photosynthesis. All representatives of
cyanobacteria contai n chloro phyll and are able to increase photoautrophy , although
photohetetrophy and chemo autotrophy and growth are common to many species.
Morphology and life cycle of this group is very complex. Combining photosynthesis and
respiration in a single compart ment is unique. Photosynthesis and respiration require electron
transport pathways catalysed by proteins complexela membranes.
The importance of photosynthesis and respiration in cyanobacteria and cyanobacteria
use as a model system for the study of nanopa rticles on biological prokaryotic or model
biological system for the production of nanoparticles has been a very exciting chapter in terms
of the information presented. Thus, nanotechnology is being developed in many areas, even in
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developing countries hav e also decided that this new technology could be an investment that
can not be ignored, bringing the future economic benefits and social welfare. For new
technologies, there is a growing concern about the possible side effects from the use of
nanoparticles . Due to increased use of nanotechnology, must be well understood risks
associated with exposure to nanoparticles, entry routes and molecular mechanisms of
cytotoxicity. Are water -soluble quantum dots with biological applications are usually
passivated by different layers of inorganic and / or organic to increase fluorescence yield
(Kloepfer et al, 2004). These coatings greatly increase the p article size, making impossible
their absorption by microorganisms.
Fluorescent semiconductor quantu m dots can be used as layers on/ off bacteria and
other living cells, they affect the transport of electrons on energy metabolism, both fototrofe
bacteria and the heterotrophic bacteria. To explain these results take into account the
physicochemical properties of quantum dots in relation to ultrastructural differences of Gram –
negative and Gram -positive and cellular localization of the main energy processes, respiration
and photosynthesis. In this respect, particular attention increasingly focuses more on the
interaction b etween quantum dots and cyanobacteria for longer periods of time because these
prokaryotes oxygen fototrofe have major contributions to the synthesis of organic matter in
aquatic environments they inhabit, the consumption of carbon dioxide a nd molecular ox ygen
production.
An important issue in all these experiments relate physical relationship between
microbial populations and different quantum dots, with special emphasis on quantum dots to
position the cell wall and cell membrane. It seems logical to assum e that the first site of
interaction between the nanoparticles and cells is the cell wall, cell wall however is quite
different structure in Gram -negative bacteria (including cyanobacteri a) and Gram -positive
bacteria.
Physical access of quantum dots to the outer cell membrane (for cell wall) is still an
open question, and the ability of nano -sized quantum dots to pass through the cell membrane
intact (or previously damaged!) To enter the cytoplasm (Ardelean and al., 2011).
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OBJECTIVES OF THE THESI S
1. Isolation of strains of cyanobacteria in natural samples collected from mesothermal
sulphurous spring Obanul Mare – Mangalia ;
2. Purification of strains of cyanobacteria using traditional methods ;
3. Improving conventional purification methods by adding carbon source before antibiotic ;
4. Gender identification at some of the strains of cyanobacteria purified on principiiile
bacterial taxonomy ;
5. Physiological study of isolated following issues :
a. determination by spectrophotometric methods the g rowth rate of aerobically grown
cyanobacteria (photosynthetic oxygenic) on different culture media – BG 0 and BG 11;
b. Determination of the growth rate of cyanobacteria by frequency of dividing cells;
c. Determination of number of cells capable of growth and multiplication using the method
described by Kogure et al (1979) ;
d. Study of redox properties by spectrophotometric methods at the population level in some
strains of cyanobacteria isolated;
e. study the cellular redox properties of some strains of cyanobacteria isolated by automatic
analysis of microscopic images ;
f. cyanobacteria with quantum dots (CdSe /ZnS );
g. study the cytotoxic effect of quantum dots on cyanobacteria .
CHAPTER 3 . MATERIALS AND METHODS
This chapter of the thesis is focused on experimental description of the study area
(being the first time the source is studied cyanobacteria mesothermal from Oban ul Mare ), how
sampling , fixation and preservation of water samples , staining methods used in studying
cyanobacteria , cyanobacteria visualization method using light microscopy and comp (epi)
fluorescence . Are also precisely described methods for isolation and cultivation of strains of
cyanobacteria from mesothermal sulphurous spring at Oban High – Mangalia (genus
Synechocystis sp., Nostoc sp., Anabaena sp., Tychonema sp., unicellular and filamentous
anoxigenic cyanobacteria, filamentous oxygenic and anoxigenic thermotolerant
cyanobacteria ) , purification of cyanobacteria isolates and obtaining axenic cultures of
diazotrophic cyanobacteria using antibiotics tienam, augmentin , nalidixic acid, cephalexin ,
and lysozyme .
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Identification of cyanobacteria strains isolated and the use of digital image analysis to
study the morphological and physiological characteristics of the cyanobac teria isolated study
studying morphological characters of cyanobacteria using CellC and ImageJ program are
described in detail in this chapter.
A B C D
E F
Figure 1 . (Figure 3.7., 3.9., 3.10) . Macroscopic (A, C, E) and microscopic ( B, D, F) view of enriched culture of
unicellular and filamentous cyanobacteria , E – macroscopic view of enriched culture grown in liquid medium
BG 11 (A) microscopic view of enriched culture grown on BG 11 liquid medium after staining with crystal violet
(original ).
There a re also presented the methods for determining the growth rate of cyanobacteria
in various conditions both in classical but also refer the identical spectrophotometric
determination and calculation of frequency of dividing cells in heterocystous cyanobacteria
strain Anabaena sp and strain Tychonema sp., determining the number of cells capable of
growth and multiplication described by Kogure et al (1979) in the strain Anabaena sp. , in
strain Synechocystis PCC 6803 and isolated strain of unicellula r cyanobacteria Synechocystis
sp., the last method being applied on natural popula tions of cyanobacteria from mesothermal
spring ;studying at the redox properties of some isolated using automated analysis of digital
images , spectrophotometric measurement of dehydrogenase activity in some populations of
cyanobacteria and quantifying individual proprităților biological redox level (filament of
cyanobacteria ) from Anabaena sp., are all methodologies well used, together with
investigating the interaction between quantum dots (CdSe /ZnS ) and populations and highlight
the fluorescence of quantum dots marked cyanobacteria and study the cytotoxic effect of
quantum dots on cyanobacteria .
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CHAPTER 4 . RESULTS AND DISCUSSION
Next chapter entitled "Results and discussion" includes original results of experiments
conducted for the isolation, cultivation and improvement of methods for purification of strains
of cyanobacteria isolates ; there was identified following types of cyanobacteria , according to
Bergey Manual 2001: Synechocystis sp. , Synechocystis sp. – anoxy genic , Synechocococcus
sp., Anabaena sp., Oscillatoria sp., Nostoc 1 sp. , Nostoc 2 sp., Tychonema sp.
Using digital image analysis algorithms obtained by combining mathematical methods
from CellC and ImageJ software made it possible for the first time , after consulting the
international literature , precise identification in a relatively short time the number of cells
analyzed in cyanobacterial filaments from digital images ta ken in light field microscopy . The
two programs allowed me to successfully achieve automatic image files backlit practical steps
by steps that are key in obtaining experimental data .
Figure 2 (Figure 4.3.). Bright field view of unicellular
cyanobacteria of the genus Synechocystis sp. by staining with
0.02% crystal violet : A – depth of field microscopic unicellular
cyanobacteria in the natural samples collected , B – detection of
cell shape and outline of cells; C – image microscopic field
with unicellular cyanobacteria , D – detail of various regions of
interest , E-shape of cyanobacteria l cells view ed using only
black and white ( Oc.10x, Ob.40x ) (10 µmscale bar ) (original).
In the Figure 3 there are presented our isolates
identified according to Bergey Manual 2001 .
