The Synthesis of Y-zeolite-modified CaCO 3 -ZnO Nanocomposites as an Antibacterial Agent

The ability of inorganic antibacterial agents like metal oxides and nanoscale inorganic materials to inhibit bacterial growth rates has yet to receive much research attention. In this study, CaCO 3 -ZnO/Y-zeolite nanocomposites were created utilizing coprecipitation and impregnation techniques with Ca(CH 3 COO) 2 , Zn(CH 3 COO) 2 ⋅ 2H 2 O, Y-zeolite precursors. Physical and chemical characteristics of nanocomposites have been investigated using XRD, FTIR, and SEM-EDX characterizations. The agar-well diffusion method tested the substance for antibacterial activity against gram-positive and gram-negative bacteria. Nanocomposites have a crystal size range of 35.46-36.53 nm and a crystallinity of 35-37 %, according to the results of XRD analysis. The carbonate groups are visible in FTIR data at wave numbers 1433, 875, and 712 cm -1 . The Zn-O absorption band was verified at wave numbers 600-400 cm -1 . The Y-zeolite absorption bands at wave numbers 1012-997 cm -1 and 745-746 cm -1 were confirmed. The particle morphology is cube-shaped with irregular sizes. The EDX result showed that the composition consists of 35.92 % calcium, 1.68 % zinc, 44.81 % oxygen, and 13.79 % carbon as elements. With the addition of 2.5 % Y-zeolite, the antibacterial activity of nanocomposites showed the best results, with an inhibition zone diameter of 7.62 mm against Escherichia coli and 6.56 mm against Staphylococcus aureus bacteria.


INTRODUCTION
The emergence of resistant microbes and persistent bacterial infections are recognized as serious public health problems.It can affect populations in developing countries as well as low-and middle-income countries 1 .Therefore, in order to prevent and control these pathogens, it is necessary to carry out early diagnosis and appropriate treatment procedures.In this regard, nanomaterial-based therapies have received a great deal of interest in the scientific community due to their favorable antibacterial properties.Nanocomposites have unique properties, such as a high surface-to-volume ratio and antibacterial properties that outperform micronsized materials 2 .
Antibacterial agents are divided into two types: organic and inorganic.The fundamental issue with commonly used organic antibacterial agents is their low stability at relatively high temperatures and pressures, whereas inorganic antibacterial agents are highly stable and powerful 3 .Organic antibacterial agents have environmentally hazardous activities along with a limited usage period, so it is preferable to use inorganic antibacterial agents since these substances are safe for the environment and easy to use 4 .
ZnO is a multifunctional inorganic material with effective antibacterial activity.One of the applications of ZnO is in the food packaging industry because it is nontoxic, has antibacterial and antifungal properties, and has high photochemical and catalytic activity 5 .As an antibacterial, ZnO has a mechanism of action that inhibits cell synthesis, damages bacterial cell walls, and interferes with cell metabolism 6 .ZnO in the form of nanoparticles can inhibit growth rates of gram-positive bacteria such as Staphylococcus aureus 7 .

