ZnO Functional Nanomaterial in Green Microalgae Growth Advancement ^†

Abstract

Nanomaterials are substances with unique properties due to the irintrinsic confinement effect and high surface area that have allowed their use in biology and medicine for sensor application. The key feature of nanomaterials in such applications is their ability to providesensitivity enhancement for sensors. On the other hand, nanomaterials possess the ability to change the biological function in cells or tissues; therefore, it is from this point of view that nanomaterials can be considered as functional. As far as biosensor application is concerned, it is important to optimize the determination of target molecules in spatial and temporal modes. The purpose of the presented work was to study the effect of functional nanomaterials on the growth (the temporal component) and morphology (the spatial component) of cell culture. The aim was to provide a culture condition where an increase in both the spatial and temporal components of configuration could be achieved in order to optimize sensor needs. Since microalgae have a wide range of possibilities for practical use in medicine, pharmacology and various industries, the study of the effect of nanomaterials on their growth and development is very important. It was found that ZnO nanomaterial, which was obtained by volumetric electrospark dispersion, revealed the concentration-dependent effect on both the grown rate and the color intensity interior of Chlamydomonas monadina microalgae culture. Therefore, ZnO functional nanomaterial achieved the optimization of target molecule formation for biosensor application.

1. Introduction

The purpose of nanotechnology is to create nanoscale systems and composite materials with specified and adjustable properties. Because of their small size, nanoparticles can easily penetrate various biological structures. The biological properties of nanoparticles can be divided into the following two groups: (1) nanoparticles that have a biocidal effect and (2) nanoparticles that lead to a change in the functions of the material. In general, the literature on nanoparticle bioassays is increasing every year. But these data are contradictory regarding the impact of nanomaterials and do not give clear ideas about the effect of nanoparticles on plant organisms, since it is difficult to compare the available results both in terms of doses and dimensions of nanoparticles, and in terms of plant species. Experimental data on the study of the effect of nanoparticles on the growth and development of microalgae are currently quite limited. Since microalgae have a wide range of possibilities for practical use in medicine, pharmacology and various industries, such research is relevant, necessary and important. Currently, the issue of developing and improving methods of mass cultivation of microalgae, including the use of modern technologies, in particular nanoparticles, is relevant. From the point of view of biological application, zinc oxide nanoparticles (ZnO NPs) attract special attention due to their biocompatibility and low toxicity. The biocompatibility, low toxicity and optical features of these NPs, in our opinion, will have a pronounced effect on photosynthesis during the growth of microalgae. Therefore the aim of our work was to investigate the possible influence of ZnO nanoparticles with specific morphology on the growth of green algae.

2. Materials and Methods

Deionized water was obtained by bidistillation. Zn powder (~99%) was purchased (Merck).

The culture of microalgae Chlamydomonas monadina var. charkowiensis was obtained from the collection of microalgae cultures of Taras Shevchenko Kyiv National University (ACKU) [1]. The strain number was ACKU 267-03. Liquid medium “K” [2] was used for cultivation.

Cultivation was carried out on a luminostat with LB-40 fluorescent lamps with 12 h alternation of light and dark phases at a temperature of +18–22 °C and mixing of cultures once a day.

The number of cells was counted once every 3 days for 4 weeks. A comparative analysis of the number of cells in experimental and control cultures was carried out at the stage of stationary growth of the culture.

During the visual assessment of the condition of the cultures (at the macroscopic level), indicators such as the color of the culture and the intensity of the color were taken into account. Morphological differences between cultures were studied using a Carl Zeiss Primo Star light microscope (objective magnification: 100×). The density of cells in the culture was determined using a hemocytometer.

The method of volumetric electric spark dispersion has established itself as one of the most effective and technologically advanced in the production of nano-sized powders of metals and their alloys. To obtain nanostructured particles of biologically active metals by volumetric electric spark dispersion in water, discharge pulses are used. As a result of such dispersion, sedimentation-resistant hydrosols of biologically active metals are obtained. Colloidal solutions obtained by this method are characterized by high sedimentation stability for several months [3]. The amplitude of discharge voltage between the Zn electrodes was varied between 200 and 600 V, the discharge pulse recurrence frequency was 5–300 Hz; the capacity of the electrical energy storage (capacitor) was varied between 10 and 50 μF. The discharge pulse length was 15–800 μs, and the discharge current amplitude was 300–1350 A.

The obtained ZnO NPs were studied using transmission electron microscopy (TEM, JEOL 1011, Tokyo, Japan; 100 kV).

3. Results

A colloidal solution of flaky ZnO NPs with sizes from 400 nm to 1100 nm in cross-sections was obtained using the volumetric electrospark dispersion method (Figure 1).