A B C
D E
Figure 3 (Figure 4.27. , 4.28, 4.29, 4.30, 4.32) . A – isolate Synechocystis sp. oxygen ic, B-isolate
Anabaena sp. ; C – isolate Oscillatoria sp., D – isolate Nostoc sp. , E-isolate Tychonema sp. (original ).
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The purpose of subchapter for determining the growth rate of cyanobacteria by
spectrophotometric method is to determine the rate of growth of cyanobacteria isolates under
study aerobic conditions using microplate reader with ultra spectrophotometer , which covers
a wide range of wavelengths , from 220 nm to 850 nm, allowing data collection and
interpretation in Excell format soon. Measurements were made at D.O. 750 nm , the first time
after 3 hours, after 22 hours, after 28 hours , after 124 hours of incubation in continuous light .
Cultures of cyanobacteria in the study were distributed to wells microplates and automated
reading was performed at 750 nm , achieving concomitant readings by 8 for each crop and
each sample for analysis .
Table 1 ( Table 4.3). Optical densities of the cultures of cyanobacteria grown BG 0 (original)
Time
(hours ) D.O.750nm
Nostoc sp. D.O.750nm
Oscilatoria
sp. D.O.750nm
Nostoc sp. D.O.750nm
Synechocystis
sp. D.O.750nm
Anabaena
sp. D.O.750nm
Synechocystis
sp.
0 0,07 0,079 0,097 0,566 0,097 0,184
22 0,091 0,126 0,108 0,784 0,127 0,212
28 0,095 0,236 0,139 1,178 0,308 0,258
124 0,266 0,294 0,246 1,413 1,222 0,297
Table 2 ( Table 4.4). Optical densities of the cultures of cyanobacteria grown in BG 11 medium (original ).
Time
(hours ) D.O.750nm
Nostoc sp. D.O.750nm
Oscilatoria
sp. D.O.750nm
Nostoc sp. D.O.750nm
Synechocystis
sp. D.O.750nm
Anabaena sp. D.O.750nm
Synechocystis
sp.
0 0,07 0,17 0,084 0,7 0,077 0,11
22 0,084 0,292 0,097 0,858 0,109 0,184
28 0,091 0,681 0,178 1,161 0,137 0,237
124 0,205 0,888 0,313 1,618 0,279 0,521
The frequency of dividing cell (FCD) is an indirect measure of growth rate in bacteria
(Hagström et al., 1979, Campbell & Carpenter , 1986; Carpenter & Campbell , 1988; Nielsen ,
2006) and this report extended and applied this technique on populations of filamentous
heterocystous cyanobacteria isolated from sulphurous mesothermal spring Oban ul Mare –
Mangalia (Sarchizian and Ardelean, 2010). This report presents the use of frequency of
dividing cells (FDC) method to calculate the growth rate in populations of filamentous
(heterocystous and nonheterocystous) cyanobacteria isolated from sulphurous mesothermal
spring from Obanul Mare (Mangalia ).Septa were counted on heat – fixed preparations of
cyanobacteria (300 cells per sample) stained with 0.02% crystal violet , using digital image
analysis using two software CellC and ImageJ to clearly observe the shape of the dividing
cells form filaments allowing us to obtain accurate data: for heterocyst forming strain
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(Anaba ena sp .) the maximum growth rate on BG 0 in light is 0.039 h-1 and for non heterocyst
forming strain ( Tychonema sp.) the maximum growth rate on BG 11 in light is 0.057 h-1.
Frequency of dividing cell (FDC) is an indirect measure of the average rate of growth
measurement in bacteria (Hagström et al., 1979, Campbell and Carpenter, 1986; Campbell
and Carpenter, 1988; Nielsen, 2006), and now is extended and applied on the research
conducted in the cyanobacteria populations from mesothermal spring Obanul Mare –
Mangalia.
In figure 4 there are pres ented the images taken at different time during light
incubation for our isolate Anabaena sp., the arrows indica ting the cell s in division within the
cyanobacterial filaments stained with crystal violet [( initial tim e (T0), after 12 hours (T1),
after 24 hours (T2) and after 60 hours (T5) ], and the digital image analysis obtained using
ImageJ and CellC software for cell counting and determination of septa .
The calculation of FDC during incubation in light and darkness clearly showed
differences between light and dark incubation, which is in strong correlation with differences
in growth rate and determined by classical methods, spectrophotometry. The main result in
this experiment refers to differences between l ight and dark incubation, the growth rate is
much higher in light compared with the rate of growth in the dark, especially after 24 hours,
the results of automated digital image analysis being accord with those obtained by the
classical method. Appearance of dividing cells in samples incubated in the dark and
corresponding growth rates almost similar in the first 24 hours could be supported by the use
of endogenous reserves accumulated in the dark during the light as a source of carbon and
energy, agreement with the biological significance of these intracytoplasmic inclusions. The
sharp decline in both F DC and the growth rate of long incubation periods in the dark is
explained by the the possible reduction of intracellular organic reserves and a change in
strategy for cells to survive in hostile conditions, decrease the frequency of cell division is one
of the most important responses of bacteria against lack of carbon and energy.
Figure 4 ( Figure 4.40). Filaments of Anabaena
sp. cell division during incub ation in light after
staining with 0.02% crystal violet (T0), after 12
hours ( T1), 24 hours ( T2) and after 60 hours of
incubation (T5), the ) digital image analysis using
ImageJ software and CellC for cell counting and
determination of division septa , arrows indicate
cells in division (Sarchizian and Ardelean , 2012 ).
9
The FDC calculated in light and darkness incubation clearly show important
differences between light and dark incubations which are in strong correlation (in agreement
with the a bove equation) with the differences in the growth rate . The main result concerns the
differences between light and dark incubations, the growing rate being much higher in light as
compared with dark conditions , especially after 24 hours. The occurrence of dividing cells in
dark incubated samples and the corresponding growth rates almost similar during first 24
hours could be sustained by the use of endogenous r eserves accumulated in previous
(continuous) light period as a source of carbon and energy in agreement with the biological
signification of these reserves. Furthermore, the absence of organic substances in the
composition of BG 0 medium constrain the cells to have access only to intracellular organic
reserves. The sharp decrease both in FDC and in the growth rate at longer incubation times in
darkness could be correlated with the diminution of intracellular organic reserves and with a
change in the strategy of the cells in order to survive during hostile conditions, the decrease
in the frequenc y of cellular division being one of the most important responses of bacteria
against shortage in carbon and energy (e.g. Roszak and Colwell, 1987) .
A B
Figure 5 . A (Figure 4.41) . – Comparison of FCD in Anabaena sp. incubation under light and dark, B (Figure
4.44) – FDC in Tychonema sp. incubation under light (blue line ) and dark (red line ) (original).
The FDC calculated for Tychonema sp. incubated in light and darkness clearly show
important differences between light and dark incubations which are in strong correlation
with the differences in the growth rate .The results obtained in this nonheterocystous strain are
in agreement with t hose obtained in Anabaena sp. , the heterocystous strain used in the thesis.
The above argumentation based on the diminution of intracellular organic reserves and/or
with a change in the strategy of the cells in order to survive during hostile conditions a re
probably valid also for Tychonema sp. 00.20.40.60.8
12 ore 24 ore 36 ore 48ore 60 oreF
C
D
Timpul (ore)FCD la lumina FDC la intuneric0.10.20.3
12 ore 24 ore 36 ore 48ore 60 oreFDC
Timpul (ore)
Frecvența celulelor aflate în diviziune la lumină (FCD)
Frecvența celulelor aflate în diviziune la întuneric …
10
A B
Figure 6.A – (Figure 4.42) Growth rate (μ) in culture Anabaena sp. incubated in light and dark (light
blue line indicates cultivation , growing black line indicates the dark ) B – (Figure 4.45.) – Growth rate ( μ) in
culture Tychonema sp. incubated in light and dark (blue line shows the rate of growth to light black line shows
the rate of growth in the dark ) (original).
