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- Summary of environmental technique doctoral thesis: Synthesis of silver, copper, iron nanoparticles and their applications in controlling cyanobacterial blooms in the fresh water body
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- MINISTRY OF EDUCATION VIETNAM ACADEMY OF
AND TRAINING SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY
---------------------------
TRAN THI THU HUONG
SYNTHESIS OF SILVER, COPPER, IRON
NANOPARTICLES AND THEIR APPLICATIONS IN
CONTROLLING CYANOBACTERIAL IN THE FRESH
WATER BODY
Major: Environmental Technique
Code: 9 52 03 20
SUMMARY OF ENVIRONMENTAL TECHNIQUE
DOCTORAL THESIS
HaNoi - 2018
- The thesis was completed at the Graduate University of Science
and Technology, Vietnam Academy of Science and Technology
Scientific Supervisor 1: Assoc. Prof. Dr. Duong Thi Thuy
Scientific Supervisor 2: Dr. Ha Phuong Thu
Reviewer 1:
Reviewer 2:
Reviewer 3:
The dissertation will be defended protected at the Council for
Ph.D. thesis, meeting at the Viet Nam Academy of Science and
Technology - Graduate University of Science and Technology.
Time: Date …… month …. 2018
This thesis can be found at:
- The library of the Graduate University of Science and Technology.
- National Library of Viet Nam.
- 1
INTRODUCTION OF THESIS
1. The necessary of the thesis
In recent years, pollution of soil, water and air has become a
serious problem not only in Vietnam but also in many parts of the
world in which the water pollution is more serious problem.
"Water blooming" is the development of microalgae outbreak,
especially cyanobacteria in fresh water bodies and often cause the
harmful effects on the environment such as: the water turbidity and
pH are increase, the levels of dissolved oxygen is reduce due to the
respiration or degradation of algae biomass and especially, the fact
that most cyanobacteria produce the toxicity high. The preventing
and minimizing the development of cyanobacteria is an important
environmental issue that need to pay the attention. The many
methods have been used such as: chemistry, mechanics, biology,
etc., but they are ineffective and expensive, affecting ecosystem
and conducting is difficult, especially in large water bodies.
Therefore, the search and development of new effective solutions
without secondary pollution and friendly with the environment are
increasingly focused research. Nanotechnology is the technology
relating to the synthesis and application of materials with
nanometer sizes (nm). At nanoscale, the material has many
advantage features such as: size is smaller than 100 nm, larger
surface to volume ratio, crystalline structure, high reactivity
potential, creating the effect of resonance Plasmon surface; high
adhesion potential and the nanomaterial was applied in various
fields such as: medical, cosmetics, electronics, chemical catalyst,
environment... For the above reasons, the thesis is proposed as:
“Synthesis of silver, copper, iron nanoparticles and their
applications in controlling cyanobacterial blooms in the fresh
water body” was selected to researched.
2. The objectives of the thesis
Research, fabricate and determine the characteristic of three
nanomaterials (silver, copper and iron) and evaluate the ability to
inhibit the cyanobacteria of nanomaterials in fresh water bodies.
3. The main contents of the thesis
- Fabricate and determine the characteristic of three
nanomaterials: silver, copper and iron.
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- Investigate the ability to inhibit and prevent cyanobacteria of
three nanomaterials.
- Assess the safety of materials and their application.
- Experimental application of materials at laboratory-scale with
the Tien lake water sample.
5. The structure of the thesis
The thesis is composed of 149 pages, 10 tables, 62 figures, 219
references. The thesis consists of three parts: Introduction (3 pages);
chapter 1: Literature review (42 pages); chapter 2: Methodology (16
pages); chapter 3: Resutl and discussion (59 pages); Conclusion and
recommendation (2 pages).
CHAPTER 1. LITERATURE REVIEW
1.1. Introduction of nanomaterial
1.2. Introduction of Cyanobacteria and Eutrophication
1.3. Introduction of the methods to treat the toxic algae
contamination
CHAPTER 2. METHODOLOGY
2.1. The research subjects
2.2. The equipment is used in study
2.3. The methods for synthesis of materials
2.3.1. Synthesis of silver nanomaterial by chemical reduction
method
The silver nanomaterial was synthesized by chemical reduction
method, ion Ag+ in the silver salt solution is reducted to Ag0 by the
reducing agent NaBH4.