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Calcium carbonate (CaCO3) is an inorganic mineral used as a filler in chemical industries such as toothpaste, paper, and paint.There are three types of crystalline forms of calcium carbonate: calcite, aragonite, and vaterite.Calcite with rhombohedral shapes includes stable phases at room temperature, while aragonite and vaterite are stabilizing phases that can transform into stable stages 8 .Calcium carbonate in the form of calcite has many applications in the field of health, such as a nutritious calcium booster and as an antiacid for the stomach 9 .Based on the production process, calcium carbonate is divided into two types: heavy and light.The heavy type is produced through the milling process of calcium stones with a high CaCO3 composition.The light type is manufactured through the process of precipitation resulting from chemical reactions so that calcium carbonate of high purity is obtained 10 .Precipitate calcium carbonate (PCC) is a calcium carbonate deposit resulting from the reaction of natural materials such as dolomite, calcite, limestone, and marble 11 .The use of PCC as an antibacterial agent offers great opportunities due to its compatible material, biodegradability, and small particle size.
Previous research has explored the antibacterial activity of combining two inorganic materials such as CuO-ZnO 3 , ZnO-MgO 12 , CaCO3-MgO 13 , and CaCO3-ZnO 9 .The CuO-ZnO nanocomposite synthesized using the sol-gel method showed antibacterial activity with an inhibition zone diameter of 2.1 mm against Staphylococcus aureus bacteria and 2.3 mm against Escherichia coli bacteria 14 .CaCO3-MgO nanocrystals synthesized using the mixing method with calcination at 800 o C have the potensial as an antibacterial agent because CaCO3-MgO nanocrystals have a crystal size between 61.85-66.11nm which can inhibit antibacterial activity 13 .The combination of inorganic materials including ZnO and CaCO3 with stabilizer is expected to produce products with nanocrystal size and good antibacterial activity against Escherichia coli and Staphylococcus aureus.Several researchers have used zeolite as a stabilizer and promoter.Zeolite is used as a material to promote and stabilize both metals and metal oxides by controlling the size of nanoscale particles.The synthesized CuO-ZnO nanocomposite with zeolite as an agent successfully controls nanoscale particle size 15 .Yzeolite is chosen as a stabilizer because it has a large Si/Al ratio compared to type A and X-zeolites.A high Si/Al ratio on zeolite produces zeolites with good stability and a large surface area 16 .
The agar-well diffusion method is a method of testing antibacterial activity by making holes in an agar medium containing test bacteria.Then the holes are injected with the samples to be tested.After the incubation process, bacterial growth was observed in the inhibition area around the hole 17 .The working principle of this diffusion method is that antibacterial compounds diffuse into the agar medium that has been inoculated with test bacteria.The zone of inhibition on microbial growth is characterized by the presence or absence of a clear zone around the hole.The advantage of the agarwell diffusion method compared to the disc diffusion method is that measuring the zone of inhibition in the agar-well diffusion method is easier to do.This is because microbes are active both on the upper and lower surfaces of the nutrient agar 18 .
Based on this explanation, this research will synthesize CaCO3-ZnO nanocomposites with a Y-zeolite modifier as a carrier using coprecipitation and impregnation methods.The CaCO3-ZnO/Y-zeolite nanocomposites were tested for antibacterial activity against gram-negative (Escherichia coli) and grampositive (Staphylococcus aureus) bacteria.

Synthesis of CaCO3-ZnO Nanocomposites
CaCO3-ZnO nanocomposites were synthesized using the coprecipitation method with modification 19 .CaCO3-ZnO nanocomposites (mole ratio 1:1) were prepared by mixing 50 mL calcium acetate 1 M and 50 mL zinc acetate dihydrate 1 M until homogeneous.Oxalic acid dihydrate solution 0.15 M was added to the mixture until a precipitate formed while stirring for 10 hours at room temperature.The precipitate was collected and washed using distilled water until the pH was equal to the pH of the solvent (pH 5).The precipitate obtained was then dried in an oven for 12 hours at 105 o C. The solid was calcined for 5 hours at 600 o C in a furnace to obtain a CaCO3-ZnO nanocomposite.The same steps were followed for the preparation of CaCO3-ZnO nanocomposites with mole ratios of 2:1 and 1:2.

Synthesis of CaCO3-ZnO/Y-zeolite Nanocomposites
CaCO3-ZnO/Y-zeolite nanocomposites were synthesized using the coprecipitation method with modification 19 .Comparison of mole ratios in the synthesis of CaCO3-ZnO with the best antibacterial activity modified with Y-zeolite.Calcium acetate and zinc acetate dihydrate solutions were mixed until homogeneous.Y-zeolite at 2.5 % was added to the calcium acetate and zinc acetate mixture.Oxalic acid dihydrate solution was added to the mixture until a precipitate formed while stirring for 10 hours at room temperature.The precipitate was collected and washed using distilled water until the pH was equal to the pH of the solvent (pH 5).The precipitate obtained was then dried in an oven for 12 hours at 105 o C. The solid was calcined for 5 hours at 600 o C in a furnace to obtain a CaCO3-ZnO nanocomposite.Furthermore, the same steps were carried out for the addition of Y-zeolite 5 and 10 %.

