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Phycobased synthesis of TiO2 nanoparticles and their influence on morphology, cyto- ultrastructure and metabolism of Spirulina platensis
Awatief F. Hifney1, Dalia A. Abdel-Wahab2,1Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut,
Egypt,715162 Botany Department, Faculty of Science (New Valley Branch), Assiut University, Egypt.
Corresponding author: E-mail:[email protected], [email protected]
Abstract Titanium dioxide nanoparticles (TiO2 NPs) are allegedly to have better chemical, physical and biological properties which make them significantly applied in enormous industrial manufacturing. Since these nanoparticles are inevitably released into environment especially aquatic ecosystem whereby human and other living organisms including algae could be impactful affected. The aim of the present work was to study the characterization of green synthesis of TiO2 NPs using Spirulina platensis (GSSp.TiO2 NPs) and the effect of these biosynthesized (phycobased synthesis) nanoparticles on morphology, growth, ultrastructure and enzymatic activates of the same blue green alga. The phycobased synthesis of TiO2 NPs has good stability and solubility in water with average size 17.3 nm. GSSp.TiO2 NPs was found to aggregate and adsorb on S. platensis membrane. Penetration and internalization of the nanoparticles into the Spirulina cells were also observed. Addition of GSSp.TiO2 NPs induced cell distortion, plasmolysis, cell wall and membrane damage. The growth of S. platensis was inhibited when the alga was exposed to more than 160 mg/l TiO2 NPs. Measurements of antioxidant enzyme activities showed that maximum catalase (CAT) and ascorbate peroxidase (APX) activities were recorded at 280 and 240 mg/l TiO2 NPs. While addition of GSSp.TiO2 NPs. stimulated lipoxygenase (LOX) activity of S. platensis compared to control. Thus, TiO2 NPs caused hazard and have harmful effect on S. platensis especially at high concentrations, so that disposal of nanoparticles may exhibit adverse effects on the ecosystem.
KeywordsMicrobial synthesis, Spirulina platensis, TEM, SEM, GSSp.TiO2 NPs, Cyto-ultrstructure
1. Introduction The use of nanoparticles (NP) is of increasing industry importance, since they are suitable for
manifold applications. In the field of medicine, for example, devices and vehicles have been
developed in the micro and nano-scale, to carry drugs to specific sites (Duncan, 2003). Among the
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various types of NPs, Titanium dioxide nanoparticles (TiO2NPs) are currently of great interest.
TiO2 NPs are mineral compounds that had been manipulated to its nano-scale dimension,
approximately 1 to 100 nm. The physicochemical properties of TiO2NPs prepared by various
methods have been intensely studied in recent years (Liu et al., 2011; Luechinger et al., 2009).
TiO2NPs are widely used for many applications such as sunscreens, paints, coatings, and consider
as active component of solar cells (Chaturvedi et al., 2012) and photocatalytic water purification
(as the most promising photo catalyst).
The annual production of TiO2 NPs is predicted to reach 2.5 million tons by 2025 (Robichaud et
al., 2009). The widely used TiO2 NPs would find their way into aquatic environments (Batley et
al., 2012)and interact with aquatic organisms (Ma and Lin, 2013). Eco-toxicity of TiO2NPs is
received worldwide research attentions (Ji et al., 2011; Kim et al., 2014).
Precautionary principle that manufacturers, importers and downstream users have to prove that
their manufactured product do not adversely affect human health and environment, before being
allowed to sell or use such products. Thousands of species of algae occur world-wide in both fresh
and marine waters. The most commonly encountered groups of freshwater algae are green algae,
diatoms, and blue-green algae (more correctly known as cyanobacteria). Algae are considered as
primary products in ecosystem and has been known for highest rate of photosynthesis on the earth,
important biological sources of high valued products that has a wide range of biotechnological
application (Hifney et al., 2016), high nutritional value as Spirulina platensis. Increasing
application of nanoparticles hence trigger the requirement for evaluation of its environmental
impact especially on aquatic ecosystem, therefore the present study aimed and designed firstly, to
investigate the characterization of microbial synthesis of TiO2 NPs from S. platensis. The second
target was to follow the effects of the biosynsizied TiO2 NPs (GSSp. TiO2 NPs) on the
morphological and cyto- ultra structure metabolic activates of S. platensis especially enzymatic
activity with especial attempt to follow how these organism get ride or accumulation these
nanoparticles.