2.3.2. Synthesis of copper nanomaterial by chemical reduction
method
The copper nanomaterial was synthesized by chemical
reduction method, ion Cu2+ in the copper salt solution is reduced to
Cu0 by the reducing agent NaBH4.
2.3.3. Synthesis of iron magnetic (Fe3O4) nanomaterial by
simultaneously precipitation method
The iron magnetic (Fe3O4) nanomaterial was synthesized by
simultaneously precipitation method of Fe2+ and Fe3+ salts by
NH4OH.
2.4. The methods for determining the characteristic of material
structure
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The morphology of the three nanomaterials is determined by a
number of methods such as: TEM, SEM, IR, XRD, UV-VIS, EDX.
2.5. The experimental setup methods
The experimental setup methods such as: culture of algae,
selection of nanomaterials, evaluation of the material toxicity, the
evaluation of the influence of nanomaterial sizes and the safety of
nanomaterials on microalgae and the experiment with the Tien lake
water sample were setup.
2.6. The methods of evaluating the effect of nanomaterials on
the growth of microalgae
To evaluate the effect of nanomaterials on the growth of
microalgae, the following methods such as: OD, chlorophyll a, cell
density, the methods for analysis of some environmental quality
indicators (NH4+, PO43-) and SEM, TEM were used.
2.7. The method of statistical analysis
CHAPTER 3. RESUTL AND DISCUSSION
3.1. Synthesis of nanomaterial
3.1.1. Synthesis of silver nanomaterial by chemical reduction
method
3.1.1.1. Effect of the concentration ratio NaBH4/Ag+
The UV-VIS spectrophotometer (Fig 3.1) showed that the
nanosilver colloid was absorbed at the wavelengths about 400 nm
and the synthesized efficiency of silver nanoparticles was
maximum achieved at a ratio 1:2. TEM images (Figure 3.2)
showed that silver nanoparticle size was less than 20 nm.
M1 M2
M3 M4 M5
Figure 3.1. The UV-VIS spectra Figure 3.2. The TEM images of
of nanosilver colloid depends on nanosilver colloid depends on
the NaBH4/Ag+ concentration the BH4-/Ag+ concentration ratio
ratios
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3.1.1.2. Effect of stabilizer concentration chitosan
The UV-VIS measurements in Figure 3.4 showed that the
nanosilver colloid is absorbed at the wavelengths 402-411 nm. The
TEM image of the silver nanoparticles depends on the
concentration of chitosan shown in Figure 3.5. The optimum
chitosan concentration of nanosilver colloid fabricating was chosen
as 300 mg/L. M6 M7
M8 M9 M10
Figure 3.4. The UV-VIS spectra Figure 3.5. The TEM images
of nanosilver colloid depends on of nanosilver colloid depends
chitosan concentrations on the chitosan concentrations
3.1.1.3. Effect of citric acid concentration
The UV-VIS measurements in Figure 3.7 showed that the
nanosilver colloid is absorbed at the wavelengths 402-411 nm. At
the rate of [Citric]/[Ag+] = 3.0 the silver nanoparticles obtained
were of the most uniform, small size and less than 20 nm, the TEM
measurement is shown in Figure 3.8.
M11 M12 M13
M14 M15 M16
Figure 3.7. The UV-VIS Figure 3.8. The TEM images of
spectra of nanosilver colloid nanosilver colloid depends on
depends on acid concentration the [Citric]/[Ag+] concentration
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Figure 3.9. The HR-TEM of nanosilver colloid was tested at
optimal ratio
The structure of silver nanoparticle at the optimum ratio indicates
that they have a typical hexagon crystal structure of metallic
nanoparticles. The HR-TEM images in Figure 3.9 showed that the
crystals has got Fcc (Face-centered cubic) structure. The silver
nanomaterial at the conditions such as: the ratio of NaBH4/Ag+ is
1/4, the [Citric]/[Ag +] is 3.0 and a concentration of chitosan
stabilizer is 300 mg/L were synthesized to experimented the effect of
material on the growth of the studied subjects in the thesis.
