A Modiﬁed Silver–Egg Shell Nanocomposite Applied for Antibacterial Activities †

: Bacterial infections have extensive impacts on public health. Therefore, ﬁnding compounds with antibacterial properties could serve as an effective method. A nanocomposite, Ag/CaO, was prepared from silver nitrate and egg shells. After calcination of the egg shells, the remaining solid, CaO, was cooled; then, silver nitrate was added and the mixture was ground to a ﬁne powder, and ﬁnally heated to 300 ◦ C. The brown solid obtained was characterized by XRD, SEM and XRF methods. The prepared Ag/CaO was examined for antibacterial activity against Gram-positive and Gram-negative bacteria, including Keleb pneumonia, staphylococcus aureus, and Escherichia coli. We previously published a similar paper in the 25th ECSOC 2021, but the current paper has two changes, including the amount of silver nitrate and calcium oxide in the synthesis route, and the size of the ﬁrst synthesized nanocomposite by grinding with a ball mill; then, we examined these two substances against the bacteria. In fact, changing the amount of silver, known as the antibacterial metal, was compared to changing the size of the nanocomposite, which could have a greater antibacterial effect.


Introduction
Egg shell is considered a source of pollution, as well as of calcium carbonate [1]. Today, synthetic methods without dangerous solvents are attractive, particularly for their environmental advantages. It is important to prepare materials that have antibacterial properties and are not harmful to the environment; moreover, the synthesis method used should be compatible with the life cycle.

Preparation of CaO from Egg Shell
CaO was prepared from collected egg shells after washing carefully, drying at room temperature, grinding in a porcelain mortar, and then calcinating at 900 • C for 5 h [2]. As a final step, it was cooled down to room temperature. The obtained CaO was used for antibacterial activity.

Preparation of Ag-NP@CaO(1)
The obtained 3 g of CaO was ground in a porcelain mortar, then added to 1 g AgNO 3 and crushed again to obtain a fine uniform powder. The powdered mixture was placed in a furnace at 300 • C for 3 h until producing a brown solid, indicating Ag@CaO [3].

Preparation of Ag-NP@CaO(2)
The obtained 2 g of CaO was ground in a porcelain mortar, then added to 2 g of AgNO 3 and crushed in the mortar. The mixture was placed in the furnace at 300 • C for 3 h until producing a brown solid, indicating Ag@CaO(2).

Preparation of Ag-NP@CaO(3)
Ag-NP@CaO(1) was ground in a ball mill for 20 min and was considered for studying its morphology and size.
In the XRD patterns, the three characteristic lines of CaO shown in Figure 1 are also present in Figure 2. The Ag-NP@CaO(1) nanocomposite was synthesized with 3 g of calcium oxide and 1 g of silver nitrate. Figure 3 shows an increase in silver nitrate compared to calcium oxide, and the synthesis of CaO and Ag(NO) 3 with an equal ratio of them. These changes can be clearly seen.

Preparation of Ag-NP@CaO(2)
The obtained 2 g of CaO was ground in a porcelain mortar, then added to 2 g of AgNO3 and crushed in the mortar. The mixture was placed in the furnace at 300 °C for 3 h until producing a brown solid, indicating Ag@CaO(2).

Preparation of Ag-NP@CaO(3)
Ag-NP@CaO(1) was ground in a ball mill for 20 min and was considered for studying its morphology and size.
In the XRD patterns, the three characteristic lines of CaO shown in Figure 1 are also present in Figure 2. The Ag-NP@CaO(1) nanocomposite was synthesized with 3 g of calcium oxide and 1 g of silver nitrate. Figure 3 shows an increase in silver nitrate compared to calcium oxide, and the synthesis of CaO and Ag(NO)3 with an equal ratio of them. These changes can be clearly seen.   The obtained 2 g of CaO was ground in a porcelain mortar, then added to 2 g of AgNO3 and crushed in the mortar. The mixture was placed in the furnace at 300 °C for 3 h until producing a brown solid, indicating Ag@CaO(2).

