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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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.

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Nội dung Text: Summary of environmental technique doctoral thesis: Synthesis of silver, copper, iron nanoparticles and their applications in controlling cyanobacterial blooms in the fresh water body

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.
2 - 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
3 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
4 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
5 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
6 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,
7 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) 2900 2800 2700 2600 2500 2400 2300 2200 d=2.089 2100 2000 1900 1800 1700 Lin (Cps) 1600 1500 1400 1300 1200 1100 d=1.808 1000 900 800 700 d=1.278 600 500 400 300 200 100 0 1 1) c) 10 20 30 40 2-Theta - Scale 50 60 70 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. 80 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.
8 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
9 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
11 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
12 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
13 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)
14 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
15 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:
16 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
17 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  0.21g/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