3.1.2. Synthesis of copper nanomaterial by chemical reduction
method
3.1.2.1. Effect of the concentration ratio NaBH4/Cu2+
The results in Figure 3.10 show that, in the XRD spectrum
appears the three peak with the intensity match for the standard
spectra of the copper metal at the side (111), (200), (220)
corresponding to angle 2θ = 43.3; 50.4 and 74.00 belong to the
Bravais network in the fcc structure of the copper metal.
M1 M2
M3 M4 M5
Figure 3.10. The XRD pattern
Figure 3.11. The SEM images
of CuNPs were tested in
of CuNPs in NaBH4/Cu2+ ratio
NaBH4/Cu2+ concentration
The SEM measurements (Fig 3.11) of the material were
performed to determine the distribution of the copper particles and
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the TEM measurement for determine the size of copper
nanoparticles (Fig 3.12).
M1 M2
M3 M4 M5
Figure 3.13. The XRD
Figure 3.12. The TEM images
spectrum of CuNPs was tested
of CuNPs in NaBH4/Cu2+ ratio
by Cu0 concentration
The TEM image results showed that, when the NaBH4/Cu2+
concentration ratio is 1: 1 and 1.5: 1, the size of synthesized copper
nanoparticles are bigger than 50 nm. The nanoparticles are
distributed rather uniformly with a size about 20-50 nm when the
NaBH4/Cu2+ ratio is 2 : 1. The nanoparticles are clumped together,
unevenly distributed with the size nanoparticle > 50 nm when the
NaBH4/Cu2+ ratio is 3: 1 and 4: 1 and match with the SEM results.
To respone the objective of this thesis, the M3 sample
2+
(NaBH4/Cu ratio is 2: 1) was chosen as the representative sample.
3.1.2.2. Effect of Cu0 concentration
XRD spectrum in Figure 3.13 showed that the of copper
nanoparticles presents the characteristic peaks of copper
nanomaterial. The characteristic peaks on the schematic have the
sharpness intensity and the wide range of the absorption peak
relatively narrow. In addition, the XRD spectrum of the material
also shows the characteristic peaks of CuO, Cu2O crystals.
The SEM (Fig 3.14) measurement results showed that, the
copper nanoparticles form of the unequal size distribution when the
concentration of Cu0 increases. At concentrations of Cu0 is 2g/L,
the copper nanoparticles are distributed rather uniformly with the
size at 20-40 nm. When the concentration of Cu0 increases to 3;
4g/L, the synthesized copper particles will clump together and
form of the particle sizes >50 nm; at Cu0 concentration is 6, 7 g/L,
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the nanoparticles distributed unevenly and match for the TEM
measurement (Fig 3.15).
N1 N2 N1 N2
N3 N4 N5 N3 N4 N5
Figure 3.14. The SEM image of Figure 3.15. The TEM image
copper nanomaterial was tested at of copper nanomaterial was
Cu0 concentration tested at Cu0 concentration
3000
a) Faculty of Chemistry, HUS, VNU, D8 ADVANCE-Bruker - Cu-51
b)
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File: ThuyVCNMT Cu-51.raw - Type: 2Th/Th locked - Start: 1.000 ° - End: 79.990 ° - Step: 0.030 ° - Step time: 0.3 s - Anode: Cu - WL1: 1.5406 - Generator kV: 40 kV - Generator mA: 40 mA - Creation: 06/10/2016 3:54:39 P
Left Angle: 42.490 ° - Right Angle: 44.350 ° - Obs. Max: 43.281 ° - d (Obs. Max): 2.089 - Max Int.: 1890 Cps - Net Height: 1668 Cps - FWHM: 0.231 ° - Raw Area: 852.6 Cps x deg. - Net Area: 440.4 Cps x deg.