2. Materials and Methods
2.1.Microorganism and culture mediaIn this investigation Spirulina platensis was isolated from Wadi El-Natron salt lake (Egypt) as
described byHifney et al. (2013). Spirulina platensis was grown in 500 ml modified Zarrouk's
medium (Zarouk, 1966). The pH of the medium was adjusted to pH 9.00 prior to autoclaving. The
cultivated flask was illuminated 24 h with continuous cool white fluorescent philips lamp at 400
W. (48.4 µ mole. m-2.s-1) at 30˚C [regular growth condition].
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2.2.Green Synthesis and characterizations of (GSSp. TiO2 NPs)
2.2.1. Green Synthesis of titanium dioxide nanoparticles from S. platensisSpirulina platensis was grown in 250 ml culture medium for one week. At the beginning of the
stationary phase 100mM of TiO2 (bulk) was added to the culture medium and left without shaking
or bubbling at room temp (~25oC).After 24h from addition of TiO2 we notice change of culture
colour to pale blue then gradually increase of the white colour in the culture medium. After one
week the sample was centrifuged and supernatant (cell free supernatant) was collected and left to
dry in the oven at 80oC. The biosynthesized creamy white powder of titanium dioxide from S.
platensis was collected for characterization and toxicity study.
2.2.2.Characterizations of the green synthesis of titanium dioxide
nanoparticles from S. platensis (GSSp.TiO2 NPs)The green synthesized TiO2 NPs from S. platensis were characterized by ultraviolet-visible
spectroscopy, transmission electron microscopy, and X-ray diffraction as following.
2.2.2.1.Ultraviolet-Visible Spectroscopy (UV-Vis):The bio-reduction of titanium dioxide solution and formation of titanium dioxide nanoparticles
were scanned in the 200–800 nm wavelength range using a double beam spectrophotometer
(PerkinElmer Lambda 950 UV/Vis).
2.2.2.2.Transmission Electron Microscope (TEM(The transmission electron microscopy was applied tomeasure the size and the morphological
characters of the biosynthesized (TiO2 NPs). A drop of synthesized titanium dioxide nanoparticles
was placed on a negative carbon coated copper grids and dried in air. TEM micrographs of the
sample were taken using the JEOL instrument 1200 EX (Electron Microscope Unit, Assiut
University, Egypt).
2.2.2.3. X-ray diffraction (XRD)The formation of TiO2 NPs from S. platensis biosynthesized (GSSp.TiO2 NPs) was checked by X-
ray diffraction technique using X-ray diffraction (XRD) patterns using Phillips PW
1830instrument Powder (Phillips, USA). The operating conditions were voltage of 40 kV and
current of 30 mA with Cu kα radiation of 0.1541 nm wavelength, in the 2θ range 10-80º angle.
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2.2.3. Assay of the effects of various concentration of GSSp.TiO2 NPs on
morphological, cyto-ultrastructure and metabolic pathwayof S.
platensis
2.2.3.1.CultivationGSSp.TiO2 NPs were added (40, 80, 120, 160, 200, 240, 280 and 320 mg/l) separately to the
culture media and left to grow under regular growth condition.
2.2.3.2. Light microscope (LM)Morphological features (shape and structure) of S. platenesis were examined and visualized by
light microscope (model Olympus CX41 micrscope equipped with a digital camera) (S30
Olympus digital camera, Japan).
2.2.3.3. Scan electron microscope (SEM)Spirulina cells were grown under various concentrations of (GSSp.TiO2 NPs) and then were
collected by gentle centrifugation at 3600×g, and fixed overnight in 3%gluteraldehyde with 0.25M
sucrose. Then the samples were washed with 0.2 M sodium cacodylatebuffer at pH 7.2. The fixed
cells were dehydrated in a graded ethanol series (30-50-70-90 and 100%ethanol), then suspended
in acetone, and dried in a Hitachi HCP-I critical-point dryer. A small portion of the dried cells was
attached to a conductive stub and sputter-coated with gold in ion coater JFC1100E (JEOL) ion
sputtering device. Samples were examined by a JSM- 5400 LV (JEOL) scanning electron
microscope.
2.2.3.4.Cyto-ultratructure of S. platenesisTEM were used to image the cyto-ultrastructure changes of S. platensis as response to its
GSSp.TiO2 NPs [as described previously (Gupta, 1983)].