Characterization
The nanocomposite products (CaCO3-ZnO and CaCO3-ZnO/Y-zeolite) were characterized by FTIR, XRD, and SEM-EDX.Functional group analysis was carried out using PerkinElmer Spectrum IR Version 10.6.1 at wave numbers 4000-400 cm -1 .Nanocomposite products were mixed with KBr powders at a ratio of 1:10 and pressed into transparent pellets.X-ray diffraction (XRD) analysis was conducted using X'Pert PRO PANalytical with Cu radiation at a wavelength of 1.5405 Ǻ on 40 kV 30 mA, scanning samples from 10-80 o .Calculation of the crystal size of CaCO3-ZnO and CaCO3-ZnO/Y-zeolite nanocomposites using the Debye-Scherrer equation 20 .The results of the crystal size calculation can be seen in Table 1.
which D is the average crystal size (nm); k is constant (0.9), λ is the monochromatic wavelength of Cu-Kα (0.54106 nm); β is the full width at half maximum (FWHM) in radians; and theta is the scattering angle (degree).
Scanning Electron Microscopy (SEM) was conducted using FEI Inspect 850 with high-energy electron beam scanning.X-rays in SEM can be used for the identification elemental composition of samples by Energy Dispersive X-ray (EDX) technique.The EDX spectrum was measured by the EDAX-AMETEK detector.

Antibacterial Activity Test Preparation of bacterial culture
Bacterial stocks were prepared by inoculating one ose of a pure culture of Escherichia coli and Staphylococcus aureus into nutrient agar, then incubated at 37 o C for 24 hours.

Preparation of nutrient agar media
As much as 10 grams of nutrient agar were dissolved in 500 mL of distilled water (20 grams/1000 mL).This nutrient agar was used as the base layer and seeding layer.The nutrient agar media that had been homogenized was sterilized using an autoclave at 121 o C for 15 minutes.Then it was cooled to a temperature of 45-50 °C.

Sterilization of equipment
Petri dishes, cork borer, spatula, and glassware that will be used in the test were sterilized using an autoclave for 15 minutes at 121 o C.

Preparation of bacterial test
Bacteria were diluted by adding one ose of Escherichia coli and Staphylococcus aureus bacterial suspensions into each test tube containing 5 mL of a 0.9 % NaCl solution.Then, they were homogenized using a vortex.The turbidity of the bacterial solution should be equal to the turbidity of the 0.5 McFarland solution.

Antibacterial test against Escherichia coli and Staphylococcus aureus
The antibacterial activity of nanocomposites was evaluated using double-layer agar-well diffusion against gram-positive (Staphylococcus aureus) and gramnegative (Escherichia coli) bacteria 12 .The base layer was made by pouring 25 mL of nutrient agar (NA) solution into a petri dish and allowing it to solidify.The seed layer was prepared by adding 15 mL of bacterial suspension that had been standardized to the 0.5 Mc Farland standard.Next, four wells with a diameter of 5 mm were formed.Then, 0.025 grams of CaCO3-ZnO sample (1:1) was filled in each hole.A petri dish was incubated at 37 o C for 24 hours.The same process was carried out on CaCO3-ZnO 2:1 and 1:2 as well as on CaCO3-ZnO/Y-zeolite 2.5, 5, and 10 %.The diameter of the clear zone formed was measured using a caliper.

Synthesis of CaCO3-ZnO and CaCO3-ZnO/Y-zeolite Nanocomposites
In the synthesis process, zinc acetate dihydrate and calcium acetate were impregnated with zeolite Y to form the complex compound Ca(C2O4)2Zn-Y zeolite.The impregnation process aims to attach the active side of the metal to the buffer material 21 .The solid was calcined at 600 o C for 5 hours to obtain CaCO3-ZnO and CaCO3-ZnO/Y-zeolite nanocomposites.Through the calcination process, complex chemical compounds such as Ca(C2O4)2Zn and Ca(C2O4)2Zn-Y zeolite can be dissolved in the presence of heat to form the CaCO3-ZnO and CaCO3-ZnO/Y-zeolite structure.Both the nanocomposite without modification and with Y-zeolite modification produced a white powder.