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01-085-1326 (C) - Copper - Cu - Y: 16.13 % - d x by: 1. - WL: 1.5406 - Cubic - a 3.61500 - b 3.61500 - c 3.61500 - alpha 90.000 - beta 90.000 - gamma 90.000 - Face-centered - Fm-3m (225) - 4 - 47.2416 - I/Ic PDF 8.9 - F4
Figure 3.16. The detail characteristics of the N1 copper
nanomaterials sample: (a) SEM image, (b) TEM image, (c) XRD
spectrum
The structure of copper nanomaterial at selected ratio showed
that, the formed copper nanoparticles have the rather
homogeneous surface (SEM image, Fig 3.16a), the uniformly size
in the range of 30 - 40 nm (TEM image, Fig 3.16b) and have the
Fcc structure with diffraction peaks of the netface (111), (200) and
(220) corresponding to angle 2θ = 43.3; 50.4 and 74.00 with high
intensity (XRD spectrum, Fig 3.16c). This material sample is
suitable with the objective of the thesis and were choosen for
further experiment.
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3.1.3. Synthesis of magnetic solution nanomaterial by co-
precipitation method
3.1.3.1. Effect of the CMC stabilizer concentration
The tested result of morphological, size and the dispersion of
material in the ratio of CMC stabilizer and precursor (Fe3O4)
respectively were 1/1; 2/1; 3/1; 4/1 and 1/2 by the SEM and
methods shown in Figure 3.17 and 3.18. The SEM result showed
that the concentration of CMC in the solution is high, the
ferromagnetic nanoparticles are unevenly and the particle size is
big, the accumulation of nanoparticles is easy to occur. At the rate
of CMC/Fe3O4 is 2/1, the obtained ferromagnetic nanoparticles are
uniformly sized and less 20 nm.
Figure 3.17. The SEM image of Figure 3.18. The TEM image of
magnetic solution nanostructure magnetic solution nanostructure
tested in ratios of CMC/Fe3O4 tested in ratios of CMC/Fe3O4
The TEM results showed that the nanoparticle size varies
considerably when the CMC concentrations changed. When the
Fe3O4/CMC is 2:1, the obtained nanoparticles were the smallest,
most uniform and less than 20nm within the superparamagnetic size
range. Therefore, the material sample has a Fe3O4/CMC ratio of 2:1
(encoded sample is FC21) selected to tested for the further factors.
3.1.3.2. The result of infrared measurement of the material
Figure 3.19. The infrared spectrum Figure 3.20. The
of Fe3O4 (a), CMC (b), FC21 (c) and magnetization hysteresis
spectrum of three samples (d) result of material FC21
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The observation in Figure 3.19 showed that the IR spectrum of
ferromagnetic nanoparticles have peaks similar with CMC and
Fe3O4, this proves that the structure of CMC is not broken by the
material synthesis conditions. Therefore, the co-precipitation
method for synthesis of material is suitable for purity as well as
efficiency.
3.1.3.3. The magnetization hysteresis result of material
The result of saturate magnetization hysteresis measurement in
Figure 3.20 showed that ferromagnetic nanoparticles are in the
form of superparamagnetic. The saturate magnetization of Fe3O4
and FC21 is 68 emu/g and 49 emu/g, corresponding to the content
of magnetic phase of the material. The result proves that the
surface interaction of the magnetic phase with the polymer
decreased the saturate magnetization and suitable with the results
of the TEM analysis.
3.2. Evaluating the ability of growth inhibition and prevent
microalgae by synthesized nanomaterials
3.2.1. Study on the selection of concentrations of three types of
nanomaterials
Table 3.1. The screening results of removal M. aeruginosa KG
cyanobacteria of fabricated nanomaterials
The growth
Experimental
No. Samples inhibition of
concentration (mg/L)
cyanobacteria
1 Ag nano 3, 5 and 10 +++
3 Cu nano 3, 5 and 10 +++
5 Fe3O4 nano 5, 10, 100, 150 and 200 -
6 Control 0 -
Notes: +++: Very strong inhibitory effect, ++: Strong inhibitory effect, +:
Normal inhibitory effect, -: Non inhibitory effect.
Figure 3.21. Effect of nanomaterials on growth of cyanobacteria M.
aeruginosa KG after for 7 days.