2.2.3.5. Growth and physiological activity of S. platensis
2.2.3.5.1. Growth measurementsThe growth of treated and untreated Spirulina platensis were daily followed by determination of
optical density at 750 nm. Chl. a content was determined according to the method described by
Marker (1972). In the late of exponential or beginning of the stationary phase. The algal cells
were harvested for estimation of some metabolites.
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2.2.3.5.1.1. Determination of phycobiliproteins (phycocyanin,
allophycocyanin and phycoerythrin)Phycobiliproteins contents were determined according to the method modified by Sharma et al.
(2014). 5 ml of Spirulina cell suspension was taken and centrifuged to obtain the pellet for 10
minute at 4000 rpm. The pellet was washed with distilled water and phycobiliproteins were
extracted from the algal pellet with 5 ml of phosphate buffer (0.05 M, pH 6.7) by three times
repeated freezing and sonication using Bandelin, sicherum gen 2XF2A ultra sonicatior (Sonopuls
Bandelin electronic Berlin, Germany) at 40K Hz to facilitate the extraction. The sample was
centrifuged for 15 min at 10,000 rpm the supernatant was collected. The absorbance was recorded
at E652, E615, and E652against phosphate buffer as blank by using Unico UV/ VIS 2100
spectrophotometer and concentration of phycocyanin (PC), allophycocyanin (APC), and
phycoerythrin (PE) were calculated by using the formula:
PC= (E615 - 0.476 E652) / 5.34 PC= c-phycocyanin
APC= (E652 - 0.208 E615) / 5.09 APC= allophycocyanin
PE= E526- 2.4 (PC) - 0.849 (APC) / 9.62 PE= phycoerythrin
Total phycobiliproteins = PC+APC+PE x volume of extract (ml)/volume of culture.
2.2.3.5.1.2.Determination of soluble carbohydratesSoluble sugar contents of S. platensis grown under various concentrations of biosynthesized
GSSp.TiO2 NPs were determined using anthrone sulphuric acid method (Fales, 1951).
2.2.3.5.1.3.Determination of soluble proteinsProtein contents were determined in the algal extract by Folin reagent according to Lowry et al.
(1951). A calibration curve was constructed using bovine serum albumin (BSA) and the data were
expressed as mg protein / ml culture.
2.2.3.5.1.4. Assay of enzymes activity
2.2.3.5.1.4.1. Preparation of enzyme extract Hundred ml of the algal culture were centrifuged at 5000 rpm and the pellet was homogenized in 5
ml of 100 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM of EDTA and 0.1 g
polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 18000 rpm for 10 min. at 4ºC
and the supernatants were collected and used for the assays of Lipoxygenase (LOX), catalase
(CAT) and ascorbic peroxidase (APX). All colorimetric measurements (including enzyme
activities) were made at 20ºC using a Unico UV/VIS -2100 spectrophotometer. The specific
activity was expressed as units/mg protein
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2.3.2.5.2. Assay of Lipoxygenase activityLipoxygenase (LOX; EC 1.13.11.12) activity was estimated according to the method of Minguez-
Mosquera et al. (1993).
2.3.2.5.3. Assay of Catalase Catalase (CAT; 1.11.1.6) activity was assayed by following the consumption of H2O2 for 1 min. as
described by Matsumura et al. (2002).
2.3.2.5.3.2. Ascorbate peroxidase:Ascorbate peroxidase (APX; EC 1.11.1.11) activity for control and treated samples was
determined according to Nakano and Asada (1987).
2.4. Statistical AnalysisAll data obtained were subjected to one-way analysis of variance (ANOVA), using the SPSS
version 19 statistic package. For comparison of the means, the Duncan’s multiple range tests (p>
0.05) were used.
The enzymatic activates, protein, carbohydrates, pigments and phycobiloproteins were subjected
to principal component analysis (PCA) using PRIMER V6 software. The values were normalized
prior to the analysis, and the first two principal components were plotted to visualize the
correlation between enzymatic activates, protein, carbohydrates, pigments and phycobiloproteins
and various concentrations of GSSp.TiO2 NPs.
3. Results and discussion
3.1.Characterizations of green synthesized titanium dioxide nanoparticles
(GSSp.TiO2 NPs)Previous reports on TiO2NPs revealed that TiO2 nanoparticles were more reactive than its bulk
form. They have a promising physical, chemical, and electrical properties due to their size and
large surface area per given mass (Karakoti et al., 2006).
