Influence of Different Concentrations of Nano-Copper Oxide on the Growth of Coelastrella terrestris

This paper conducted on the effect of nanoparticles on the growth of green alga Coelastrella terrestris which was firstly used to find out the effect of nanoparticles on its growth. Four concentrations of copper were used, represented by a proper concentration of CuSO4 that exists in the Chu-10 medium 0.08 mg/l, as control treatment and the rest was CuO nanoparticles represented by (0.04, 0.08 and 0.16) mg/l. Results of exposure of the Coelastrella terrestris to the copper nanoparticles showed a decrease in the growth rate, chlorophyll a,b and carotenoids in comparison to the control treatment which gave the best results of the characteristics. Results were confirmed by the results of the statistical analysis at P<0.05. Although the Copper plays a vital role in fundamental physiological processes. However, CuO in nanoparticles form becomes toxic, causing growth inhibition of alga due to adverse impacts on the same cellular processes.


Introduction
CuO is the simple form of copper compounds and has a range of potential physical properties, like electron correlation effects, high-temperature superconductivity, and spin dynamics [1]. It is a semiconductor compound with a monoclinic structure. CuO has crystal structure with a narrow band gap therefore possesses useful photovoltaic and photoconductive properties [2]. Copper is one of the essential elements for maintaining homeostasis [3]. It may cause toxicity when exceeding the physiological tolerance range [4]. A nanoparticle can be defined as a small object that behaves as a whole unit in terms of its transport and properties. Nanoparticles used in a lot of materials, because it possesses physical properties such as uniformity, conductance or special optical properties [5].
Copper is microelement, essential for plant and algal metabolism, however, their higher concentrations have toxic effects. Copper acts as a structural element in regulatory proteins and participates in photosynthetic electron transport, oxidative stress responses, mitochondrial respiration and hormone signalling and cell wall metabolism. It is an ion, that act as cofactors in many enzymes, such as amino oxidase, plastocyanin, laccase, polyphenol oxidase and Cu/Zn superoxide dismutase cytochrome c oxidase. It also plays an essential role in cellular level such as oxidative phosphorylation and iron mobilization and signalling of transcription and protein trafficking machinery [6].
Algae are unicellular or multicellular organisms exist in different habitats such as marine water, freshwater, or surface of moist soil and rocks. It is an economically and an ecologically important group of the photosynthetic organism [7]. They play an important role in agriculture, medicine, aquaculture, pharmaceutical, and cosmetics applications. It is a source for different commercial products such as biofuels and natural dyes [8]. Previous studies indicate that the addition of 0.67-4 mg/1 of Cu nano-carboxylates resulted in the increase in Chlorella biomass; however, their concentrations ranging from 20 to 40 mg/l strongly inhibited algae growth after the 12th day of cultivation.
The addition of 2-4 mg/l of Cu nano-carboxylates caused an initial increase in chlorophyll a fluorescence parameters as maximal quantum yield of photosystem II photochemistry and in the light-adapted [9]. Zhang et al [10] investigated the toxicity of CuO engineered nanoparticles (ENPs)to Chlorella Pyrenoidosa growth Inhibition. The effect of CuO ENPs on growth was analyzed by exposed the algal cells to the different concentration of CuO ENPs. The growth inhibition was significant with the CuO ENPs concentration increasing. In the fourth day, 10 mg/l CuO ENPs inhibited 5.59% cells, while 30 mg/l CuO ENPs inhibited 33.51% cells. The inhibition effect became significant with the increase of the exposure time.
Much research on the effect of Nano-copper oxide has been done, green synthesized copper oxide nanoparticles show growth inhibition against Microcystis aeruginosa. The inhibition efficiency was 31.8 % at lower concentration and 89.7 % at a higher concentration of copper oxide nanoparticles, respectively. The chlorophyll a and b and carotenoid content of M. aeruginosa declined in a dose-dependent manner with respect to induction of copper oxide nanoparticles [11]. Another research has investigated the effect of polymer coating on CuO NP toxicity in the green alga Chlamydomonas reinhardtii, by comparing bare and polymer-coated CuO NPs, prepared from the same CuO nanopowder. Both CuO NP suspensions were toxic to Chlamydomonas reinhardtii after 6 hours treatment to concentrations of 0.005-0.04 g/l. These results indicate that the high toxicity of polymer-coated CuO NPs in algal cells results of intracellular interactions between NPs and the cellular system [12]. The focus of recent research has been on acute toxic effects of nano-CuO (N-CuO), micro-CuO (M-CuO) and Cu2+ on Chlorella sp. after 96 hours, the toxicities decreased in an order of Cu2+＞N-CuO＞M-CuO, Where that the effect of exposure to 96 hours EC50 of Cu2+ on Chlorella sp. was 1.06 mg/l, and of N-CuO it was 74.61 mg/l, while no pronounced toxicity was observed when the concentration of M-CuO was lower than 160 mg/l [13].
The main objective of this study to evaluate the effect of Nano-copper oxide on the growth of Coelastrella terrestris according to their concentration in the Chu-10 medium.