- 10
The concentration screening tests were conducted to rapidly
assess inhibition effect to M. aeruginosa KG for 7 days. The
results in Table 3.1 and Figure 3.21 showed that the two silver and
copper nanomaterials inhibited the growth and development of
cyanobacteria M. aeruginosa KG after 6 days (Table 3.1 and Fig
3.21a, b), whereas that the ferromagnetic nanomaterial were not
effective against M. aeruginosa KG (Table 3.1 and Fig 3.21c).
3.2.2. Effect of silver nanoparticles on growth and development
of cyanobacteria M. aeruginosa KG and green algae C. vulgaris
3.2.2.1. Effect of silver nanoparticles on growth and development
of cyanobacteria M. aeruginosa KG
The experiments were conducted with the concentrations of
silver nanoparticles increasing from 0; 0.001; 0.005; 0.01; 0.05; 0.1
to 1 ppm in 10 days. The evaluation parameters include: optical
density (OD), chlorophyll a and cell density at 0, 2, 6 and 10 days
(Fig 3.22a, b). The toxicity of silver nanoparticles on growth of the
cyanobacteria M. aeruginosa KG as measured by the concentration
of supplementary material into the culture medium that affected
50% of the individuals (EC50) was 0.0075 mg/L.
Figure 3.22. Effect of silver Figure 3.23. Effect of silver
nanomaterial on growth of the nanomaterial was measured by
cyanobacteria M. aeruginosa the cell density (a) and the
KG after 10 days was growth inhibition efficiency on
measured by (OD) (a), cyanobacteria M. aeruginosa
chlorophyll a (b) KG (b)
The cell density and chlorophyll a showed that, the cell density
and biomass in the control sample increased from the first day (D0)
(110,741 ± 6,317 cells/mL and 1.98 ± 0.06 μg/L, respectively) to the
end of experiment (D10) (5,475, 556 ± 541,274 cells/mL and 23.4 ±
2.96 μg/L, respectively) (Fig 3.23a). All five tested concentration
ranges are toxic to cyanobacteria M. aeruginosa KG. The growth
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inhibition efficiency (Fig 3.23b) > 75% appears in only 4 tested
concentrations from 0.01; 0.05; 0.1 and 1 ppm.
The SEM image result of cell surface structure after 48h exposed
to silver nanoparticles at the concentration of 1 ppm is shown in
Figures 3.24a (the control sample) and 3.24b (the sample exposed to
the concentration of 1ppm silver nanoparticles). In the control sample,
the morphological of cyanobacteria M. aeruginosa KG cells
maintained a round and had a spherical shape with a smooth exterior
surface (Fig 3.24a). In the experimental sample, the cells were
changed to with a distorted and shrunk cell after exposure to silver
nanoparticles (Fig 3.24b). It is said that the silver nanoparticles have
significantly altered the morphology of the cell.
a) b) a) b)
Figure 3.24. Scanning Electron Figure 3.26. Transmission
Microscopy (SEM) micrograph of Electron Microscopy (TEM)
M. aeruginosa KG micrograph of M. aeruginosa KG
The SEM combined with EDX analysis was used to
characterize the chemical composition and the location of AgNPs
on the cell surface of M. aeruginosa KG. The EDX result in Figure
3.25 showed that the silver nanoparticles appear on the surface of
the cyanobacteria M. aeruginosa KG with 0.37% Ag by weight.
The TEM image in the control sample (Fig 3.26a), the M.
aeruginosa KG ultrastructure image had clearly cell wall and the
organelle lie neatly in the cell. When exposed to silver
nanoparticles at a concentration of 1ppm after 48 hours, the
cyanobacteria cells were destroyed (Fig 3.26b). It is proved that the
silver nanoparticles was affected to structure of the cyanobacteria
M. aeruginosa KG cell.
Elements % Weight % Element
CK 38.69 55.90
OK 30.59 33.18
Na K 1.95 1.47
Al K 6.02 3.87
Cu L 11.82 3.23
Ag L 0.37 0.06
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Totals 100.00
Figure 3.25. The EDX spectrum and the element composition
appear on the cell surface of M. aeruginosa KG after 48 h of
exposure with AgNPs (1ppm)
3.2.2.2. Effect of silver nanoparticles on growth and development
of green algae Chlorella vulgaris
The experiments were conducted with the concentrations of
silver nanoparticles increasing from 0.005; 0.01; 0.05; 0.1; 1 to 5
ppm in 10 days. The evaluation parameters include: optical density
(OD), chlorophyll a and cell density at 0, 2, 6 and 10 days (Fig
3.27 b). The toxicity of silver nanoparticles on growth of the green
algae C. vulgaris as measured by the concentration of
supplementary material into the culture medium that affected 50%
of the individuals (EC50) was 0.017 mg/L.