Preparation of Ag-NP@CaO(3)
Ag-NP@CaO(1) was ground in a ball mill for 20 min and was considered for studying its morphology and size.
In the XRD patterns, the three characteristic lines of CaO shown in Figure 1 are also present in Figure 2. The Ag-NP@CaO(1) nanocomposite was synthesized with 3 g of calcium oxide and 1 g of silver nitrate. Figure 3 shows an increase in silver nitrate compared to calcium oxide, and the synthesis of CaO and Ag(NO)3 with an equal ratio of them. These changes can be clearly seen.   In XRF analysis of CaO, shown in Table 1, we can see the percentage of pure CaO equal to 97.78% which can be acceptable.
In the XRF analysis of CaO, shown in Table 2, we can see that the percentage of pure CaO equals 97.7%. The XRF analysis of Ag@CaO(1) is given in Table 2 and shows 15.67% Ag in the composite. In Table 3, showing the synthesis with an increasing percentage of silver nitrate compared to calcium oxide, which has an equal ratio of these two substances, the amount of silver reaches 46.9% in the Ag@CaO (2).
The SEM micrographs of the four samples, CaO, Ag@CaO(1), Ag@CaO(2), and Ag@CaO(3) are shown in Figure 4. The flake morphology of CaO can be clearly observed in Figure 4, and it can be seen that the edges of the flakes become round, but in Ag@CaO, the Ag particles are settled on the planes of CaO. In Ag@CaO (3), only ball mill grinding was used, and the same composite (1) was placed in the ball mill for 15 min. The particle size approached from 7.559 nm to 6.735 nm and in Ag@CaO(2), a 1:1 ratio of silver nitrate and calcium oxide was applied. The nanocomposite has a slightly more amorphous shape. In XRF analysis of CaO, shown in Table 1, we can see the percentage of pure CaO equal to 97.78% which can be acceptable.
In the XRF analysis of CaO, shown in Table 2, we can see that the percentage of pure CaO equals 97.7%. The XRF analysis of Ag@CaO(1) is given in Table 2 and shows 15.67% Ag in the composite. In Table 3, showing the synthesis with an increasing percentage of silver nitrate compared to calcium oxide, which has an equal ratio of these two substances, the amount of silver reaches 46.9% in the Ag@CaO(2).

Antibacterial Activity
The antibacterial activity of CaO [4,5], Ag@CaO(1), Ag@CaO(2), and Ag@CaO(3) composite against Gram-positive and Gram-negative bacteria were tested. The bacteria include Keleb pneumonia, Staphylococcus aureus, and Escherichia coli. The results are shown in Figure 5a-c and are summarized in Table 4. In all cases, the way in which the inhibition zone diameter of Ag@CaO(1), Ag@CaO(2), and Ag@CaO(3) nanocomposite changes can be seen.
Chem. Proc. 2022, 12, 80 4 of 6 The SEM micrographs of the four samples, CaO, Ag@CaO(1), Ag@CaO(2), and Ag@CaO(3) are shown in Figure 4. The flake morphology of CaO can be clearly observed in Figure 4, and it can be seen that the edges of the flakes become round, but in Ag@CaO, the Ag particles are settled on the planes of CaO. In Ag@CaO(3), only ball mill grinding was used, and the same composite (1) was placed in the ball mill for 15 min. The particle size approached from 7.559 nm to 6.735 nm and in Ag@CaO(2), a 1:1 ratio of silver nitrate and calcium oxide was applied. The nanocomposite has a slightly more amorphous shape.

Antibacterial Activity
The antibacterial activity of CaO [4,5], Ag@CaO(1), Ag@CaO(2), and Ag@CaO (3) composite against Gram-positive and Gram-negative bacteria were tested. The bacteria include Keleb pneumonia, Staphylococcus aureus, and Escherichia coli. The results are shown in Figure 5a-c and are summarized in Table 4. In all cases, the way in which the inhibition zone diameter of Ag@CaO(1), Ag@CaO(2), and Ag@CaO(3) nanocomposite changes can be seen.

Conclusions
In this work, a waste material was converted to a bioactive product against many types of bacteria. Moreover, its composite with metallic silver showed a more effective antibacterial effect. When the synthesized nanocomposite with two different ratios and its ground form by a ball mill were examined, not only did the particle sizes change, but

Conclusions
In this work, a waste material was converted to a bioactive product against many types of bacteria. Moreover, its composite with metallic silver showed a more effective antibacterial effect. When the synthesized nanocomposite with two different ratios and its ground form by a ball mill were examined, not only did the particle sizes change, but they also behaved differently against five types of applied bacteria.
Author Contributions: M.A. and F.M. contributed equally to this manuscript. All authors have read and agreed to the published version of the manuscript.