*3.4. In Vitro Testing*

## Microbial Viability Assay (WST)

Figure 9 demonstrates the antibacterial test conducted against the CA/AlN composites material with *S. epidermidis* using WST method. It is clearly observed that absorbance level increases until 15 wt. % and start to reduce at 20 wt. % for 12 h. Besides, the same samples were tested with the laser microscope, where the biofilm production was clearly seen on the surface as shown in Figure S1. Almost similar results were obtained for 12 h, the biofilm formation reduces slightly from the control sample and the antibacterial effect was not clearly observed. Whereas, for 24 h, the absorption shows the similar trend as 12 h, but at 20 wt. % shows a very lower absorption than control. The biofilm formation shows a very clear trend at 24 h where, at control it was observed the full area covered by patches of biofilm and it start to reduce as the concentration of AlN increases and at 20 wt. % and very low amount of biofilm was detected.

To reconfirm the antibacterial property, the samples were analyzed using Crystal violet (CV) staining method. The stained samples were analyzed with WST and Viability Staining, and ImageJ Analysis and the results were shown in Figure 9 The CV-stained samples show high absorbance at 12 h and start to reduce slightly at higher concentration especially at 15 and 20 wt. %, at 24 h the absorbance does not increase and was maintained as 0 wt. % sample but at 20 wt. % it decreases drastically. When compare with viability staining method, it shows the similar trend as the previous WST method where the biomass increases up to 15 wt. % and decreases at 20 wt. % for 12 h, whereas, at 24 h the biomass shows slight increment up to 15 wt. % and drastic drop at 20 wt. %.

For the *E. coli* bacteria, the WST measurement was not precise as the absorbance increases at 12 and 24 h even at 20 wt. % as shown in Figure 10. However, at 24 h, clear trend of decrement was observed especially at 20 wt. %. Comparing with the result of biofilm formation, the same trend was observed where the biofilm increases as the concentration increases. However, at 24 h the reduction of biofilm was clearly observed at 20 wt. % which in agreement with WST result. Although, laser micrographs images in Figure S2 (Supplementary Materials) shows the reduction of the biofilm formation as the concentration of AlN increases at 24 h.

In CV-stained experiment, the absorbance of *E. coli* increases as the concentration increases even at 20 wt. % as illustrated in Figure 11c,d. However, the absorbance shows a decrement at 20 wt. % compared with other concentrations. In the viability test, the biomass of *E. coli* reduces as the concentration increases but shows sudden spike at 20 wt. % for 12 h. However, at 24 h, a clear trend was observed where the biomass starts to reduce after 5 wt. % and lowest at 20 wt. %.

biomass shows slight increment up to 15 wt. % and drastic drop at 20 wt. %.

patches of biofilm and it start to reduce as the concentration of AlN increases and at 20

To reconfirm the antibacterial property, the samples were analyzed using Crystal violet (CV) staining method. The stained samples were analyzed with WST and Viability Staining, and ImageJ Analysis and the results were shown in Figure 9 The CV-stained samples show high absorbance at 12 h and start to reduce slightly at higher concentration especially at 15 and 20 wt. %, at 24 h the absorbance does not increase and was maintained as 0 wt. % sample but at 20 wt. % it decreases drastically. When compare with viability staining method, it shows the similar trend as the previous WST method where the biomass increases up to 15 wt. % and decreases at 20 wt. % for 12 h, whereas, at 24 h the

wt. % and very low amount of biofilm was detected.

**Figure 9.** WST adsorption after 12 and 24 h of testing with *Staphylococcus epidermidis* on the CA/AlN composites, as a function of the fraction of AlN. **Figure 9.** WST adsorption after 12 and 24 h of testing with *Staphylococcus epidermidis* on the CA/AlN composites, as a function of the fraction of AlN. *Antibiotics* **2021**, *10*, x FOR PEER REVIEW 12 of 17

**Figure 10.** WST adsorption after 12 and 24 h of testing with *Escherichia coli* on the CA/AlN composites, as a function of the fraction of AlN. **Figure 10.** WST adsorption after 12 and 24 h of testing with *Escherichia coli* on the CA/AlN composites, as a function of the fraction of AlN.

In CV-stained experiment*,* the absorbance of *E. coli* increases as the concentration in-

decrement at 20 wt. % compared with other concentrations. In the viability test, the biomass of *E. coli* reduces as the concentration increases but shows sudden spike at 20 wt. % for 12 h. However, at 24 h, a clear trend was observed where the biomass starts to reduce

after 5 wt. % and lowest at 20 wt. %.

**Figure 11. (a**) *Staphylococcus epidermidis* biofilm formation after 12 and 24 h evaluated by quantitative measurement of crystal violet staining as an indicator of biomass accumulation on CA/AlN composites, (**b**) Quantitative comparison of accumulated biomass in (**a**), (**c**) *Escherichia coli* biofilm formation after 12 and 24 h evaluated by quantitative measurement of crystal violet staining as an indicator of biomass accumulation on CA/AlN composites, (**d**) Quantitative comparison of accumulated biomass in (**c**). **Figure 11.** (**a**) *Staphylococcus epidermidis* biofilm formation after 12 and 24 h evaluated by quantitative measurement of crystal violet staining as an indicator of biomass accumulation on CA/AlN composites, (**b**) Quantitative comparison of accumulated biomass in (**a**), (**c**) *Escherichia coli* biofilm formation after 12 and 24 h evaluated by quantitative measurement of crystal violet staining as an indicator of biomass accumulation on CA/AlN composites, (**d**) Quantitative comparison of accumulated biomass in (**c**).