Figure 3.28. Effect of silver
Figure 3.27. Effect of nanomaterial to the green algae
silver nanomaterial on growth C. vulgaris was measured by
of the green algae C. vulgaris and the growth inhibition
a) OD and b) cell density efficiency (a) and the cell
density (b)
After 48h exposure to silver nanoparticles, the cell density
decreased from 195,925 ± 18,770 (D0) to 82,778 ± 41,384 (D10)
cells/mL (Fig 3.27a). At concentrations of 0.005 and 0.01 ppm,
AgNPs did not affect the growth of the green algae C. vulgaris, the
cell density after 2, 6 and 10 days increased linearly with control
samples. Figure 3.28b shows the analysis results of the chlorophyll
a, in the control sample and the experimental samples
supplemented with 0.005 and 0.01 ppm silver nanoparticles, the
content of chlorophyll a increased from 2.0604 ± 0.3505 μg/L (D0)
and reached to the highest value at the end of the testing period
27.285 ± 4.6893 µg/L (D10). The growth inhibition efficiency of
silver nanomaterial concentrations after 10 days is shown in Figure
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3.28a. At the tested concentrations from 0.05 to 1 ppm, the
inhibition efficiency was achieved > 90%.
a) b) a) b)
Figure 3.31. TEM
Figure 3.29. SEM micrograph of
micrograph of the green
the green algae C. vulgaris
algae C. vulgaris
The SEM image result of cell surface structure after 48h
exposed to silver nanoparticles at the concentration of 1 ppm is
shown in Figures 3.29a (the control sample) and 3.29b (the sample
exposed to the concentration of 1ppm silver nanoparticles). In the
control sample, the green algae cells had spherical or elliptical
shape with a smooth exterior and the organelles were seen clearly
(Fig 3.29a). The cell was distorted with a rough and clumpy
exterior surface after exposure with AgNPs (Fig 3.29b). This
suggests that silver nanoparticles have significantly altered the
morphology of the cell.
The SEM-EDX results in Figure 3.30 confirm that silver
nanoparticles appeared and attached to the surface of green algae
with 5.76% Ag by weight. The ultrastructure TEM image of C.
vulgaris cell (Fig 3.31 a) showed that, in the control sample, the
cells had spherical or elliptical, smooth and the organelle in cells
can be seen clearly. When exposed to silver nanoparticles at a
concentration of 1ppm after 48 hours, the cyanobacteria cells were
slightly distorted, rough and clustered with other (Fig 3.31b). It is
proved that the silver nanoparticles was affected to structure of the
green algae C. vulgaris.
Elements % Weight % Elements
CK 41.56 50.84
OK 52.68 48.38
Ag L 5.76 0.78
Totals 100.00
Figure 3.30. The EDX spectrum and the element composition
appear on the cell surface of the green algae C. vulgaris after 48 h
of exposure with AgNPs (1ppm)
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3.2.3. Effect of copper nanoparticles on growth and development
of cyanobacteria Microcystis aeruginosa KG and green algae
Chlorella vulgaris.
3.2.3.1. Effect of copper nanoparticles on growth and development
of cyanobacteria Microcystis aeruginosa KG
The similar experiments were conducted with copper
nanomaterial to test the effect of materials on the growth and
development of the cyanobacteria M. aeruginosa KG. The results
are shown in Figure 3.32.
