· Web viewThe operating conditions were voltage of 40 kV and current of 30 mA with Cu kα...

32
Phycobased synthesis of TiO 2 nanoparticles and their influence on morphology, cyto- ultrastructure and metabolism of Spirulina platensis Awatief F. Hifney 1 , Dalia A. Abdel-Wahab 2 , 1 Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut, Egypt,71516 2 Botany Department, Faculty of Science (New Valley Branch), Assiut University, Egypt. Corresponding author: E-mail:[email protected], [email protected] Abstract Titanium dioxide nanoparticles (TiO 2 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 TiO 2 NPs using Spirulina platensis (GSSp.TiO 2 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 TiO 2 NPs has good stability and solubility in water with average size 17.3 nm. GSSp.TiO 2 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.TiO 2 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 TiO 2 NPs. Measurements of 1

Transcript of  · Web viewThe operating conditions were voltage of 40 kV and current of 30 mA with Cu kα...

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

1

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].

2

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.

3

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.

4

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

5

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

6

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).

7

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)

8

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.

9

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).

10

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.

11

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

12

attempt to degrade what is perceived by the cell as foreign or aberrant, similar to bacteria and

other pathogens (Shcharbin et al., 2006).

13

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.

14

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

15

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,

16

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).

17

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

18

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.

5. ReferencesAL-Rubaee, E.A., Abd, S.T., Kadim, N.M., 2015. The effect of titanium dioxide nanoparticles on

salivary alkaline phosphatase activity. Europ J. Mol. Biotec. 4, 1813- 1819,https://doi.org/10.13187/ejmb.2015.10.188

Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373-399,https://doi.org/10.1146/annurev.arplant.55.031903.141701

Aruoja, V., Dubourguier, H.-C., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ. 407, 1461-1468,https://doi.org/10.1016/j.toxlet.2008.06.088

Asada, K., 1984. Chloroplasts: Formation of active oxygen and its scavenging. Methods Enz. 105, 422-429,https://doi.org/10.1016/s0076-6879(84)05059-x

Ayatallahzadeh Shirazi, M., Shariati, F., Keshavarz, A.k., Ramezanpour, Z., 2015. Toxic Effect of Aluminum Oxide Nanoparticles on Green Micro-Algae dunaliella salina. Int. J. Environ. Res. 9, 585-594, http://www.ijer.ir/pdf_933_98c4f4e09daf835f91cd518d3a10eb04.html.

Batley, G.E., Kirby, J.K., McLaughlin, M.J., 2012. Fate and risks of nanomaterials in aquatic and terrestrial environments. Accounts of Chem. Res. 46, 854-862,https://doi.org/10.1021/ar2003368

Chaturvedi, S., Dave, P.N., Shah, N., 2012. Applications of nano-catalyst in new era. J. Saudi Chem.Soc. 16, 307-325,https://doi.org/10.1016/j.jscs.2011.01.015

Chen, L., Rahme, K., Holmes, J.D., Morris, M.A., Slater, N.K., 2012a. Non-solvolytic synthesis of aqueous soluble TiO2 nanoparticles and real-time dynamic measurements of the nanoparticle formation. Nanoscale Res. Lett. 7, 1-10,https://doi.org/10.1186/1556-276x-7-297

19

Chen, L., Zhou, L., Liu, Y., Deng, S., Wu, H., Wang, G., 2012b. Toxicological effects of nanometer titanium dioxide (nano-TiO2) on Chlamydomonas reinhardtii. Ecotoxicol. Environ. Saf. 84, 155-162,https://doi.org/10.1016/j.ecoenv.2012.07.019

Duncan, R., 2003. The dawning era of polymer therapeutics. Nature Reviews Drug Discovery 2, 347-360,https://doi.org/10.1038/nrd1088

Fales, F.W., 1951. The assimilation and degradation of carbohydrates by yeast cells. J. Biol. Chem. 193, 113-124,

Gao, F., Liu, C., Qu, C., Zheng, L., Yang, F., Su, M., Hong, F., 2008. Was improvement of spinach growth by nano-TiO2 treatment related to the changes of Rubisco activase? Biometals 21, 211-217,https://doi.org/10.1007/s10534-007-9110-y

Gupta, S., 1983. Autologous mixed lymphocyte reaction in health and disease states in man. Vox Sang. 44, 265-288,https://doi.org/10.1111/j.1423-0410.1983.tb04483.x