In the current study GSSp. TiO2 NPs showed good solubility, stable in water and no obvious large
aggregation or precipitation was observed with time. Xie et al. (2012) reported that chemically
synthesized TiO2 NPs showed similar properties, but the phycosynthesis of TiO2 NPs is more
effective with low cost.
The size and morphological characters of the biosynthesized TiO2 NPs using cell free supernatant
of S. platenesis were examined using (TEM image and XRD measurements). The pycosynthesized
nanoparticles exhibited a particle size less than 10.9 nm and was found to be spherical or
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hexagonal like structure shape (Fig 1). Sadiq et al. (2011) found two shape like structure in TiO2
NPs ranged between 10-20nm in size. The larger particles were found to be spherical shape,
whereas, the smaller particles were in hexagonal and spherical shapes. From X-ray diffraction, the
average particle size was 17.3 nm (Fig. 2). The XRD pattern of nano-titania was matched with the
database of international center for diffraction data (ICDD) with card file No (04-012-6345).
The UV-Vis spectra of these nanoparticles showed that the characteristic absorption peaks were
232, and 240 while other beaks observed at 340 nm shown (Fig. 3). AL-Rubaee et al. (2015)
recorded that TiO2 NPs show small and sharp beak around 220 and 240 nm. Chen et al. (2012a)
indicated the absorbance intensity of GalA-TiO2 NPs dispersion in the UV region is <300 nm.
These data should be taken into account in assessing the potential effects on aquatic life.
Figure 1. Transmission electron microscopy (TEM) image of titanium dioxide nanoparticles synthesized from Spirulina platensis (GSSp.TiO2 NPs).
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Figure 2. X-ray diffraction (XRD) pattern of crystalline titanium dioxide nanoparticles synthesized by Spirulina platensis (GSSp.TiO2 NPs).
Figure 3. UV-Visible absorption spectra of titanium dioxide TiO2 NPs synthesized from Spirulina platensis (GSSp.TiO2 NPs)
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2θ degree
Inte
nsity
(a.u
.)
3.2.Effect of various concentrations of (GSSp.TiO2 NPs) on morphology and
ultra-structure of S. platensisAlgae are the first link in the aquatic food chain. They have an important role in aquatic
ecosystems and an essential role in self-purification of polluted waters. Therefore, they are
considered as model organisms in testing toxicity of nanoparticles (Ji et al., 2011). Any change in
the density, biomass and algae population affects the food chain. Therefore, studying the response
of algae to disposal nanoparticles have a special importance (Ayatallahzadeh Shirazi et al., 2015).
Morphological and cyto-ultra structure changes of S. platensis with response to various
concentrations of its biosynthesized TiO2 NPs were visualized and studied using light microscope
(LM) (Fig. 4), (SEM) as in (Fig. 5) and (TEM) as in (Fig.6).
3.2.1. Light microscope (LM)Under light microscope S. platensis composed of vegetative cells undergo binary fission in a
single row with an easily noticeable transfer cross- walls in control sample (Fig. 4a) compared
with treated one (Fig.4 b, c).The trichome enveloped by a thin sheath with more or less
pronounced construction on the cross- walls, clearly obvious in control in comparison to the
treated samples (Fig. 4).
Figure 4. Light micrographs of Spirulina platensis treated with various concentrations of its biosynthesized titanium dioxide nanoparticles (GSSp.TiO2
NPs). Where (a) control, (b, c) expressed as 200 and 320 mg/l GSSp.TiO2 NPs treated cultures, respectively.
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3.2.2. Scan electron microscope (SEM)Further characterization and effects of GSSp.TiO2 NPs in the cells of S. platensis was carried out
with SEM (Fig.5). S. platensis is a filamentous cyanobacterium (Fig. 4, 5a). The main
morphological feature of this genus is the arrangement of the multicellular cylindrical trichomes in
an open left-hand helix along the entire length. The cylindrical trichomes of S. platensis were
strongly damaged after exposure to more than 160 mg/l GSSp. TiO2 NPs. They became broken in
many places (Fig.5 d-f).The untreated cells shape showed a smooth surface with an
uncompromised cell membrane (Fig 5a). Exudates released from the treated culture were observed
(Fig. 5b) which seem to be caused the attachment of the algal cells. While the application of high
concentrations of GSSp.TiO2NPs caused damage and distortion in the cell membrane and in the
cross walls (Fig.5d-f). Numerous TiO2 NPs aggregates were observed tightly attached to various
treated algal cell surfaces (Fig.5 d-f). The deformities of the cell membrane were clearly observed,
combined with abnormalities resemble to notches (Fig. 5 d). Application of 320 mg/l of
GSSp.TiO2 NPs caused disappearance of cross wall or at least distorted the structure (Fig. 5f).