FTIR Characterization
FTIR spectrophotometer was used to identify the functional groups of CaCO3-ZnO and CaCO3-ZnO/Yzeolite nanocomposites.FTIR analysis was conducted at wave numbers 4000-400 cm -1, as shown in Figure 1.The FTIR analysis results in Figure 1 show the absorption band associated with the stretching vibration of the C-O bond of the carbonate group.The characteristic peaks of CaCO3-ZnO nanocomposites at wave numbers 1433, 875, and 712 cm -1 indicate the presence of carbonate groups from CaCO3 compounds 22 .Then, in the CaCO3-ZnO/Y-zeolite nanocomposites, there was a shift of the carbonate groups at wave numbers 1392, 874, and 712 cm -1 .The Zn-O absorption band was confirmed at 600-400 cm -1 with low peak 23 .There is a shift in the absorption peak along with an increase in the amount of ZnO that is composited.In CaCO3-ZnO/Y-zeolite nanocomposites, asymmetrical absorption bands of Si-O-Si and O-Al-O appear at wave numbers 1012, 1000, and 997 cm -1 while symmetrical absorption bands appear at wave numbers 745 and 746 cm -1 which are characteristic of Y-zeolite 24 25 .

XRD Characterization
X-ray diffraction (XRD) analysis was used to analyze the phase and crystal size of the CaCO3-ZnO and CaCO3-ZnO/Y-zeolite nanocomposites.XRD analysis was performed at 2θ angles between 10-80 o .The diffractogram of the synthesized nanocomposites can be seen in Figure 2.
As a result of the X-ray diffraction analysis (Figure 2), the diffraction patterns were matched with the Joint Committee on Powder Diffraction Standards (JCPDS) for comparison.ZnO diffraction patterns appear to peak on 2θ = 31; 34; 36; 56; 72; and 77 ° 26 , whereas CaCO3 diffraction patterns appear to peak on 2θ = 23; 29; 39; 43; 47; 48; 57; and 60° 9 .The diffraction pattern of ZnO corresponds to JCPDS 36-1451 with a hexagonal wurtzite structure 27 , and CaCO3 corresponds to JCPDS 47-1743 with a rhombohedral crystal structure, the phase of calcite 28 .At a calcination temperature of 600°C, the characteristic peak of CaCO3 has a very high intensity at peak 2θ = 29.4°,indicating the calcite phase.In the CaCO3-ZnO/Y-zeolite nanocomposites, there are characteristic peaks of Yzeolite shown at 2θ = 26.96;31.29; and 33.96° by JCPDS 38-0240 29 .Both in CaCO3-ZnO and CaCO3-ZnO/Yzeolite nanocomposites, there is a small amount of CaCO3 converted to CaO.This occurs due to the process of releasing CO2 from CaCO3 to form CaO 30 .
Table 1 shows that the CaCO3-ZnO and CaCO3-ZnO/Y-zeolite nanocomposites are already nanosized, less than 100 nm.The CaCO3-ZnO nanocomposite with a mole ratio of 1:2 has the smallest crystal size with large  crystallinity.This shows that the more ZnO that is composited with CaCO3 has an impact on the size of the crystals produced.The combination of ZnO with lower CaCO3 can increase activity as the crystal size decreases.Similar results were reported that CaCO3-ZnO with a mole ratio of 1:2 has better activity 19 compared to the mole ratios 1:1 and 2:1.The crystal size of CaCO3-ZnO was increased after being modified with Y-zeolite.This might be explained by the active sites on CaCO3-ZnO being covered by the presence of Y-zeolite, thus causing an increase in the crystal size of the modified CaCO3-ZnO.
The crystallinity of CaCO3-ZnO was obtained by comparing the area of the crystal with the total area of amorphous and crystalline crystals.The crystallinity of CaCO3-ZnO increases as the amount of added ZnO increases.The addition of ZnO can have a positive effect on the formation of a more regular crystal structure.The peaks shown in the nanocomposites have a high intensity and a clear peak separation pattern.The use of Y-zeolite generally reduces the crystallinity of CaCO3-ZnO.The addition of Y-zeolite in large quantities can reduce the number of active sites due to the closure of the active side by Y-zeolite at high loading, which can reduce crystallinity.