Direct viable count method for the quantification of living cells (DVC) was initially
developed to distinguish viable heterotrophic bacterial cells (Kogure et al.,1979 ; Kogure et
al.,1984 ) from cells unable to grow and multiplication in natural samples . This subchapter
presents the results concerning the quantification of cyanobacterial cells capable of cellular
growth and multiplication using the direct viable count method (DVC). During incubation of
cyanobacterial samples in the presence of nalidixic acid as inhibitor of DNA replication , all
other metabolic properti es remain active. Viable cells may continue to metabolize nutrients
and grow but will not be able to divide , thus becoming more elongated after incubation
whereas inactive cells do not elongate during the incubation period. Measurements of cells
size were performed using two automatic software Image J to measure cell leng th and CellC
software for cell quantification in light microscopy, compared with manual counting and
measuring. The results show that the medium cell size increases from 1.81 µm to 3.35 µm in
84 hours of light incubation with nalidixic acid . Up to our knowledge this is the first report
on DVC applied to filamentous cyanobacteria. The method was also used on the unicellular
cyanobacterium Synechocystis PCC 6803 where the results were compared with growth rate
calculated taking into account the increase in optical density (Sarchizian and Ardelean,2012) .
Figure 7
(Figure 4.47). The evolution of the mean and maximum size of cyanobacterial cells during incubation in the
presence of nalidixic acid. of Anabaena sp. at T0, after 24 , 48, 72, 84 hours of (Sarchizian and Ardelean , 2012) .
-0.0100.010.020.030.040.05 Rata de creștere
Timpul (ore)-0.0200.020.040.060.08
T0 12
ore24
ore36
ore48ore 60
oreRata de creștere
Timpul (ore) µ la lumină µ la întuneric
1.8192.293.0853.623 3.354 3.4693.7746.7957.1028.133
T0 24 hours 48 hours 72 hours 84 hoursCell's lenghth (µm)
Time(hours )
11
Analyzing the experimental data we found that 64 % of the cells are capable of growth
and division , 7% of cells belonging to the first class of scale (<1 m) and 57 % (89% -32%) of
cells in class 1-3 μm can be found at the end of the experiment in class size 3 – 6 μm (67 % –
4% = 63%) and 6 -9 μm (1%).
Table 3 ( Table 4.9). Cell size distribution of cyanobacterial filaments during incubation time with nalidixic acid.
(original ).
Direct viable count method applied to Syneccocystis PCC 6803. In Figure 8 is
presented the cells’ shape and size in cultures of Syneccocystis PCC 6803 after criystal violet
0,02% staining, obtained automatically with ImageJ software. Digital image analysis allowed
us to corectelly identify the sizes of cells. Every digital image was analyed with ImageJ in
bright field microscopy and then we utilised the graticula attached to the microscope to
measure the size of cells , we observed increase in cell size during the experiment , and their
elongation .
Figure 8 (Figure 4.48) . Microscopic appearance of cells of
Synechocystis PCC 6803 during incubation with nalidixic acid
after staining with 0.02% crystal violet and cell shape
determination using ImageJ software , using the scale of size
10 µm (Sarchizian and Ardelean , 2012) .
Through the analysis of cell size by size class (percentage values ) we found a
significant increase in the number of cells in class 2 – 3 µm after 2 hours of incubation in light
in the presence of nalidixic acid, along with enlargement of number of cells class 1 to 2 μm,
reaching a value of 47% at the end of 3 hours of incubation.
According to the calculation presented above 71% of the cells are able to grow and
multiply; 71% of the cells from the first size class increase their size, thus increasing the Time
(hours) Cell size distribution (%)
< 1 µm 1-3 µm 3-6 µm 6-9 µm
T0 7% 89% 4% 0%
12 0% 94% 6% 0%
24 0% 90% 10% 0%
36 0% 92% 8% 0%
48 0% 52% 46% 2%
60 0% 80% 19% 1%
72 0% 19% 79% 2%
84 0% 32% 67% 1%
12
7
percentage of cells in larges size classes : 26% increases in class 1 -2 µm, 41 % increase in
class 2 -3 µm and 4 % increase in class 3 -4 µm.
Figure 9 (Figure 4.49) . Size class distribution of Synechocystis PCC 6803 cells incubated in the absence or
presence of nalidixic acid at the beginning of the experiment (T0 – 0 hours) and at the end of 60 hours of
incubation (Sarchizian and Ardelean , 2012) .
As one can see in figure 9, there is a clear difference in size distribution of cells grown
in the absence of nalidixic acid or in its presence. Whereas, at different times (T0 – 0 hours and
T3-60 hours) the distribution is practically the same in populations growing in the absence o f
nalidixic acid, there is a clear shift towards larger cells in populations grown in the presence
of nalidixic acid . The monitoring of cell size distribution in popu lations without nalidixic acid
is needed for accurate quantification of cells capable of growth and division by DVC method
(Kogure et al., 1979). In our experiments, practically the same size distribution at different
times (e.g. 0 hours and 60 hours) in populations growing in the absence of nalidixic acid is
determined by the fact that t he work is done on a pure strain during asincron cultivation, the
increase in length being undoubtedly continue throughout the cell cycle (Sargent, 1975). The
increase in optical density of cultures is practically the same in the absence and in the
presence of nalidixic acid .
Figure 10 ( Figure 4.50) .
Evolution of the optical density in
cultures of Synechocystis PCC 6803
increased year presence or absence
of nalidixic acid (Sarchizian and
Ardelean , 2012) .
In the section of quantifying the redox properties of some strains of cyanobacteria
experiments followed spectrophotometrically measuring of dehydrogenase activity in some
populations of cyanobacteria , analyzing color changes at both the filament and the filament
individual cell level . Preliminary experiments performed to quantify the reduction claim MTT
or 2.6 diclorphenol indophenol (alone or in the presence of a lipophilic electron carrier – 0.30.350.40.450.5
0 24 ore 48 ore 60 oreD.O.
Timpul (ore)
Synechocystis PCC 6803 incubare în prezența acidului nalidixic
Synechocystis PCC 6803 incubare în absența acidului nalidixic
020406080
T0 incubare în absența
acidului nalidixic după 60 ore de incubare în
absența acidului nalidixic după 60 ore de incubare în
prezența acidului nalidixic Numărul de celule Timpul (ore)
< 1
µm
1 -2
µm
13
phenazin metosulfat or 2.6 dichloro benzoquinon ) while reducing MTT cellular level ,
measured i n numerical decline in the blue channel .
Dehydrogenase activity spectrophotometric measurement was performed in the
presence of DCPIP and DCPIP and FMT separately in the presence and in Table 4.18 are
calculated reduction rates in cultures of cyanobacteria .
Table 4 (Table 4.18) . Reduction rates in cultures of cyanobacteria under study (original ).