Hartmann, N., Von der Kammer, F., Hofmann, T., Baalousha, M., Ottofuelling, S., Baun, A., 2010. Algal testing of titanium dioxide nanoparticles—testing considerations, inhibitory effects and modification of cadmium bioavailability. Toxicology 269, 190-197,https://doi.org/10.1016/j.tox.2009.08.008

Hifney, A.F., Fawzy, M.A., Abdel-Gawad, K.M., Gomaa, M., 2016. Industrial optimization of fucoidan extraction from Sargassum sp. and its potential antioxidant and emulsifying activities. Food Hydrocolloids 54, 77-88,https://doi.org/10.1016/j.foodhyd.2015.09.022

Hifney, A.F., Issa, A.A., Fawzy, M.A., 2013. Abiotic stress induced production of β-carotene, allophycocyanin and total lipids in Spirulina sp. J. Biol. Earth Sci. 3, B54-B64,

Iswarya, V., Bhuvaneshwari, M., Alex, S.A., Iyer, S., Chaudhuri, G., Chandrasekaran, P.T., Bhalerao, G.M., Chakravarty, S., Raichur, A.M., Chandrasekaran, N., 2015. Combined toxicity of two crystalline phases (anatase and rutile) of Titania nanoparticles towards freshwater microalgae: Chlorella sp. Aquat. Toxicol. 161, 154-169,https://doi.org/10.1016/j.aquatox.2015.02.006

Ji, J., Long, Z., Lin, D., 2011. Toxicity of oxide nanoparticles to the green algae Chlorella sp. Chem. Eng. J. 170, 525-530,https://doi.org/10.1016/j.cej.2010.11.026

Jing, G., Wang, J.J., Zhang, S.X., 2012. ER stress and apoptosis: a new mechanism for retinal cell death, Exp. Diabetes Res., p. 11,https://doi.org/10.1155/2012/589589

Karakoti, A., Hench, L., Seal, S., 2006. The potential toxicity of nanomaterials—the role of surfaces. J. MIN. MET. MAT. S. 58, 77-82,https://doi.org/10.1007/s11837-006-0147-0

Kim, J., Lee, S., Kim, C.-m., Seo, J., Park, Y., Kwon, D., Lee, S.-H., Yoon, T.-H., Choi, K., 2014. Non-monotonic concentration–response relationship of TiO2 nanoparticles in freshwater cladocerans under environmentally relevant UV-A light. Ecotox. Environ. Safe. 101, 240-247,https://doi.org/10.1016/j.ecoenv.2014.01.002

20

Liu, J., Qiao, S.Z., Hu, Q.H., 2011. Magnetic nanocomposites with mesoporous structures: synthesis and applications. Small 7, 425-443,https://doi.org/10.1002/smll.201001402

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J biol Chem 193, 265-275,

Luechinger, N.A., Grass, R.N., Athanassiou, E.K., Stark, W.J., 2009. Bottom-up fabrication of metal/metal nanocomposites from nanoparticles of immiscible metals. Chem. Mater. 22, 155-160,https://doi.org/10.1021/cm902527n

Ma, S., Lin, D., 2013. The biophysicochemical interactions at the interfaces between nanoparticles and aquatic organisms: adsorption and internalization. Environ. Sci. Process Impacts 15, 145-160,https://doi.org/10.1039/c2em30637a

Marker, A., 1972. The use of acetone and methanol in the estimation of chlorophyll in the presence of phaeophytin. Freshwater Biol. 2, 361-385,https://doi.org/10.1111/j.1365-2427.1972.tb00377.x

Matsumura, T., Tabayashi, N., Kamagata, Y., Souma, C., Saruyama, H., 2002. Wheat catalase expressed in transgenic rice can improve tolerance against low temperature stress. Physiol. Plant. 116, 317-327,https://doi.org/10.1034/j.1399-3054.2002.1160306.x

Minguez-Mosquera, M., Jaren-Galan, M., Garrido-Fernandez, J., 1993. Lipoxygenase activity during pepper ripening and processing of paprika. Phytochemistry 32, 1103-1108,https://doi.org/10.1016/s0031-9422(00)95073-8