SEM-EDX Characterization
Scanning Electron Microscopy (SEM) analysis was aimed to determine the surface morphology of CaCO3-ZnO and CaCO3-ZnO/Y-zeolite nanocomposites.The results of the surface morphology analysis of CaCO3-ZnO and CaCO3-ZnO/Y-zeolite nanocomposites (Figure 3) exhibit cube-like shapes particles with uneven and irregular sizes.This is in accordance with the results confirmed in the FTIR and XRD analyses, which showed the presence of CaCO3 compounds in each sample.In the CaCO3-ZnO 1:1 and 2:1 samples, some particles are bound together as aggregates.In the CaCO3-ZnO 1:2 sample, the aggregate size tends to be smaller and in small quantities.This is in accordance with research conducted by Rahmawati et al. 31 , which shows that increasing the molar ratio of Ca/Zn affects increasing the size of agglomerates.Smaller grain and aggregate sizes provide a higher specific surface area 32 .With the addition of 2.5 % Y-zeolite, the aggregate size tends to be smaller than with the addition of 10 % Y-zeolite, which has larger aggregates.This indicates that the more zeolite Y added, the larger the aggregate produced.
Figure 4 shows the results of the elemental composition analysis on CaCO3-ZnO and CaCO3-ZnO/Y-zeolite nanocomposites.The CaCO3-ZnO nanocomposites consist of 45 % calcium, 4.76 % zinc, 42.37 % oxygen, and 7.15 % carbon elements, which indicate the presence of CaCO3 and ZnO compounds 9 .The CaCO3-ZnO/Y-zeolite nanocomposites consist of 35.92 % calcium, 1.68 % zinc, 44.81 % oxygen, 13.79 % carbon, 0.95 % silicon, 1.20 % aluminum, and 1.66 % Y.The confirmed presence of Si, Al, and Y elements indicates that the CaCO3-ZnO/Y-zeolite nanocomposites contain Y-zeolite compounds.Through EDX analysis, the composition of Ca and Zn in the CaCO3-ZnO modified by Y-zeolite changes the composition of the elements formed.Even though the amount is small, it can be seen in the measurement results that it is homogeneous.