Cyanobactetrial culture Reduction rate DCPIP
(µmoli/min/ D.O.750 nm) Reduction rate DCPIP
+ FMT ( µmoli/min/ D.O.750 nm)
Synechocystis sp . 3,141 6,666
Synechocystis PCC 6803 1,047 3,044
Anabaena sp. 3,455 4,14
The purpose subchapter showing quantification redox properties at the individual
biological (cyanobacteria filament of Anabaena sp.) i s to investigate the possibility of
Anabaena sp strain to reduce a artificial acceptor of electrons, with particular emphasis on
quantitative determinations within a single cell using automated image analysis for accurate
color measurement cells within filaments of cyanobacteria, as up to this time the first report
on the use of automated image analysis to measure the reduction of artificial redox carrier
within a single cell in cyanobacteria. In this section are presented the quantitative results
concerning biotechnological potential of filamentous cya nobacteria strain Anabaena sp. the
ability to reduce a electron acceptor added artificial extracellular composition of the
individual cells of a filament cianobacterian. A particular focus of this chapter is on
quantitative determination by automated digit al image analysis of cells in each filament
capacity to reduce MTT, an artificial electron acceptor. Our results showed a strong decrease
(about 4 times in 24 hours) blue signal during MTT reduction by each individual cell
analyzed, as a consequence of ora nge light absorbed by reduced MTT. After consulting
international literature, this is the first report on the use of automatic digital image analysis to
measure the ability to reduce artificial electron acceptor at a cellular level, the filaments of
cyanob acteria. This paper argues for the importance of particular mathematical methods of
digital imaging in bright field, a precise methodology for analyzing detailed and objective
measurement of color intensity of each individual cell (Sarchizian et al., 2011) .
A new way of M icrobiology at the cellular level approach is automatic image analysis
classical individual bacterial cells obtained using different types of microscopes, to quantify
important parameters such as cell enumeration, calculation of cell volume and frequency
division cells, in situ classification of bacteria, active bacteria enumeration in terms of
breathing and characterization of bacterial growth on a solid medium, biofilm viability and
14
physiological activity (eg, Yang et al., 2000. Lehm ussola et al., 2008,. Chavez de Paz, 2009,
Edelstein et al., 2010). Figure 11 presents successively macroscopic appearance of
cyanobacteria in suspension culture Anabaena sp. in the presence of MTT at T0 – initial time,
T4 – after 4 hours of incubation in light, T6 – after 6 hours incubation in light, T24 – after 24
hours of incubation in light at 28 ° C. Highlighting MTT reduction was visualized using
bright -field optical microscopy, which can be observed in filaments of cyanobacteria cells that
changes co lor as a result of MTT reduction.
T0 T4 T6 T24 A B
Figure 11 (Figure 4.55) . Macroscopic appearance of cyanobacteria l suspension during incubation in light for
studying MTT reduction (T0, initial time, T4, after 4 hours of incubation in the light, T6 – after 6 hours
incubation at light T24 – after 24 hours of incubation at light ) A – (Figure 4.56) . – Digital image of Anabaena sp
filaments without addition MTT , B – the view of culture Anabaena sp. after 24 hours of incubation in light in the
presence of MTT (Sarchizian , Cîrnu , Ardelean , 2011) .
Figure 12 presents the results of automatic analysis of digital color images , while for
10 consecutive cells of a filament of cyanobacteria in the presence of MTT and the average
results of pixels in the three RGB channels .
Figura 12 (Figura 4.57). Analiza automată de imagine digitală pentru determinarea schimbării de culoare în
timp (T0, T2, T6, T24) pentru câte 10 celule consecutive dintr -un filament de Anabaena sp. tratată cu MTT și
rezultatele automate ale medii lor pixelilor în trei canale de culoare (Sarchizian, Cîrnu, Ardelean, 2011) .
These type of images were further used to measure reduction of MTT occurring at
single cell level by automated color image analysis taking into account the change in color
due to formation of purple formazan (reduced, insoluble MTT). We observed that all images
taken in bright field microscopy can be analyzed with Image J software and can detect any
region of interests (ROI’s) and after that measure the mean of pi xel for each cells from
15
cyanobacterial filament. This steps used in our study allowed to determine the mean of pixel
in channel red, green and blue (RBG) for analyzes cells and also to study the different aspect
of MTT reduction in cyanobacterial filaments in time.
In Figure 12 there is a decrease in time, the total intensity of light that passes through
every cell of the filament (scale decreases from 20,000 pixels to 1000 pixels at time zero after
24 hours of incubation with MTT ), suggesting that analyzed each cell absorbs and / or reflects
incident light more therefore less light is available to go through each cell. Since the reduced
MTT is colored (purple ) and insoluble , both processes (increasing ) the absorption of light by
colored compounds and light scattering (increasing ) the reduced MTT crystals should be
considered for light diminished passing through each individual cell of cyanobacteria .
Figure 13 ( Figure 4.58).
Evolution arithmetic mean of
pixels in the three color channels :
red, green and blue during the
incubation period of suspension of
cyanobacteria in the presence of
MTT (0.5 mg / mL) (Sarchizian ,
Cîrnu , Ardelean , 2011) .
The dramatic decrease in blue channel could logically be attributed to the absorption
of the complementary color, orange, by the reduced, purple, MTT; the same for the decrease
in red channel as a consequence of absorption of green light by the reduced MTT . When it
comes to the decrease in the green channel, its signification is under investigation being
probably related to the occurrence of multiple light processes (absorption, reflection,
transmission) whose interaction with different (colored) cell compo nents, and processes, is not
yet understood (Sarchizian, Cîrnu , Ardelean , 2011) .
To quantify the redox properties of individual cell level filament Anabaena sp. results
obtained by digital image analysis of cells in filaments cation analyzed for each experimental
time to light during cultivation were found under UserGuide regions of interest were analyzed
ImageJ and color histograms of each cell . The results of automated digital image analysis of
cells in a filament of cyanobacteria at time T0 , T1, T2, T3, T4 was considered filaments
composed of respectively 13 , 17, 24 , 19, 16 cells that were defined regions of interest (ROI )
as UserGuide ImageJ and were analyzed color histograms of each cell , resulting in the
following rezutate , summarized in Figure 14.
30,000130,000
T0 o oră 2 ore 6 ore 24 oreM
e
d
i
ap
i
x
e
l
i
l
or
Time(hours) canalul roșu canalul verde
canalul albastru Linear (canalul roșu)
16
T0
T1
T2
T3
70,00090,000110,000130,000150,000170,000190,000
Celula
1Celula
2Celula
3Celula
4Celula
5Celula
6Celula
7Celula
8Celula
9Celula
10Celula
11Celula
12Celula
13Media pixelilor din canalul
de culoare
Celula din filament
Canal roșu Canal verde Canal albastru
100,000110,000120,000130,000140,000150,000160,000170,000180,000190,000Media pixelilor din canalul de
culoare
Celula din filament
60,00080,000100,000120,000140,000160,000
70,00090,000110,000130,000150,000170,000
Canal roșu Canal verde Canal albastru
17
T4
Figure 14 (Figure 4.67) . Automatic analysis of cyanobacteria cell level of filaments studied (original ).
In conclusion, these results show the importance of mathematical methods for image
processing and light signals, useful for microbiological research at the cellular level.
Our results show a strong decrease in signal blue during MTT reduction by each individual
cell analyzed, as a consequence of orange light absorption by reduced MTT. This is the first
report on the use of automated digital image analysis to measure the reduction of artificial
electron carriers at the cellular level in filament ous cyanobacteria. Also, these results are
important for basic research in microbiology at the cellular level, but also for biotechnological
research linking redox properties of cyanobacteria by using them as light energy into
electricity converters and their use as biosensors.
Using quantum dots to study aims to investigate the cytotoxicity cyanobacteria
interac tion betw een quantum dots (CdSe/ ZnS) and cyanobacteria in natural samples collected
from mesothermal sulphurous spring, as well as enrichment cultures unicellular and
filamentous cyanobacteria, while with digital image analysis of cyanobacteria (Armaselu et
al., 20 11). Digital images taken with epifluorescence microscope showed that unicellular
cyanobacteria were stained with quantum dots, and in terms of filamentous cyanobacteria,
quantum dots have migrated inward and remained attached to sheath their digital image
analysis performed successively digital images obtained from a video attachment
cyanobacterial filaments quantum dots tried to explain this change of color of filamentous
cyanobacteria labeled with quantum dots by adding additional quantities of quantum d ots in
enriched cultures of cyanobacteria. Adding quantum dots 0559 the spiral filaments of
cyanobacteria have found that adding a second after the quantum dots, the filament of
cyanobacteria has deep red color due to the natural fluorescence of chlorophyl l color overlay
over color quantum dots, and after about 40 seconds filament cyanobacteria broken, noting the
toxic effects of cyanobacteria on quantum dots under study. 050,000100,000150,000200,000
18
Each image taken by the video recording was automatically analyzed three color
channe ls RGB and subject to statistical analysis of data with Microsoft Excel. Were analyzed
11 images taken every 10 seconds using Microsoft MovieMaker software. Thus, we found
that the green channel pixel value increases with quantum dots attached filament
cyanobacteria, increases with decrease in year blue channel.