#### **4. Discussion 4. Discussion**

The results of the laser microscope, FTIR and Raman spectroscopy clearly show the surface morphology and chemical composition of the CA/AlN composites. The surface roughness increases as a function of AlN concentration. Raman spectra clearly showed the presence of AlN particles which were hard to observe by FTIR. There was no observable alteration in the chemical bonds of both CA and AlN during the casting process which were clearly seen from FTIR and Raman spectroscopy. The results of the laser microscope, FTIR and Raman spectroscopy clearly show the surface morphology and chemical composition of the CA/AlN composites. The surface roughness increases as a function of AlN concentration. Raman spectra clearly showed the presence of AlN particles which were hard to observe by FTIR. There was no observable alteration in the chemical bonds of both CA and AlN during the casting process which were clearly seen from FTIR and Raman spectroscopy.

The mechanical properties of the composites are affected by AlN concentration: the ultimate strength, the elongation and the toughness increase with a low concentration of AlN of 5 wt. %, then decreases with AlN concentration of 10 wt. % and above. The Young modulus of the composite also increases with AlN contents of 5 and 10 wt. %, then decreases when the AlN concentrations reach 15 and 20 wt. %. The mechanical behaviors of the CA/AlN composites could be explained by the phenomenon called "mechanical percollation" [33]. Generally, in the polymer/filler composite system, the mechanical properties of the composite will increase until the filler concentration reaches a critical value, and then decreases with further filler content. The high concentration of the AlN particles could lead to the formation of agglomerates in the polymer matrix, affect the homogeneity of the CA/AlN composites, and causing lower mechanical properties. The mechanical properties of the composites are affected by AlN concentration: the ultimate strength, the elongation and the toughness increase with a low concentration of AlN of 5 wt. %, then decreases with AlN concentration of 10 wt. % and above. The Young modulus of the composite also increases with AlN contents of 5 and 10 wt. %, then decreases when the AlN concentrations reach 15 and 20 wt. %. The mechanical behaviors of the CA/AlN composites could be explained by the phenomenon called "mechanical per-collation" [33]. Generally, in the polymer/filler composite system, the mechanical properties of the composite will increase until the filler concentration reaches a critical value, and then decreases with further filler content. The high concentration of the AlN particles could lead to the formation of agglomerates in the polymer matrix, affect the homogeneity of the CA/AlN composites, and causing lower mechanical properties.

The thermal properties were affected by the presence of reinforcing powders: the Tm and Tg values shows an increment. These increments could be attributed to the presence of AlN particles in the composite system. Due to the agglomeration of the filler particles, The thermal properties were affected by the presence of reinforcing powders: the T<sup>m</sup> and T<sup>g</sup> values shows an increment. These increments could be attributed to the presence of AlN particles in the composite system. Due to the agglomeration of the filler particles,

the mobility of the polymer chains is reduced. In order to mobilize the polymer chains,

the mobility of the polymer chains is reduced. In order to mobilize the polymer chains, more energy is required, leading to the increase in the T<sup>m</sup> and T<sup>g</sup> values [34]. Whereas the degree of crystallinity of the composite material reduces when there is presence of AlN. However, the concentration does not affect the degree of crystallinity. The value was almost similar from 5 wt. % to 20 wt. %. Most importantly, this shows AlN can be a good reinforcing material.

The antibacterial properties were clearly shown in the Results chapter; however, it was difficult to observe the effect played by the increasing concentration of AlN which due to the releasing of active antibacterial component from the substrate. At higher concentrations, the antibacterial effect for both Gram-positive (*S. epidermidis)* and Gram-negative (*E. coli*) at 24 h was clearly observed. Secondly, the surface roughness at 20 wt. % might be the reason of the antibacterial effect which is expected to the liberation of ammonia (NH3) from the surface of composite material into the aqueous solution upon exposing to water. The high surface roughness promotes more surface area which causes the bacteria to become exposed to the AlN and die. In total, 1 mole of AlN reacts with 3 mole of water and produces 1 mole of aluminum hydroxide and 1 mole of (NH3). This reaction was considered as the overall hydrolysis reaction as given below [11,31,32,35–38]:

$$\text{AlN} + \text{3H}\_2\text{O} \rightarrow \text{Al} \text{ (OH)}\_3 + \text{NH}\_3 \tag{2}$$