Our results agreed with Iswarya et al. (2015), who showed that exudates released by the algal
cells helped in the aggregation and the attachment of cells together as a result of NPs stress. Nano
TiO2 formed characteristic aggregates entrapping algal cells that contribute to the toxic effect of
nano TiO2 to Pseudokirchneriella subcapitata (Aruoja et al., 2009).
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Figure 5. Scan electron micrographs (SEM) of a trichome of axenic Spirulina platensis culture treated with various concentrations of its biosynthesized titanium dioxide nanoparticles (GSSp.TiO2 NPs). Where (a) control and (b-f) expressed as 80, 160, 240, 280 and 320 mg/l GSSp.TiO2 NPs treated cultures, respectively. Showed that trichome segment into cells by cross walls (S), aggregation and accumulation of nanoparticles (NPs), and formation of notches like structure (n.s).
3.2.3. Transmission electron microscope (TEM)TEM photographs of the sliced S. platensis treated with 80, 160, 240, 280, 320 mg/l GSSp.TiO2
NPs compared to control (0 mg/l GSSp.TiO2 NPs) were depicted in (Fig. 6). Generally penetration
and internalization of the nanoparticles into the S. platensis cells were also observed especially
with high concentration.
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TiO2 NPs penetrated into the cytoplasm in all the treated samples (Fig 6 b-e) implied that NPs
inside the cell may be activated to be more toxic to cells which may be essential to asset the risk
of TiO2 transfer through the tropic chain. The untreated cells were compact and with its typical
characteristic organelles. The most distinct feature of the cell cytoplasm is the photosynthetic
lamellae, which are composed of stacks of thylakoid membranes. Small intrathylakoid membranes
were observed. In addition, there are numerous gas vacuoles, which are found in clusters at the
cell periphery (Fig. 6a). Additionally, NPs internalization was very clearly observed in cells where
the NPs were accumulated in a vacuole space region (Fig.6 b-f). Cell membrane damage occurred
with complete destruction of the internal organelles (Fig. 6d).Gas vacuoles are drastically reduced
to stalk at higher concentration (Fig. 6 d- f).
Amazing and clearly visible notches –like structure (outgrowth) were induced by the addition of
240 mg/l GSSp.TiO2 NPs from the cell wall of treated sample (Fig.5 d) and a giant cavity
structure were seen in TEM image in the same position near cell wall and cross walls of the
trichome (Fig.6 d, f). While (Fig. 6 b–f) showed that cell damage caused by exposure to
GSSp.TiO2 NPs is more severe when concentration dose is increased. [Photosynthetic lamellae are
the first structures affected by GSSp.TiO2 NPs the disintegration and disorganization of thylakoid
membranes clearly observed (Fig. 6 e, f)], a slight increase of gas vacuoles is also observed at 80,
160 and 240 mg/ l GSSp.TiO2 NPs (Fig 6 b, d). Disruption of the thylakoid may generate
intermediate signals involved in programmed cell death and induces the apoptosis of the cells
(Apel and Hirt, 2004; Van Breusegem and Dat, 2006). Our investigation sought to estimate that
addition of 320 mg/l GSSp.TiO2 NPs caused a complete damage of the cell wall and the
membrane and finally plasmolysis (Fig 6 f).
Our results showed that aggregation of nano particle into the cavity of S. platensis may happen
with the addition of 80 mg /l of GSSp.TiO2 NPs and the sequester of these nanomaterial were
highly recorded at 240mg/l treated sample may be an attempt from the cell to degrade or disposal
of these materials into that cavity following to its inference to the cytoplasm. We postulated that
the accumulation of these nanomaterials into vesicles may behave as a mechanism protection
against foreign and harmful substances to get ride it outside the cell (autophagy or lysosomes,
outgrowth). During autophagy (also known as macroautophagy),cytoplasmic components are non-
selective and enclosed within a double-membrane vesicle known as the autophagosome and
delivered to the vacuole/lysosome for degradation of toxic components and recycling of needed
nutrients (Mizushima et al., 2011).