Figure 15 (Figure 4.77) . Evolution of
pixels in the green channel and blue
(original).
Cytotoxicity studies were conducted and cyanobacteria isolated from the Black Sea,
viewed as the natural fluorescence and bright field, quantum dots in the presence or in the
absence thereof 0560 . In this case was intended effect on the color of fluorescent quantum
dots under study cyanobacteria . 7,00057,000107,000
0 sec 20 sec 40 sec 60 sec 80 sec 130 secMedia pixelilor
Timpul (secunde)Canalul verde Canalul albastru
19
A. a. b. c. d. d.
B. a. b. c. d.
C. a. b. c. d.
D. a. b. c. d.
Figure 16 (Figure 4.78) . Evolution of cyanobacteria filament fluorescence : A-natural fluorescence of cyanobacteria , B-colored cyanobacteria filament after one second from
the addition of quantum dots 0560 , C-color filament of cyanobacteria after a minute after the addition of quantum dots 0560 , D-color cyanobacteria filament after 15 minutes
after the addition of quantum dots 0560 , to digital image red channel b – channel digital image green digital image channel c-blue d-digital image of the region of interest
analyzed after extraction stages image substrate (Armaș elu et al., 2011) .
20
Evolution after addition of fluorescent color , step by step, 0560nm quantum dots is
shown in Fig. 16 (Fig. 4.78) . Color natural fluorescence of chlorophyll a in cyanobacteria
filament is red fluorescent color representing adequate cyanobacteria. After the addition of
quantum dots in cultured cyanobacteria , we found that they preferentially migrated to the
filament of cyanobacteria , remaining attached to them . An explanation of the natural
fluorescence color change from red to purple color would be overlap , such as natural
fluorescence of chlorophyll a red with green fluorescent quantum dots used in the experiment ,
and purple is an overlap with the blue red . These findings that have revealed directly by
fluorescence microscopy were subsequently explained by digital image analysis performed on
digital microphotographs captured during the experiment .In automatic digital analysis using
ImageJ software , the software options for determining the intensity of the primary colors red,
green and blue color histograms by analyzing each digital image analysis and data processing ,
we found that the intensity of green color of digital images analyzed increase from T0 to T3
(after 30 minutes of adding quantum dots repeated suspension cyanobacteria ), while
increasing the intensity of blue color , while red intensity showed a downward curve .
Figure 17 ( Figure 4.79).
Digital analysis of the evolution of
fluorescent color in RGB (Armaș elu et
al., 2011) .
Consulted the literature , we found that this is the first report of automatic digital
analysis showing color change fluorescent filaments of cyanobacteria , following the
submission , step by step, quantum dots filaments of cyanobacteria (Armaș elu et al., 2011) .
Figure 18 (Figure 4.80) . Color
evolution of cyanobacteria
filament (region of interest
automatically analyzed ) in each
color channel : red channel , green
channel and the blue channel after
addition of quantum dots constant
in 0560 extracting suspension of
cyanobacteria and substrate each
image (original ).
010203040
T0 1 minut 15 minute 30 minuteM
e
a
n
o
fp
i
x
e
l
Time (min utes )
canalul roșu canalul verde canalul albastru
0100200300
T0 T1 (1 min.) T2 (15 min) T3 (30 min.)M
e
a
n
o
fp
i
x
e
l
Time(min utes )
canalul roșu canalul verde canalul albastru
21
To note is that the green channel intensity increases by about 5 times after adding the
quantities of quantum dots. After adding a second quantity of quantum dots, the green channel
intensity increased further, and after adding m ultiple quantities of quantum dots, green
channel intensity remains almost constant. Regarding the blue channel fluorescence intensity
is higher than the red channel or even unprocessed green channel (blue channel was observed
in digital images digital ana lysis early in filamentous cyanobacteria automatic digital).
Cytotoxicity of quantum dots fluorescence at 490nm, 520nm, 560nm and 600nm was
investigated in different species of unicellular cyanobacteria, such as Synechocystis PCC 6803
culture collection an d cultured unicellular cyanobacteria isolated from mesothermal
sulphurous spring, denoted Synechocystis sp . It is known that quantum dots affects electron
transport related to energy metabolism, both fototrofe bacteria and the heterotrophic bacteria.
The Light (Figure 19) and the dark (Figure 20) for Synechocystis PCC 6803 collection
culture differences were observed in the first 3 hours after adding resazurin when column 9
(quantum dots added before adding resazurinei) shows a slight change color as a re sult of
reducing resazurinei for all quantum dots used in the study (483nm, 522nm, 559nm, 609nm
and), these differences being more evident after 7 hours of reaction (results not shown). More
differences between the effects of QD incubated together with cya nobacteria either in light or
in dark appeared after 24 hours. In light, at 24 hours of incubation in the presence of
resazurine in column 1 and 9 the reduction is more advanced then at 7 hours, whereas very
low changes in color are visible in other wells, arguing that in light with all types of QD the
suppression of metabolic activity by these nanoparticles is very severe.
Figure 19 (Figure 4.81) . Reducing resazurinei (pink-purple) by Synechocystis PCC 6803 incubated
with 200 pg dots light cuantice/200μL suspension of cyanobacteria (A-483nm quantum dots, quantum dots B-
522nm, 559nm quantum dots C-, D-609nm quantum dots ) at time zero and after 24 hours of incubation in light
(Ardelean et al., 2011) .
In darkness after 24 hours in column 1 and 9 the reduction is more advanced then at 7
hours; moreover different degrees of reduction are visible in other wells arguing that even in
longer incubation times in darkness with all types of QD the ability to red uce resazurin to its Timpul zero: la lumină După 24 de ore: la lumină
A
B
C
D 1 3 2 6 4 5 9 7 8 10
A
B
C
D 1 3 2 6 4 5 9 7 8 10
22
20
pink, semi reduced form (resorufin) is present in all experimental conditions. This is an
important difference as compared with light incubation, suggesting that the cytotoxicity of
these QD against Synechocystis PCC 6803 is stronger in light than in darkness. Up to our best
knowledge this is the first report concerning the higher citotoxicity of QD against
cyanobacteria in light than in darkness, results which suggest that the interactions between
photosynthetic cel ls and QD is stronger in light than in darkness .
Figure 20 (Figure 4.82) . Reducing the Synechocystis PCC 6803 resazurinei incubated in the dark with 200 pg
dots cuantice/200μL suspension of cyanobacteria (A-483nm quantum dots, quantum dots B-522nm, 559nm
quantum dots C-, D-609nm quantum dots) at time zero and after 24 hours of incubation in the dark ( Ardelean et
al., 2011) .
Given the well -known higher chemical reactivity to light these semiconductor
nanoparticles may believe that the higher reactivity could be involved in higher cytotoxicity
them to light. Whether there is an interaction of light with quantum dots photosynthetic
metabolism of intact cyanobacteria is another interesting question. One can believe that
quantum dots located on t he cell wall or cell membrane tilacoidele should interact with
membranes located in interiorulul cytoplasm by an unknown mechanism and / or because of
very small diameters, 4 -6 nm of these quantum dots their penetration within the cytoplasm
could also be c onsidered (Ardelean et al., 2011).