Figure 3.32. The growth of cyanobacteria M. aeruginosa KG at
different concentrations CuNPs (0.01; 0.05; 0.1; 1 and 5 ppm):
(OD) (a); chlorophyll a (b); cell density (c)
During the first two days of testing, the results showed that no
significantly difference in growth between the control and five
samples in which supplemented with CuNPs. At the tenth day
(D10), in the experimental samples were recorded the biomass
content of cyanobacteria M. aeruginosa KG larger than the control
sample (Fig 3.32a, b).
a) b)
Figure 3.33. The growth Figure 3.34. SEM image of the
inhibition efficiency of cyanobacteria M. aeruginosa
cyanobacteria M. aeruginosa KG: a) control sample and b)
KG after 10 days the sample with 1 ppm after 48h
The chlorophyll a (D0) in the experimental samples in which
supplemented with 1 and 5 ppm CuNPs were achieved 1.845 ±
0.1569 μg/L and 2.295 ± 0.1155 μg/L. At the last day (D10), this
value was only 1.068 ± 1.001 μg/L and 0.11168 ± 0.0501 μg/L,
respectively. In contrast, the chlorophyll a content in the control
sample increased from 2.485 ± 0.135 μg/L (D0) to 7.1501 ± 0.9766
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μg/L (D10). This result showed that CuNPs do not affect the growth
of cyanobacteria M. aeruginosa KG at concentrations from 0.01 to
0.1 ppm. The inhibition effect of the copper nanomaterial on the
growth of cyanobacteria M. aeruginosa KG after 10 days (Fig 3.33)
at the concentration 1 and 5 ppm were 90.1% 93.7%, respectively.
The calculation results of the optical density (OD) recorded the
efficiency concentration of 50% (EC50) of CuNPs on growth of
cyanobacteria M. aeruginosa KG were 0.7159 mg/L.
The SEM image in Figure 3.34 showed that, when exposed to 1
ppm CuNPs after 48 hours, the cyanobacteria M. aeruginosa KG
cells are slightly distorted and clustered. The SEM-EDX result was
used to characterize the chemical composition and the location of
CuNPs on the cell surface of the cyanobacteria M. aeruginosa KG
cells. The results confirm that copper nanoparticles appeared and
attached to the surface of green algae with 11.63% Cu by weight.
Elements % Weight % Elements
CK 57.97 69.85
OK 30.40 27.50
Cu L 11.63 2.65
Totals 100.00
Figure 3.35. The EDX spectrum and the element composition
appear on the cell surface of the cyanobacteria M. aeruginosa KG
after 48 h of exposure with CuNPs
The result of TEM image (Fig 3.36) showed that the cell wall of
the M. aeruginosa KG in which exposed to copper nanoparticles
was broken, the organelle were destroyed. The membrane and cell
wall are not intact compared to the cells in the control sample.
a) b) Figure 3.36. TEM micrograph
of the cyanobacteria M.
aeruginosa KG: (a) control
sample and (b) the sample with
1 ppm CuNPs after 48h
3.2.3.2. Effect of copper nanoparticles on growth and development
of the green algae C. vulgaris
The similar experiments were conducted with copper
nanomaterial to test the effect of materials on the growth and
development of the green algae C. vulgaris. Three parameters:
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optical density (OD) at 680 nm, chlorophyll a and cell density were
analyzed at 0, 2, 6 and 10 days. The results are shown in Figure 3.37.
Figure 3.37. The growth of the green algae C. vulgaris at different
CuNPs concentrations: OD (a); chlorophyll a (b); cell density (c)
The results of the three tested parameters are similar each other.
At all test concentrations, the biomass increased linearly with the
CuNPs concentration by the time and reached the maximum value
at the end of the experiment period (D10). The average value of
optical density (OD) was 0.012 ± 0.002 at the first day (D0) and
0.514 ± 0.117 at the last day (D10) (Fig 3.37a). The content of
chlorophyll a increased in all experimental samples, the biomass
density after 10 days increased from 0.0121 ± 0.0019 μg/L (D0) to
0.5137 ± 0.17171 μg/L (D10) (Fig 3.38b). The cell density also
shows the same result (Fig 3.37c).
Figure 3.38a shows that, in the control sample, the cells had
clearly cell wall and the organelle lie neatly in the cell. When
exposed to silver nanoparticles at a concentration of 1ppm after 48
hours, the cell wall of the green algae C. vulgaris was shrunk but
the cell was not broken (Fig 3.38b).