However, according to Bowen et al. [39] the hydrolysis of AlN in room temperature, the reaction can be classified into three processes as stated in following equations:

$$\text{AlN} + 2\text{H}\_2\text{O} \rightarrow \text{AlCOOH}\_{\text{(amorphous)}} + \text{NH}\_3 \tag{3}$$

$$\text{NH}\_3 + \text{H}\_2\text{O} \rightleftharpoons \text{NH}^+\_4 + \text{OH}^-\tag{4}$$

$$\text{AlOOH}\_{\text{(amorphous)}} + \text{H}\_2\text{O} \to \text{Al} \text{ (OH)}\_3 \tag{5}$$

The release of ammonia reacts with the water to produce ammonium ions and hydroxide ions. The ammonium ions and ammonia release are responsible for the antibacterial property for the composite. There are few studies that have shown the antimicrobial property of ammonium salts. On the other side, the volatile ammonia (NH3) gas release is expected to directly attack the structure of DNA of microorganisms [40–43]. Kleiner focused on the review of transportation of ammonia in bacteria and fungi which explains why bio membranes are highly permeable to free ammonia [39]. In another comprehensive study, the author claimed the ammonium ion, (NH<sup>4</sup> + ) can only diffuse into the cytoplasmic space through ion channels and the tiny (NH3) molecules can freely penetrate through the membrane [36,40–43]. Therefore, based on the previous studies and the results obtained, it can be speculated that the mechanism of antibacterial action is the elution of ammonia (NH3) and ammonium ion (NH<sup>4</sup> + ) during hydrolysis of AlN, as shown in Equations (1)–(3), diffuses into the bacterial cell and damages the DNA as well as causing cell lysis [35–43].

The amount of ammonium ion (NH<sup>4</sup> + ) and ammonia (NH3) released will determine the level of toxicity, thus a small preliminary experiment was conducted to measure the amount of NH<sup>4</sup> <sup>+</sup> and NH<sup>3</sup> using a Quick Ammonia Meter AT-2000 instrument and the results were shown in Figure S3. The experiment was conducted at different time intervals of 2, 6, 12 and 24 h using the 20 wt. % of CA/AlN composite. The results demonstrate the release of NH<sup>4</sup> <sup>+</sup> and NH<sup>3</sup> are very limited, and even at 24 h the maximum level of NH<sup>4</sup> <sup>+</sup> and NH<sup>3</sup> are 0.44 mg/L and 0.28 mg/L respectively. Figure S4 shows the differences of the amount of NH<sup>4</sup> <sup>+</sup> and NH<sup>3</sup> release by pure AlN powder and 20 wt. % of CA/AlN composite. The results clearly show the release of NH<sup>4</sup> <sup>+</sup> and NH<sup>3</sup> is controlled by the CA substrate and the generation of aluminum hydroxide on the surface when exposed with water as shown in Equations (2)–(5) play a role to slow down the release. Therefore, the level of toxicity is much lower when compared to pure AlN powder. Secondly, the intended application of these CA/AlN composite is not for ingestion or biomedical devices: it is intended to be used as tablecloth or food wraps which are not in direct contact with the human body environment.

#### **5. Conclusions**

The surface characterization of the composite material shows an increment in surface roughness due to presence of AlN, that could also be identified by Raman.

The mechanical strength of the composite was reduced at AlN fractions >10 wt. %. On the other hand, the Young's modulus showed an increase up to 10 wt. % and a decrease at a higher concentration. In addition, a clear decrement observed in toughness upon increasing concentration of AlN was observed.

The melting temperature (Tm) and glass transition temperature (Tg) increased with increasing AlN concentration, showing that the thermal properties of the CA/AlN composites were improved in the presence of AlN.

The CA/AlN composites showed antibacterial effects for both the Gram-positive and Gram-negative bacteria at higher concentration of AlN due to the reaction of AlN with water to produce ammonia (NH3) and ammonium ions, which caused lysis by disruption of bacterial cell membrane.

In conclusion, this could be a promising material to replace plastic bags, food packaging or other plastic products thanks to improved antibacterial and thermal property.

Future research will focus on the degradability and stability of this composite material.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/antibiotics10111292/s1, Figure S1: Microscopic images of Staphylococcus epidermidis biofilm formation on the different CA/AlN composites after 12 h (a–e), 24 h (f–j), Figure S2: Microscopic images of Escherichia coli biofilm formation on the different CA/AlN composites after 12 h (a–e), 24 h (f–j), Figure S3: Quantification of Ammonium ion and Ammonia released during hydrolysis process of AlN at different time interval, Figure S4: Comparison of Ammonium ion and Ammonia released by AlN powder and 20 wt. % CA/AlN composite.

**Author Contributions:** Conceptualization, T.P.M.S.; methodology, T.A.; validation, T.A., G.P., E.M. and W.Z.; formal analysis, T.P.M.S., T.H. and H.N.D.; investigation, F.B., T.P.M.S. and T.H.; resources, T.P.M.S.; data curation, H.N.D., F.B., E.M. and W.Z.; writing—original draft preparation, T.P.M.S.; writing—review and editing, G.P., E.M., H.N.D., F.B. and W.Z.; visualization, K.K.; supervision, E.M., W.Z. and G.P.; project administration, T.P.M.S., E.M. and G.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research project did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