Gross dehydrogenase activity . Preliminary experiments shown that after 21 hours of
incubation in darkness or in light with all the 4 types of QD used in these experiments at a
concentration of 1 pg QD/ 1 μL the abi lity of either Synechocystis PCC 6803 or Synechocystis
sp. to reduce DCPIP alone or in the presence of PMS is completely abolished, showing the
cytotoxic effect of these QD in theses experimental conditions. Following these results, new
experiments have been designed to measure the cytotoxic effect -if any – at shorter incubation
time, namely one or two hours. The incubation of cell suspension with QD were performed
in light as well as in darkness in order to further test the interaction in light as compared with
the dark incubation, as suggested by microplate assays done with resazurin. Incubation of
Synechocystis PCC 6803 cultures in darkness together with QD for one or two hours induces
interesting effe cts on the ability of these cells to reduce DCPIP in the presence of PMS .
A
B
C
D 1 3 2 6 4 5 9 7 8 10
A
B
C
D 1 3 2 6 4 5 9 7 8 10
Timpul zero: la întuneric După 24 de ore : la întuneric
23
20
It noted total inhibition of DCPIP reduction in the presence of FMT by Synechocystis
sp. incubated for 1 -2 hours at light Proving strong cytotoxic effect in light of quantum dots.
Interestingly, the incubation light of Synechocystis PCC 6803 culture or the culture of
Synechocystis sp. quantum dots with one or two hours, completely eliminate the ability of
these cells to reduce DCPIP in the presence of FMT again claiming higher cytoto xicity of
quantum dots to light than in the dark, in these species of cyanobacteria.
When it comes to the mechanism(s) responsible for the inhibitory effects of QD no
original experiments have been done but, in agreement with the literature, one could thin k that
the interactions between cells and QD causes the production of reactive oxygen species (ROS)
but other mechanism(s) could also be involved. One important task in all these experiments
concerns the physical relationship between QD and different microbial populations, with
special emphasis on the position of QD towards cell wall and cell membrane. It seems
logically to assume that the first site of interaction between these nanoparticles and cells is at
the level of cell wall; however cell wall ha s rather different structure in Gram – negative
bacteria (including cyanobacteria) and Gram -pozitive bacteria. The physical access of QD at
the external face of the cell membrane (toward cell wall) is still an open question as well as
the ability -if any – of these CdSe/ZnS core -shell quantum dots (with long chain amine capping
agent) with dimensions in the range of few nanometers to pass through the intact (or
previously damaged!) cell membrane to enter the cytoplasm .
24
20
CONCLUSIONS :
1.The thesis presented the isolation, purification and ident ification at genus level samples of 8
strains of cyanobacteria from sulphurous mesothermal spring from Oban -Mare (Mangalia).
2. Improved methods for purification of cyanobacteria strains have established the optimal
conditions to remove heterotrophic contaminants adding the carbon source before antibiotic;
antibiotics t ienam, augmentin, cephalexin, nalidixic acid had bactericidal effect on
heterotrophic bacteria in cultures this is important fundamanet for making a method of
obtaining axenic cultures of cyanobacteria ; augmentin was used for the first time on
cyanobacteria l cultures in these experiments, having also a strong bactericidal effect on
heterotrophic bacterial tested.
3. Determination of the growth rate of cyanobacteria under aerobic conditions in light
whatever using atmospheric nitrogen as the only nitrogen source (BG 0 culture medium) or
nitrate (BG 11 culture medium) led me to obtain the generation time for cyanobacte rial strains
(Nostoc 1 sp., Nostoc 2 sp., Synechocystis sp., Oscillator ia sp., Anabaena sp., Synechocystis
sp. anoxigenic).
4. Determination of the growth rate by calculating the frequency of dividing cells (FDC)
revealed: calculating growth rate in two po pulations of filamentous c yanobacteria isolated
from Obanul Mare (Mangalia), grown in the laboratory to light: the strain of Anabaena sp. ,
the maximum growth rate in BG 0 culture medium is 0.039 hours-1, and for unformatted strain
of heteroc ysts Tychonema sp. maximum growth rate in BG 11 culture medium is 0.057 h-1,
based on data from the literature we can say that this is the first report on the use of the
method for determining the growth rate using frequency of dividing cells (FDC) applied on
filamentous cyanobacteria ( heterocysts forming or not) and also, this is the first report on the
use of the method for determining the growth rate by calculating FCD coupled with
automated image analysis of digital images obtai ned from bright field microscopy .
5. Usin g the direct viable count method described by Kogure et al (1979) on filamentous
cyanobacteria led me to the following conclusions from experiments : that 64% and 71% of
cells are capable of growth and division. The method could be successfully used for dir ect
determination of cells capable of growth and reproduction in natural samples containing
filamentous cyanobacteria, including cells within a single filament or to differentiate
individual filaments are increasing (containing at least one cell capable of growth and
multiplication) to the rest of the filament (which do not contain any such cell), only some cells
from filaments analyzed changes its size during the experiment, suggesting that under natural
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conditions, only certain cells made cell growth, after the consultations of international
scientific literature this method were not applicated on filamentous cyanobacteria, only one
reporting unicellular cyanobacteria (Lucilla et al., 1996), also we combined bright field
microscopy techniques with digital image analysis in the study of filamentous cyanobacteria
treated with nalidixic acid.
6. Studying the redox properties with spectrophotometric methods at the population level in
some strains of cyanobacteria isolated led me to obtain different values of overall
dehydrogenase activity.
7. During incubation in light of cyanobacteria in the presence of artificial electron acceptor
(MTT) changes in color intensity level filament are significant compared to initial time during
the channel red, green and blue, decreases as the 91.7%, 89 , 8% and 86.8%, in line with
higher speed of reduction of MTT in conditions of light during incubation in the dark in color
intensity changes at filametent are very small compared to the initial time (decrease of up to
95-97%) c ompared with the results obtained during incubation in light, consistent with very
low speed to reduce MTT in the dark. Existence of variability for each individual filament of
cyanobacteria suggests that metabolic intensity is different for different fila ments of
cyanobacteria (at individual biological level ) individual cell level for all cells in the filament,
there is great variability in the values obtained during incubation at light, suggesting that
metabolic intensity is different for the individual biological cells, represented by
cianobacterian filament, these results represent the first report on the use of automated digital
image analysis to measure the reduction of artificial electron at the cellular level in
filamentous heterocysts forming cyan obacteria.
8. The use of quantum dots (CdSe/ZnS) in cyanob acteria was found that the intensity of green
color of digital images analyzed time increases from initial time to 30 minutes of adding
quantum dots repeated suspension of cyanobacteria, as a result of accumulation on the surface
of quantum dots on cyanobacterial filaments, while increasing the intensity of blue color,
while red intensity showed a declining path as the original time average pixel values in the
three color channels, observing to one minute that these values have changed in the 3 color
channels. Pixel intensity value of red channel decreased very slowly after the first treatment
with quantum dots, but after the second treatment this value drops to half and then becomes
stationary. T his behavior can be attributed to changes in red fluorescence of chlorophyll a in
filamentous cyanobacteria. Green channel can be considered as the next channel variation in
the intensity of green fluorescence of quantum dots, experimental methods are successfully
applied both on freshwater cyanobacteria and marine cyanobacteria.
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9. The s tudy of the cy totoxic effect of quantum dots on cyanobacterial culture i n light and in
dark cultivation led us to the following conclusions: the cytotoxicity of quantum dots on the
culture of Synechocystis PCC 6803 is stronger during incubation in the light than in the dark.