Researchers have observed nanomaterial-induced lysosomal dysfunction (Stern et al., 2012),there
also remains the likely possibility that autophagy induction by nanomaterials may simply be an
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attempt to degrade what is perceived by the cell as foreign or aberrant, similar to bacteria and
other pathogens (Shcharbin et al., 2006).
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Figure 6. Transmission electron micrographs (TEM) of section in Spirulina platensis after exposure to its biosynthesized titanium dioxide nanoparticles (GSSp.TiO2 NPs). Where (a), control (b-f) expressed as 80, 160, 240, 280 and 320 mg/l GSSp.TiO2 NPs treated cultures, respectively. Cell wall (C.W). Cross walls (S). Photosynthetic lamellae are well differentiated consisting of stacks of thylakoid membranes (Th), numerous gas vacuoles (G.V).
3.2.4. The effect of GSSp. TiO2 NPs on the growth of S. platensisAddition of more than 160 mg/l of GSSp.TiO2 NPs to the growth medium of S. platensis caused
noticeable inhibition in the growth parameter with the time elapsed, Fig (7). Ji et al. (2011)
showed that large aggregates of nanoparticles entrapping the algal cells were observed under the
treatment of nano-ZnO and HR3 (TiO2) which may reduce the light and nutrient available to the
entrapped algal cells and thus inhibit their growth. Some researches as Aruoja et al. (2009); Chen
et al. (2012b); Hartmann et al. (2010) indicated that nanoparticles inhibited algal growth.
Addition of 320 mg/l of GSSp.TiO2 NPs caused a significant growth inhibition and even cell
death.
Figure 7. Growth parameter (Absorbance) of Spirulina platensis treated with various concentrations of its
biosynthesized titanium dioxide nanoparticles (GSSp.TiO2 NPs) as discussed in materials and methods.
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1 3 5 8 120
0.2
0.4
0.6
0.8
1
1.2
1.4
Cont. 40 80 120 160200 240 280 320
Time (days)
Abso
rban
ce a
t (7
50 n
m)
3.2.5. The effect of GSSp.TiO2 NPs on the phycobiloprotein of S. platensisPhycocyanin and total phycobiliprotein content of S. platensis treated with (80, 120, 160 mg/l) of
GSSp.TiO2 NPs were significantly enhanced compared with control, Fig (8). While concentrations
more than 160 mg/l of GSSp.TiO2NPs reduced phycocyanin and total phycobiliprotein but the
values are not significantly different with control, Fig (8). Only 40 and 80 mg/l of the
biosynthesized TiO2 NPs caused alleviation of carotenoides content (data not shown) the rest
applied concentration has no noticeable effect as accordance with Chen et al. (2012b).
Figure 8. Bilioprotein content of Spirulina platensis treated with various concentrations of its biosynthesized
titanium dioxide nanoparticles (GSSp.TiO2 NPs) as discussed in materials and methods.
3.2.6. The effects of GSSp.TiO2 NPs on enzymes activity of S. platensisLike other photosynthetic organisms S. platensis increases internal reactive oxygen species (ROS)
when it confronts of adverse environmental conditions. ROS increase may also go with disorder
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of photosystem under nutrient deprivation, salinity and heavy metals conditions. Under normal
conditions, concentration of oxygen radicle remains low because of the activity of protective
enzymes including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)
(Asada, 1984), in resistant forms. Stress conditions may enhance the protective processes such as
accumulation of compatible solutes and increase in the activities of detoxifying enzymes.
Another factor that indirectly inhibit NMs effects is the production of reactive oxygen species
(ROS) by nanoparticles, which can cause chemically-induced toxicity (Petit et al., 2010). The
generation of ROS is a consequence of the physical structure and reactive surface chemistry of
very small particles. Light illumination conditions can stimulate photocatalytic activity of NPs to
generate hydroxyl radicals, which are responsible for the toxicity of the material (Oukarroum et
al., 2012).
Gao et al. (2008) documented promotion of total antioxidant capacity with application of TiO2
NPs. The toxicity of TiO2 NPs was dependent on ROS derived from internalization and/or
photoactivity; therefore, we examined the responses of the antioxidant defense system in S.
platensis to its biosynthesized TiO2 NPs (GSSp.TiO2NPs).