Isolation and identification of alga
The sample containing Coelastrella terrestris was collected from wastewater, which is belonging to the class of Chlorophyta, for obtaining single alga, adopted the streaking on agar method [14]. To obtain an axenic culture of this alga used antibiotics methods which was described in [15]. Then the alga transferred to the sterile flask 250 ml containing 100 ml of modified Chu 10 sterilized medium [16] and incubated in the growth chamber at 25 °C and 40 μmol m−2 s−1 Periodic illumination in 8:16 light: dark cycle. The alga classified according to the classification key [17] and genetic analysis.

Preparation of oxide nanoparticles
The nanoparticles were purchased as copper oxide from approved US company, Sky Spring Nanomaterials Inc. With 99% purity and size 40 nm for ensuring they are nanoparticles, it was examined by Atomic Force Microscopy (AFM) (figure 1). Stock Solution of copper oxide nanoparticles was prepared for use instead of CuSO4.5H2O copper salts exist in the Chu-10 3 medium in three contractions on, 0.04, 0.16, 0.08 mg/l which was dissolved in deionized water and store in the refrigerator under 4°C until use. For treatment, the alga with nanoparticles 10 ml of Coelastrella terrestris grown before, were add to four flasks, three of which contained different concentrations of Nano-copper oxide solution (0.04, 0.16, 0.08 mg/l) the fourth represented by chu medium without any addition of nanoparticles as a control. Figure1. Atomic force microscopy (AFM) image of CuO nanoparticles.

Alga Biomass and Growth Rate Estimation
The Biomass of alga was, estimated using the standard methods [18] based on the spectrophotometer measurement at a wavelength of 540 nm. Whereas the specific growth rate (μ) of the microalgae was calculated according to the following formula [19]; = ln (x1/ x0)/ t1-t0 where X1 and X0 are the cell densities at times t1 and t0

Chlorophylls and Carotenoids Estimation
To estimate the chlorophyll a, b and carotenoids, content of the alga, about 10ml of the culture of Coelastrella terrestris, was centrifuge at 5000 cycles/min for 5 minutes. The process was repeated several times by using double distilled water. The supernatant was then removed and added 5 ml of acetone 90% to the precipitate and vortexed for 90 seconds then placed in a water bath at 25 °C, after that centrifuge at 6000 cycles/minute for 10 and the supernatant measured at wavelengths (664, 647,630) using a spectrophotometer. Chlorophyll a and b contents of the microalga were estimated according to the [20]

Statistical analysis:
SPSS 24 software was used for all analyses by using ANOVA table one way and least significant differences (LSD) at P<0.05.