The concentration of ZnO NPs in the obtained solution was 5 g/L. This solution was further used to obtain modified nutrient media.

To determine the effect of ZnO nanoparticles on the growth of C. monadina culture in a standard K medium, Zn²⁺ was proportionally replaced by ZnO nanoparticles in the amounts necessary to achieve the specified concentration (1 g/L, 0.5 g/L, 0.2 g/L, and 0.02 g/L). Also, the following two control lines were created: a culture on standard “K” medium, in which Zn²⁺ was present, and a culture on “K” medium without zinc.

During the visual assessment of the state of the C. monadina culture with a concentration of ZnO NPs in the medium of 0.5 g/L, it was established that the experimental culture had a more intense color than the control culture. At the light-optical level, the cells of the experimental and control lines did not differ morphologically. The density of cells at the stage of stationary growth increased by 20% compared to the control.

In the culture with a concentration of ZnO NPs in the medium of 0.2 g/L, the experimental line had a visually less intense color compared to the control line. At the light-optical level, the cells of the experimental and control lines did not differ morphologically. The density of cells at the stage of stationary growth was 31.5% higher than in the control.

At the stage of stationary growth in the C. monadina culture with a concentration of ZnO NPs of 0.02 g/L, no visual difference between the experimental and control lines was observed. At the optical level, the cells of the experimental and control lines did not differ morphologically. The density of cells in the culture at the stage of stationary growth decreased by 10% compared to the control.

The reason for the observed effects connected to the concentration of ZnO NPs is the dissociation rate of Zn²⁺ ions. The key point in algae growth is the presence of Zn²⁺ ions in solution at an appropriate concentration [4]. The low concentration of ZnO NPs in solution causes the low concentration of Zn²⁺ ions insufficient for normal cell growth. On the other hand, the high concentration of ZnO NPs in solution causes the association of Zn²⁺ ions with the nanoparticles’ surface. Therefore, the optimal conditions for algae growth include a moderate concentration of ZnO NPs.

The dynamics of changes in the number of C. monadina cells in the culture depending on the content of ZnO NPs is presented in Figure 2.

4. Discussion

When cultivating C. monadina on medium “K”, in which Zn²⁺ was proportionally replaced by ZnO NPs, the experimental line did not differ in the intensity of coloring from the control line, which was grown on standard medium “K”. The second control line (grown on medium “K”, devoid of zinc) had a less intense color compared to the previous two. Morphologically, the cells of the experimental and control lines did not differ.

After counting the number of cells, it was found that their number in the experimental line increased by 30% compared to the line grown on the standard “K” medium and by 35% compared to the line grown on the “K” medium without zinc.

The dynamics of changes in the number of cells in culture on standard and modified “K” media is presented in Figure 3.

Thus, this research demonstrated that the effect of ZnO NPs on the growth of C. monadina culture is concentration dependent. In particular, high (1 g/L) and low (0.002 g/L) concentrations of ZnO NPs suppress cell growth, while a moderate concentration of nanoparticles (0.5 g/L and 0.2 g/L) leads to an increase in the number of cells in the culture. The density of cells in the culture increases, and their color becomes more intense. This is possibly due to an increase in the chlorophyll content of the cell.

As for the experiment with the modified “K” medium, in this case, it was found that the number of cells increased on the medium where Zn²⁺ was proportionally replaced by ZnO NPs, compared to the C. monadina culture grown on the standard “K” medium.

ZnO NPs can be described by the simplified structural formula ZnO&H₂O/Zn²⁺, where the zinc ion is in equilibrium dissociation–association (dissolution of zinc and its deposition on solid-phase ZnO), forming a defined concentration of zinc ions in an aqueous solution depending on the concentration of ZnO NPs. High and low concentrations of zinc ions lead to the inhibition of culture growth.

5. Conclusions

Flaky ZnO NPs obtained by the method of volumetric electrospark dispersion have an effect on the growth of microalgae Chlamydomonas monadina on medium “K” during its cultivation.

A high (1 g/L) or low (0.02 g/L) concentration of ZnO NPs inhibits the growth of Chlamydomonas monadina cells.

A moderate concentration of ZnO NPs (0.2–0.5 g/L) leads to an increase in the number of Chlamydomonas monadina cells in the culture.

Chlamydomonas monadina grows more efficiently on modified “K” medium (with ZnO NPs instead of Zn²⁺) than on standard medium.

An increase in the number of algae cells caused by ZnO nanoparticles alongside an increase in the chlorophyll concentration cause an increase in O₂ concentration in water environments, supporting the optimal functionality of aquatic ecosystems.