Mizushima, N., Yoshimori, T., Ohsumi, Y., 2011. The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27, 107-132,https://doi.org/10.1146/annurev-cellbio-092910-154005

Nakano, Y., Asada, K., 1987. Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol. 28, 131-140,https://doi.org/10.1016/s0005-2728(00)00256-5

Oukarroum, A., Bras, S., Perreault, F., Popovic, R., 2012. Inhibitory effects of silver nanoparticles in two green algae, Chlorella vulgaris and Dunaliella tertiolecta. Ecotox. Environ. Safe. 78, 80-85,https://doi.org/10.1016/j.ecoenv.2011.11.012

Pérez-Pérez, M.E., Lemaire, S.D., Crespo, J.L., 2012. Reactive oxygen species and autophagy in plants and algae. Plant Physiol. 160, 156-164,https://doi.org/10.1104/pp.112.199992

Petit, A.-N., Eullaffroy, P., Debenest, T., Gagné, F., 2010. Toxicity of PAMAM dendrimers to Chlamydomonas reinhardtii. Aquat. Toxicol. 100, 187-193,https://doi.org/10.1016/j.aquatox.2010.01.019

Robichaud, C.O., Uyar, A.E., Darby, M.R., Zucker, L.G., Wiesner, M.R., 2009. Estimates of upper bounds and trends in nano-TiO2 production as a basis for exposure assessment. Environ. Sci. Technol. 43, 4227-4233,https://doi.org/10.1021/es8032549

21

Sadiq, I.M., Dalai, S., Chandrasekaran, N., Mukherjee, A., 2011. Ecotoxicity study of titania (TiO2) NPs on two microalgae species: Scenedesmus sp. and Chlorella sp. Ecotox. Environ. Safe. 74, 1180-1187,https://doi.org/10.1016/j.ecoenv.2011.03.006

Shahandashti, S.S.K., Amiri, R.M., Zeinali, H., Ramezanpour, S.S., 2013. Change in membrane fatty acid compositions and cold-induced responses in chickpea. Mol. Biol. Rep. 40, 893-903,https://doi.org/10.1007/s11033-012-2130-x

Sharma, G., Kumar, M., Ali, M.I., Jasuja, N.D., 2014. Effect of carbon content, salinity and pH on Spirulina platensis for phycocyanin, allophycocyanin and phycoerythrin accumulation. J. Micro. Bioch. Techn. 2014,https://doi.org/10.4172/1948-5948.1000144

Shcharbin, D., Jokiel, M., Klajnert, B., Bryszewska, M., 2006. Effect of dendrimers on pure acetylcholinesterase activity and structure. Bioeletrochem. 68, 56-59,https://doi.org/10.1016/j.bioelechem.2005.04.001

Stern, S.T., Adiseshaiah, P.P., Crist, R.M., 2012. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part. Fibre Toxicol. 9, 1,https://doi.org/10.1186/1743-8977-9-20

Tucci, P., Porta, G., Agostini, M., Dinsdale, D., Iavicoli, I., Cain, K., Finazzi-Agró, A., Melino, G., Willis, A., 2013. Metabolic effects of TiO2 nanoparticles, a common component of sunscreens and cosmetics, on human keratinocytes. Cell Death Dis. 4, 549-559,https://doi.org/10.1038/cddis.2013.76

Van Breusegem, F., Dat, J.F., 2006. Reactive oxygen species in plant cell death. Plant physiol. 141, 384-390,https://doi.org/10.1104/pp.106.078295

Wang, Z., Lee, Y.-H., Wu, B., Horst, A., Kang, Y., Tang, Y.J., Chen, D.-R., 2010. Anti-microbial activities of aerosolized transition metal oxide nanoparticles. Chemosphere 80, 525-529,http://dx.doi.org/10.1016/j.chemosphere.2010.04.047

Xie, Y., Qian, H., Zhong, Y., Guo, H., Hu, Y., 2012. Facile Low-Temperature Synthesis of Carbon Nanotube/Nanohybrids with Enhanced Visible-Light-Driven Photocatalytic Activity. Int. J. Photoenergy 2012, https://doi.org/10.1155/2012/682138

Zarouk, C., 1966. Contribution A L’etude D’une cyanophyceae. Influence de divers facteurs physiques el chimiques sur la croissance et la photosynthese de Spirulina maxima (Setch et Gardna) Geitler, Ph. D.

22