Interestingly, the incubation in light with quantum dots of Synechocystis PCC 6803 culture or
our isolated culture Synechocystis sp. with one or two hours, completely eliminate the ability
of these cells to reduce DCPIP in the presence of PMS once again supporting higher
cytotoxicity of quantum dots in light than in the dark, in these species of cyanobacteria. In
light cultivation quantum dots inhibit total gross dehydrogenase activity both in culture
Synechocystis PCC 6803 and Synechocystis sp, e ven after an hour of incubation. Referring to
the literature, this is the first report on quantum do ts high toxicity against cyanobacteria
incubated in light, compared to incubation in the dark. At dark, after 1 -2 hours of incubation
with quantum dots, is induced in Synechocystis PCC 6803 culture a strong increase of
dehydrogenase activity (from 260% to 1000%!) w hile in Synechocystis sp. culture there is a
decrease of 60 -90%.
All results presented in the doctoral thesis conducted us to say that the aims of the
study and objectives have been achieved , the results are original, novelty nationally and som e
even internationally.
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31
ANEXX
Scientific papers published in ISI journals :
Sarchizian , I., Cîrnu , M., Ardelean, I.I., 2011. Isolation of a heterocyts – forming
Cyanobacterium and quantification of its biotechnological potential with respect to redox
properties at single cell level , Romanian Biotechnological Letters, Vol. 16, No.6,
Supplement, p.3 -9.
Armașelu A., Popescu A., Apostol I., Ardelean I. , Damian V., Iordache I., Sarchizian
I., Apostol D., 2011. Passive nonspecific labeling of cyanobacteria in natural samples using
quantum dots, Opt oelectronics and Advanced Materials -Rapid Communications , 5(10),
p. 1084 -1090 .
Scientific papers published in the proceedings of international conferences ISI:
Ardelean, I., Sarchizian , I., Manea, M., Damian, V., Apostol, I., C îrnu, M.,
Armaselu , A., Iordache, I., Apostol, D., 2011. CdSe/ZnS quantum dots citotoxicity against
phototrophic and heterotrophic bacteria, Proceeding of NANOCON 2011 , Brno , Czech
Republic, 21. – 23. 09. 2011. ISBN 978 -80-87294 -27-7, pp 608 -617
Sarchizian, I., Ardelean, I.I., 2012. Frequency of dividing cells and growth rates In
population of filamentous cyanobacteria isolated from sulphurous mesothermal spring Obanul
Mare (Mangalia), Proceedings 12th International Multidisciplinary Scientific GeoConference
SGEM 2012, ISSN 1314 -2704,vol 5, pp.423 -430.
Sarchizian, I., Ardelean, I.I., 2012. Quantification of cells capable of growth and
multiplication using direct viable count method in filamentous and unicellular cyanobacteria,
Proceedings 12th International Multidisciplinary Scientific GeoConference SGEM 2012, ISSN
1314 -2704,vol.5, pp.655 -662.
Scientific papers published in the proceedings of international conferences organized by
international professional societies (indexed BDI) :
Ardelean I.I., Dıaz -Pernil D., Gutierrez -Naranjo M.A., Francisco Pena -Cantillana,
Raul Reina -Molina, Iris Sarchizian , 2012. Counting Cells with Tissue -like P Systems.
Proceedings of the Tenth Brainstorming Week on Membrane Computing, January 30 –
February 3, 2012, Sevilla (Spain), vol. I, p.69 -78.
Articles published in journals recognized by the Romanian CNCSIS – B +:
Ardelean I.I., Ghiță S., Sarchizian I ., 2009. Epifluorescent method for quantification of
planktonic marine prokaryotes . Proceedings of the 2nd International Symposium “New
Research in Biotechnology” serie F, Bucharest, ISSN 1224 -7774, p: 288 -296.
Ardelean I.I, Ghiță S., Sarchizian I. ; 2009. Isolation of oxygenic phototrophic and oxic
heterotrophic bacteria with potential for gasoline consumpt ion. Proceedings of the 2nd
International Symposium “New Research in Biotechnology” serie F, Bucharest, ISSN 1224 –
7774, p: 278 -287.
Ghiță S., Sarchizian I. , Țuțuianu A., Ghiță D., Abdulcherim E., Ardelean I.I.,2010.
Quantification of actively growing hydr ocarbon -oxydizing /tolerant bacteria in marine
microcosms supplemented with gasoline . Proceedings of the 3nd International Symposium of
Biotechnology „SimpBTH 2010” serie F, Bucharest, ISSN 1224 -7774, p: 204 -212.
Articles published in journals recognized by the Romanian CNCSIS – other categories:
32
Sarchizian I., Ardelean I.I., 2010. Axenic culture of a diazotrophic filamentous
cyanobacterium isolated from mesothermal sulphurous springs (Obanul Mare –
Mangalia), Rom. J.Biol – Plant Biol., vol. 5 5, No.1, p. 47 -53.
Sarchizian I. , Ardelean I.I., 2010. Improved Lysozyme Method to Obtain
Cyanobacteria in Axenic Cultures, ROM. J. BIOL. – PLANT BIOL., vol. 55, No 1, p. 47 –53.
Ghiță S., Sarchizian I. , Ardelean I.I., 2010. Utilization of epifluorescence microscopy
and digital image analysis to study some morphological and functional aspects of prokaryotes .
Ovidius University Annals – Biology -Ecology Series. Vol. 14, No. 1, ISSN -1453 -1267, p.
127-137.
Articles published in proceedings of national conferences :
Ghiță S., Sarchizian I., Ardelean I.I.; Enumerarea și evidențierea celulelor bacteriene
din medii marine poluate cu hidrocarburi – recomandări metodologice pentru aplicații în
cercetarea de laborator. Sesiune științifică „Dezvoltare du rabilă în regiunea Mării Negre”
Univ. Maritimă Constanța, ISSN 2069 -248X, Ed. Nautica, p: 109 -117, (2010).
Sarchizian I., Ghiță S., Ardelean I.I.; Aplicații ale analizei de imagine digitală pentru
măsurarea și enumerarea bacteriilor heterotrofe și fotosin tetizante utilizând microscopia de
epifluorescență. Sesiune științifică „Dezvoltare durabilă în regiunea Mării Negre” Univ.
Maritimă Constanța, ISSN 2069 -248X Ed. Nautica, p: 102 -108, (2010).
Participation i n international scientific meetings :
Sarchizian, I., 2011. Automated Analysis of Unicellular and Filamentous
cyanobacteria in bright field and fluorescence microscopy – preliminary results and
perspectives , First International School on Biomolecular and Biocellular Computing, Escuela
Univer sitaria de Osuna, Sevilla, Spain, 5 -7 Sept.2011 ( prezentare orala ).
Abstracts published :
Ardelean, I.I. , Cîrnu, M., Pascu, D., Sarchizian, I. , Damian, V., Apostol, I., Iordache,
I., Apostol , D., 2012. Metalic Nanoparticle ( Gold, Silver, Aluminium) Citotoxicity against
Phototrophic and Heterotrophic Bacteria, NANOCON 2012 , 23 – 25. 10. 2012 , Brno , Czech
Republic.
Ardelean, I., Sarchizian , I., Manea, M., Damian, V., Apostol, I., C îrnu, M.,
Armaselu , A., Iordache, I., Apostol, D., 2011. CdSe/ZnS Quantum dots citotoxicity against
phototrophic and heterotrophic bacteria, NANOCON 2011 , 21 – 23. 9. 2011, Brno , Czech
Republic.
Sarchizian I., Ghiță S., Manea M., Ignat M., Moisescu C., Ardelean I. 2010. Isolation
of axenic cultures of cyanobacteria from sulphurous spring and marine environments;
screening for the biotechnological signification of the isolates . International Symposium on
Phycological Research, Varanasi 221005, India, ABT29 .