The results of TEM (Fig 3.40) showed that the cells in the control
sample are spherical or elliptical, smooth and the organelle in cells
such as chloroplasts, thylakoid, granules and the cell wall can be
seen clearly by TEM technique (Fig 3.40a). When exposed to copper
nanoparticles, the cell wall of the green algae C. vulgaris was
slightly distorted, the cell surface is rough but the cell remains intact,
unbroken (Fig 3.40b).
a) b a) b)
)
Figure 3.38. SEM image of the Figure 3.40. TEM of the green
green algae C. vulgaris: a) algae C. vulgaris: (a control
control sample and b) the sample sample and (b) the sample with
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with 1 ppm CuNPs after 48h 1 ppm CuNPs after 48h
The SEM-EDX result was used to characterize the chemical
composition and the location of CuNPs on the cell surface of the
green algae C. vulgaris cells. The results confirm that copper
nanoparticles appeared and attached to the surface of green algae
with 0% Cu by weight.
Elements % Weight % Elements
CK 51.48 58.56
OK 48.52 41.44
Cu L 0.00 0.00
Totals 100.00
Figure 3.39. The EDX spectrum and the element composition
appear on the cell surface of the green algae C. vulgaris after 48 h
of exposure with CuNPs (1ppm)
The EC50 results of the two materials (Table 3.2) showed that,
both AgNPs and CuNPs have effected on the growth inhibition of
microalgae. However, the copper nanomaterial have the potential
to prevent algae more selectively than silver nanomaterial. This
material is toxic to the cyanobacteria M. aeruginosa KG but has
negligible effect on the development of the useful C. vulgaris
(Table 3.2). Therefore, copper nanomaterial was selected for
further studies.
Table 3.2. The toxicity of silver and copper nanomaterials on
growth of the cyanobacteria M. aeruginosa KG and the green algae
C. vulgaris
EC 50 Ag nano (mg/L) Cu nano (mg/L)
C. vulgaris 0.017 -
M. aeruginosa 0.0075 0.7159
3.2.3.3. Size effect of copper nanoparticles on growth and
development of the cyanobacteria M. aeruginosa
The experimental results of the growth inhibition of M.
aeruginosa KG cyanobacteria strain under the affection of copper
nanoparticle solution concentrations (0; 0.01, 0.05, 0.1; 1 and 5
ppm) with three forms of different particle sizes ( 50 nm) on D0, D1, D3, D6 and D10 days are shown in
Figure 3.41.
- 18
In all three types of particle size, the highest inhibition ability
was observed at concentrations 1 and 5 ppm, the growth of
cyanobacteria was recorded as time-dependent and as the
nanomaterial concentration were added to the medium. The optical
density (OD) increased insignificantly and reached 13÷18% (at the
concentration 1ppm) or decreased many times than the initially
value -42%÷-66% (at the concentration 5 ppm). In addition, there
was no difference in growth and biomass of cyanobacteria in the
experimental samples in which supplemented with nanoparticles
size of 25-40 and > 50 nm. In experiments to test the growth
inhibition ability on the cyanobacteria of CuSO4 material, the
results showed that the cyanobacteria cells die immediately after
exposure to copper sulphate solution, the cell biomass decreases
with time compared to the first day D0 (0.63 0.21g/L) and
reached the lowest value at D10 (0.48 0.075 g/L).
Figure 3.41. The growth of the cyanobacteria M. aeruginosa KG
under the impact of solution concentrations and different copper
particle sizes (nm) a) size 50
In the experiments with the big size nanomaterials (30 nm ÷ 40
nm and ≥ 50 nm), the optical density and the content of chlorophyll
a were increased over time with the measured values at the end of
the experiment. This value increased approximately 5 ÷ 6 times
compared with the original value and 20% to 30% higher than the
control sample, respectively. Meanwhile, at particle size ≤10 nm,
these values have the same trend in both sizes, but the inhibition
ability of the M. aeruginosa KG is more clearly showed when the
parameters of OD and chlorophyll a are lower and achieved only
15% at the same time (Fig 3.42). This value is still lower than the
biomass of the cyanobacteria cells on D10 in samples that
supplemented nanomaterial with the sizes of 25 ÷ 40 and > 50 nm.
With copper particle size
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