Measurements of antioxidant specific enzyme activities showed that lipoxygenase (LOX) activity
was stimulated with all applied concentrations of GSSp.TiO2 NPs. The highest specific activity
values were observed at 320 mg /l (Fig. 9). Lipoxygenase (LOX EC 1.13.11.12) are responsible
for membrane degradation, because they catalyze the deoxygeneration of fatty acids producing
hydroperoxy saturated fatty acids which are toxic to the cell (Shahandashti et al., 2013). The
depletion of antioxidant enzymes with a concomitant increase of LOX levels and ROS posed a
hazard to the membrane integrity. Cell wall was damaged especially with addition of 240 and /or
320 mg/l GSSp.TiO2 NPs (Fig. 5F & 6F).
The increase in the enzyme activities of the algal cells may be attributed to the early stress
response induced by excessive intracellular ROS generation. Catalase (CAT) activities were
subsequently increased with increasing TiO2 NPs, and then inhibited at 320 mg/l GSSp.TiO2 NPs
(Fig.9). From the data depicted in Fig. 9 it was noticed that the ascorbate peroxidase (APX)
activities were fluctuated with the applied TiO2 NPs: firstly decreased then induced recorded
significantly increased at 240 mg/l GSSp.TiO2 NPs compared to control.
Reactive oxygen species and autophagy have been historically associated with cell death.
However, more recent evidence indicates that both ROS and autophagy play important roles in
signaling and cellular adaptation to stress (Pérez-Pérez et al., 2012). Plants and algae can activate
several defense systems simultaneously for the efficient scavenging of different ROS, but in some
conditions, excess ROS can still be generated, causing massive damage in the cell. In this case,
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more aggressive mechanisms must be induced in the cell in order to remove damaged components
and maintain ROS under control (Pérez-Pérez et al., 2012).
Figure 9. Enzymes activities of Spirulina platensis treated with various concentrations of its biosynthesized
titanium dioxide nanoparticles (GSSp.TiO2NPs) as discussed in materials and methods.
3.2.7. Correlation
PCA analysis in Fig 10 showed that low concentrations (40-200 mg/l) of GSSp.TiO2 NPs had
positive correlation with proteins and phycobiloprotein. High concentration (280 mg/l TiO2) has a
positive correlation with SC (soluble carbohydrates),LOX,CAT and a negative correlation with
proteins and phycobiloprotein, indicating inhibition of protein and phycobilliproteins with high
concentration of GSSp.TiO2 NPs.Wang et al. (2010) found that NP-cell attachment may inhibit the
movement of substances in and out of bacterial cells, thereby causing homeostatic imbalance,
cellular metabolic disturbance and even cell death. Addition of NPs caused damage not only to the
DNA, but also to the proteins, lipids and other metabolites in cells (Tucci et al., 2013)during the
TiO2 photocatalysis. All these may lead to the release of proapoptotic factors and cause
programmed cell death (Jing et al., 2012).
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Figure 10. Principle component analysis (PCA) of different enzymes activities, protein, soluble carbohydrates and pigments of Spirulina platensis treated with various concentrations of its biosynthesized titanium dioxide nanoparticles (GSSp.TiO2 NPs).
4. Conclusion
Protect our environment from damage caused by the valley of chemical, biological or nanoscale
materials ….etc. and known the effect of them has to be the main goal for us before throwing
remnants of such product in the environments especially with highly commercialization products
as nanoparticles materials. To take the necessary precautions to protect our self and other living
creature from the potential risk associated with the manufacture, uses and the disposal of such
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products. Spirulina platensis (blue green algae with widespread habitats), exposed to such wastes
so it was chosen as a model to study the positive and negative impact of nanotechnology and the
ability to create nanoparticle from Spirulina. Spirulina platensis can be considered as a TiO2 NPs
creative microorganism with particle size around 17.3 nm and highly stable product. Spirulina
platensis bears the concentrations to 160 nm , the organism deal with it by trying assembled of
these foreign NPs in what looks like vesicles (may be in the lysosomes ) within the cell, and then
removes them throughout by notches like structure as a protocol for disposal and toxicity
protection. High concentration causes severely cell damage eventually led to decay the cell, (cell
wall damage, plasmolysis, morphogenesis and affect cell metabolism), so we have to manage our
wasted before disposal.
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