Ardelean I.I., Sarchizian I., Damian V., Apostol I., Manea M., Ghiță S., Armaselu
A., Iordache I., Apostol D. 2011. Interaction of quantum dots with phototrophic and
heterotrophic bacteria: nonspecific labeling and citotoxicity. EURONANOFORUM
Budapesta .
Ghiță S., Sarchizian I., Țuțuianu A., Ghiță D., Abdulcherim E., Ardelean I.I. 2010.
Quantification of actively growing hydrocarbon -oxydizing /tolerant bacteria in marine
33
microcosms supplemented with gasoline. Proceedings of the 3nd International Symposium of
Biotec hnology „SimpBTH 2010” serie F, Bucharest, ISSN 1224 -7774, p: 204 -212 .
Ardelean I.I., Ghiță S., Sarchizian I ., 2009. Epifluorescent method for quantification
of planktonic marine prokaryotes . Proceedings of the 2nd International Symposium “New
Research in Biotechnology” serie F, Bucharest, ISSN 1224 -7774, p: 288 -296 .
Ardelean I.I, Ghiță S., Sarchizian I., 2009. Isolation of oxygenic phototrophic and
oxic heterotrophic bacteria with potential for gasoline consumption . Proceedings of the 2nd
International S ymposium “New Research in Biotechnology” serie F, Bucharest, ISSN 1224 –
7774, p: 278 -287.
Ardelean I.I., Sima L.E., Ghiță S., Sarchizian I., Popoviciu D.R., Lăzăroaie M.M,
2009. Quantification of marine bacteria in pure culture and microcosms by epifluorescence
microscopy and flow cytometry . FEMS 2009 -3rd Congress of European Microbiologists
Gothenburg, Sweden June 28 -July 2.
Ardelean I.I, Damian V., Sarchizian I., Ghiță S., Armaselu A., Apostol D., 2010.
Visualisation of axenic and nonaxenic cul tures of cyanobacteria using semiconductor
quantum dots . International Symposium on Phycological Research, Varanasi 221005, India,
AEE28 .
Ardelean I.I., Ghiță S., Popoviciu D.R., Damian V., Sarchizian I., Manea M., Apostol
I., Iordache I., Armaselu A., Ap ostol D., 2010. Microbial dynamics and diversity in marine
microcosms studied by the use of quantum dots and fluorescent molecules. 14th Evolutionary
Biology Meeting at Marseilles september 21st -24th 2010, poster 22, Association pour l’etude
de l’evolution biologique .
Ardelean I.I., Damian V., Sarchizian I., Ghiță S., Manea M., Apostol I., Popoviciu
D.R., Iordache I, Armaselu A., D. Apostol D., 2010. The use of quantum dots to visualize
heterotrophic and photosynthetic bacteria in pure cultures and microc osms. IBB sept. 2010,
poster 36 .
Sarchizian I. , Ardelean I.I., 2012. Frequency of dividing cells and growth rates In
population of filamentous cyanobacteria isolated from sulphurous mesothermal spring obanul
mare (Mangalia), Proceedings 12th Internationa l Multidisciplinary Scientific GeoConference
SGEM 2012, ISSN 1314 -2704 .
Sarchizian I. , Ardelean I.I., 2012. Quantification of cells capable of growth and
multiplication using direct viable count method in filamentous and unicellular cyanobacteria,
Proceedings 12th International Multidisciplinary Scientific GeoConference SGEM 2012,
ISSN 1314 -2704 .
Sarchizian I., Ardelean I.I.; Evidentierea celulelor capabile de crestere si diviziune
in populatii naturale de cianobacterii filamentoase din izvorul sulfuros mezotermal de la
Obanul Mare (Mangalia); A XXI-a Sesiune de Comunicari stiintifice, Univ. Ovidius
Constanta, 25 -26 martie 2011.
Ghiță S., Sarchizian I., Ardelean I.I.; Utilization of epifluorescence microscopy and
digital image analysis to study some morphological and functional aspects of prokaryotes .
Ovidius University Annals – Biology -Ecology Series. Vol. 14, No. 1, ISSN -1453 -1267, p:
127-137, (2010).
34
10
Participation in national scientific meetings :
Sarchizian I., Ardelean I.I., Ghiță S. Perspective ale analizei de imagine digitală a
cianobacteriilor unicelulare și filamentoase pentru studierea proprietăților redox la nivel
celular, Sesiune de comunicări științifice „Calitatea și Monitoringul Mediului Înconjurător” ,
Univ. Maritimă Constanța, 31 octombrie 2011 (prezentare orala ).
Sarchizian I.; Izolarea unei cianobacterii diazotrofe din izvorul sulfuros mezotermal
de la Obanul Mare (Mangalia); A XIX-a Sesiune de Comunicari știintifice, Univ. Ovidius
Constanța, 27 -28 martie 2009 ( prezentare orală ).
Sarchizian I., Ghiță S., Ardelean I.I.; Obținerea culturilor axenice de cianobacterii.
Sesiune de comunicări științifice „Calitatea și Monitoringul Mediului Înconjurător”, Univ.
Maritimă Constanța, 31 octombrie (2009) (prez entare orală ).
Sarchizian I., Ghiță S., Ardelean I.I.; Aplicații ale analizei de imagine digitală pentru
măsurarea și enumerarea bacteriilor heterotrofe și fotosintetizante utilizând microscopia de
epifluorescență. Sesiune științifică „Dezvoltare durabilă în regiunea Mării Negre” Univ.
Maritimă Constanța, ISSN 2069 -248X Ed. Nautica, p: 102 -108, (2010) (prezentare orală ).
Ghiță S., Sarchizian I., Ardelean I.I.; Cuantificarea procariotelor marine în condiții de
microcosmos. Sesiune de comunicări științifice „Calitatea și Monitoringul Mediului
Înconjurător”, Univ. Maritimă Constanța, 31 octombrie (2009) (prezentare orală ).
Ghiță S., Sarchizian I., Ardelean I.I.; Enumerarea și evidențierea celulelor bacteriene
din medii marine poluate cu hidrocarburi – recomandă ri metodologice pentru aplicații în
cercetarea de laborator. Sesiune științifică „Dezvoltare durabilă în regiunea Mării Negre”
Univ. Maritimă Constanța, ISSN 2069 -248X, Ed. Nautica, p: 109 -117, (2010) (prezentare
orală ).
Ardelean I.I, Ghiță S., Sarchizian I., Moldoveanu M., Popoviciu D.R.; Studiul
descriptiv al microbiotei marine în sisteme microcosmos: de la rezultate microscopice în
microbiologia marină la perspective predictive în oceanografia microbiologică. Sesiune
științifică națională cu participare internațională “Biodiversitate și impact antropic în Marea
Neagră și în ecosistemele litorale ale Mării Negre” 21 – 22 octombrie 2011 ( prezentare
orală ).
Sarchizian I., Ardelean I.I., Ghiță S.; 2011. Perspective ale analizei de imagine
digitală a cianobacteriilor unicelulare și filamentoase pentru studierea proprietăților redox la
nivel celular , Simpozionul “Calitatea si monitoringul mediului marin”, Ed. Nautica ISSN
2069 -248X. (prezentare orală).
Participation in national and international research contracts :
Sistem de producere cu laser de nanoparticule pentru biotehnologii (Laser -based
manufacturing system for biotech nanoparticles production) (BIO.NANO.LAS) program
„PARTENERIATE IN DOMENIILE PRIORITARE”.
Contract nr. 159/28/10/2011 proiectul PN -II-ID-PCE-2011 -3-0742 “Biodiversitate si
distributie cronologica a microorganismelor in straturile de gheata perena din ghetarul
Scarisoara (Romania) – program IDEI.
Am obținut bursă pentr u participarea la First International School on Biomolecular and
Biocellular Computing la Universitatea din Sevilia, Spania, în septembrie 2011.
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