Antibacterial Activity Test
The antibacterial activity tests of CaCO3-ZnO and CaCO3-ZnO/Y-zeolite were conducted using the agar-well diffusion method against Escherichia coli and Staphylococcus aureus bacteria.Measuring the diameter of the inhibition zone or clear zone is used as an indicator of the effectiveness of the sample in inhibiting the activity of bacterial growth 33 .Based on the inhibition zone diameter (Figure 5), it can be seen that CaCO3-ZnO and CaCO3-ZnO/Y-zeolite nanocomposites have the ability to inhibit the growth of gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli).The negative control used distilled water because distilled water is the solvent of the CaCO3-ZnO nanocomposites.
Table 2 shows the antibacterial activity test results of CaCO3-ZnO and CaCO3-ZnO/Y-zeolite nanocomposites against gram-positive and gramnegative bacteria.In the nanocomposite before modification, CaCO3-ZnO with a ratio of 1:2 has the largest inhibition zone diameter of 6.6 mm against grampositive bacteria and 5.9 mm against gram-negative bacteria.After being modified with Y-zeolite, the CaCO3-ZnO nanocomposite with the addition of 2.5 % Y-zeolite has the largest inhibition zone diameter of 6.56 mm against gram-positive bacteria and 7.62 mm against gram-negative bacteria.Based on the inhibitory strength category, CaCO3-ZnO and CaCO3-ZnO/Y-zeolite have antibacterial properties with medium strength against the bacteria.The nanocomposites are bacteriostatic because they only inhibit the growth rate of bacteria 34 .
Unmodified CaCO3-ZnO nanocomposite showed better inhibition against gram-positive bacteria.This could be due to differences in cell wall structure in bacteria.Gram-positive bacteria are composed of a thick peptidoglycan layer and teichoic acid, while gramnegative bacteria are composed of a thin peptidoglycan layer.Teichoic acid and lipoteichoic acid in gram-  35 shows that the inhibitory activity of Ag nanoparticles on bacterial growth is related to the release of positive ions.
Positive ions can interact with the negative charge of the plasma membrane and stimulate the formation of reactive oxygen species (ROS), causing damage and the death of bacterial cells.Although the peptidoglycan layer in gram-negative bacteria is thin, it contains an outer membrane.This outer membrane consists of lipopolysaccharide (LPS), which is important for the integration of bacterial structures.The lipopolysaccharide layer on the outer membrane can be an obstacle to antimicrobial substances, causing gramnegative bacteria to be more resistant to antibacterial agents 36 .
The CaCO3-ZnO nanocomposite, with the addition of 2.5 % Y-zeolite, showed better inhibitory activity against gram-negative bacteria.This could be due to differences in cell wall structure in bacteria.Yzeolite can loosen the lipopolysaccharide layer on gramnegative bacteria and facilitate the penetration of metal ions so that zeolite particles and Ca 2+ and Zn 2+ metal ions easily enter the bacterial cells.In addition, Y-zeolite also has a strong adsorption ability, so it can attract and bind important molecules in bacteria and interfere with their performance.Gram-positive bacteria have a thick peptidoglycan layer with complex cell walls, so Yzeolite is not easily absorbed into bacterial cells.This causes gram-positive bacteria to be more resistant to antibacterial agents 37 .
The antibacterial activity of CaCO3-ZnO and CaCO3-ZnO /Y-zeolite nanocomposites is related to the crystal size produced.Based on the results, it can be seen that the antibacterial activity of CaCO3-ZnO and CaCO3-ZnO /Y-zeolite nanocomposites increases along with the smaller crystal size produced.The nanoscale crystal size will facilitate molecules to penetrate and damage the cell membrane of bacteria.The mechanism of nanocomposites as antibacterials is to interact with bacterial membranes, which causes changes in permeability and damage to bacterial membranes to inhibit bacterial growth 38 .In addition, the antibacterial activity of CaCO3-ZnO may be due to the formation of superoxide and other reactive oxygen species (ROS), such as H2O and H2O2 from the surface of CaCO3-ZnO, which produce free radicals that damage the bacterial cell membrane 39 .ROS generated on the surface of nanocomposites can cause oxidative stress by damaging cell membranes, DNA, and cellular proteins so that antibacterial activity increases 36 .
Table 3. shows the comparison of antibacterial activity.The results showed that the combination of two materials with the addition of a stabilizer has better antibacterial activity compared to pure nanoparticles.These results are in accordance with previous study that ZnO-MgO nanocomposite is better at preventing bacterial growth than pure ZnO and MgO nanoparticle 12 .The agar-well diffusion method chosen in this study showed a better bacterial growth inhibition zone compared to the disc method because the sample had an efficient osmosis process so it could effectively inhibit bacterial growth 18 .

Y-zeolite-modified
CaCO3-ZnO nanocomposites have been successfully synthesized using coprecipitation and impregnation methods.The FTIR spectra obtained showed the presence of carbonate, metal oxide, and zeolite Y groups, interpreting the CaCO3-ZnO/Y-zeolite nanocomposite.The average crystal size of CaCO3-ZnO/Y-zeolite was 35.46-36.53nm, with crystallinity 35.03-37.66%.The surface morphology of CaCO3-ZnO/Y-zeolite nanocomposite formed resembles cubes particles with uneven and irregular sizes.EDX results confirmed the presence of CaCO3, ZnO, and Y-zeolite with different elemental percentages.The antibacterial activity test showed that the CaCO3-ZnO nanocomposite was better at inhibiting the growth of gram-positive bacteria (Staphylococcus aureus).In comparison, the CaCO3-ZnO/Y-zeolite nanocomposite was better at inhibiting the growth of gram-negative bacteria (Escherichia coli).CaCO3-ZnO/Y-zeolite nanocomposite has the potential to be an antibacterial agent with medium strength.In further research, the CaCO3-ZnO nanocomposite will be modified with natural materials to investigate its antibacterial activity further.

Figure 1 .
Figure 1.FTIR spectra of CaCO3-ZnO and CaCO3-ZnO/Y-zeolite nanocomposites . The absorption tape at wave numbers 720-650 cm -1 and 780-720 cm -1 is an internal and external T-O symmetrical (Si-O/Al-O) vibration.The absorption of T-O curvature vibrations in a range of wave numbers 475-450 cm -1 corresponds to the T-O curve vibrations of the faujasitetype zeolite

Table 3 .
Literature comparison of antibacterial activity of CaCO3-ZnO/Y-zeolite nanocomposite