Results and discussion
In this study, we tried to demonstrate the effect of Cu NPs on Coelastrella terrestris growth Compared with the control treatment (without nanoparticles addition) we chose three concentration of Cu NPs (0.04, 0.16, 0.08 mg//) the 0.08 mg/l concentration was the same as the CuSO4 in Chu 10 medium, while rest of the concentration was less and higher of it. In this work, the effect of CuO NPs on growth rate was analyzed in table (1) and figure (2) The algal cells were exposed to different concentrations of CuO NPs (0.04, 0.16, 0.08 mg/l) and the growth inhibition was, significant with the CuO NPs concentration, increasing. Also, the results showed that the growth rate of Coelastrella terrestris gave a high level in control unit represented by CuSO4.5H2O 0.08 mg/l followed by CuO NPS 0.08mg/l, CuO NPS 0.04mg/l and CuO NPS 0.16mg/l. Statistically, the three concentrations of CuO NPs and control treatment differed significantly at P<0.05.
Many published reports mention that nano CuO effected on growth rate of algae [9], and found that strongly inhibited algal growth after the 12th day of cultivation for Green Algae Chlorella Vulgaris [21]. They examined the toxicity of CuO NPs to Pseudokirchneriella subcapitata and discovered that 30 nm CuO NPs led to more toxic effects than their bulk formulations. However, the exploration of toxic concentration of CuO NPs (30-40 nm) showed a significantly higher value with 150.45 mg/l CuO NPs for 72 h and less than 100 mg/l CuO NPs for Chlamydomonas reinhardtii [22]. Thus, different algae express different sensitivity to CuO NPs. Based on the investigation of CuO NPs toxicity on the green alga Chlamydomonas reinhardtii, both bare and polymer-coated CuO NPs suspensions were toxic to Chlamydomonas reinhardtii at concentrations of 5-40 mg/l, and decreased the photosystem II activity. Furthermore, it was found that coated CuO NPs were more toxic than the uncoated, which may be due to the increased penetration ability [12]. Another study explained that CuNP at a concentration of 40 mg/l and above reduced the growth of Chlorella vulgaris [23]. Further experiments, with effect of nanometal, induced metal resistance on microalgae, revealed that biochemical attributes of microalgae were increased at 20 mg L-1 concentration. Chlorophyll content of metal nanoparticle exposed microalgae was higher (1.14 mg/l) than wild strain (0.52 mg/l) revealing 54% increase in metal resistant strain (MNPS), however, the content of carotenoid was higher in wild strain. Similarly, the addition of lower concentrations of copper nano-carboxylates (20 to 40 mg/l) had stimulated the growth of Chlorella and increase in biomass along with chlorophyll content of the microalgae [9].       Carotenoids are fat-soluble pigments. It acts as provitamin A and biological antioxidants, protecting cells and tissues from the damaging effects of free radicals and singlet oxygen. Thus, carotenoids are applied in pharmaceuticals, health food, dietary supplements, cosmetics, and as a feed additive [25]. The results showed that decrease in carotenoids content of alga Coelastrella terrestris, when exposed to three concentration of the CuO NPs, compared to the control treatment that recorded the highest content of carotenoids (table 4., figure 5). Statistical analysis showed significant differences between the three concentrations of CuO NPs and control treatment. These results agreed [11] that the increasing of the concentrations of copper oxide nanoparticles (12.5, 25, and 50 mg/l) inhibition the Microcystis aeruginosa growth with respect to the he chlorophyll (a and b) and carotenoid content, when compared with control, the dose-dependent copper oxide nanoparticles treated group showed decreased chlorophyll a, chlorophyll b, and carotenoids contents at 24 and 48 h, whereas a prompt reduction was observed at 72-and 96-h treatment. At 96-h treatment with lower concentration (12.5 and 25 mg/l), the chlorophyll a, chlorophyll b, and carotenoid contents recorded 23.8, 14.7, and 3 % (29.4, 6.37, 6.43 mg/l) and 59.8, 43.7, and 44.1 % (15.5, 4.20, 3.70 mg/L), respectively. Furthermore, higher concentration (50 mg/L) at 96-h exposure of copper oxide nanoparticles, the pigment contents of M. aeruginosa were recorded as 80.8, 82.9, and 73.9%(7.4, 1.27, and 1.73 mg/l), The chlorophyll (a and b) and carotenoid content of M. aeruginosa declined in dosedependent manner with respect to induction of copper oxide nanoparticles.

Conclusions
In conclusion, Copper is a micronutrient required for algal growth and development because it plays a vital role in fundamental physiological processes. However, CuO in nanoparticles form becomes toxic, causing growth inhibition of alga due to adverse impacts on the same cellular processes. The green alga Coelastrella terrestris was firstly used to find out the effect of nanoparticles on its growth. A current study revealed that used of CuO NPs leading to decrease Coelastrella terrestris growth rate and concentrations of chlorophyll a, b and the carotenoids content. furthermore, the control treatment gives a good consequence in growth rate and Chl. a, b and carotenoids.