Antibacterial efficacy of surface aluminum oxide nanostructures produced by hot water treatment

This study utilizes a hot water treatment (HWT) method for introducing antibacterial properties to aluminum (Al) surfaces, which has relevance in several industries ranging from food packaging and ventilation systems to biomedical materials. The HWT process can produce a nanostructured oxide layer on a wide range of metallic materials by simply immersing the metal in water at temperatures ranging from 75 °C to 95 °C. In this work, Al foil was treated in deionized (DI) water for 5 min at various temperatures, including 75 °C, 85 °C, and 95 °C. Concentrations of Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus epidermidis (S. epidermidis) were placed on Al foil for different times, ranging from 30 seconds to 60 min The survival time was measured, and the analysis of the results indicates a direct correlation between when the bacteria was exposed to HWT Al foil and the number of bacteria killed. As the temperature of the HWT increased, there was an increase in antibacterial activity. This finding is consistent with our expectations; at higher HWT temperatures, more prominent nanostructures are produced, causing increased inactivation of bacteria. Our results show the nanostructured HWT Al foil was superior at inactivating Gram-negative (E. coli) and Gram-positive (S. epidermidis) bacteria compared to the untreated control Al foil. HWT Al foil treated at 75 °C, 85 °C, and 95 °C was 58%, 64%, and 73% more effective in killing the Gram-negative bacteria, respectively, after only 30 seconds of contact time compared to untreated control Al foil, while the antibacterial efficacy was enhanced 88%, 92%, and 94% for the Gram-positive bacteria, respectively. The HWT nanostructures synthesized at 95 °C, after 60 min of contact time, were able to inactivate 97% of the gram-negative bacteria and 100% of the gram-positive bacteria, demonstrating the efficacy of its antibacterial properties. This research presents a novel, inexpensive, and environmentally friendly method of producing nanostructures that inhibit bacterial activity.


Introduction
With the increase in bacterial infections and biofilm formation, which can and often do grow on medical devices such as crutches, walkers, and surgical instruments, as well as the losses attributed to foodborne illnesses, nosocomial infections, and emerging antibiotic resistance, it has become imperative to find alternative mechanisms to combat this issue. Biocides have been used for many decades, and due to overuse without complete deactivation, bacteria have been able to develop resistance. This resistance causes that same biocide to become ineffective at killing the newly developed strains of bacteria [1,2]. The waste products of biocides also present an environmental danger, as well as an imminent health danger to humans, in the form of runoffs. Irradiation and heat are commonly used to sterilize items, while metals and their antimicrobial properties are often overlooked. Even though irradiation and heat are frequently used to kill bacteria, there are drawbacks. Irradiation does not guarantee food safety as it does not eliminate all toxins and poses health risks such as chromosomal abnormalities and various cancers. It is also expensive for manufacturers to install irradiation equipment, and it often has a deleterious effect on the nutrients in food [3]. Heat is used in the industry, but it has multiple disadvantages, such as some bacteria being thermophiles, meaning they can survive at extremely high temperatures. And, as with irradiation, heat may compromise the flavor and freshness of food [4].
Bacterial growth on wheelchairs and walkers has received very little attention, though research has repeatedly shown the extent of this issue. Multiple types of bacteria, including MRSA (Methicillin-resistant Staphylococcus aureus), have been found on these medical devices [5]. Attention also needs to be given to the continuation and escalation of bacterial resistance, which has led to the nosocomial (healthcare-acquired) infection rate remaining higher than expected in healthcare facilities; MRSA is one such example [6,7].
Another pressing concern is bacterial attachment to surfaces and the formation of biofilms, creating a protective environment conducive to the growth of bacteria, leading to infection and contamination. Preventing bacterial attachment is the most effective methodology to combat these issues [5]. Hot water-treated (HWT) aluminum (Al) destroys the bacteria as it begins to attach to surfaces [7,8]. HWT is simple, low-cost, scalable, environmentally friendly, and does not release environmental pollutants [9]. The antimicrobial properties of metals have been beneficial to humanity for thousands of years; however, the toxicity of some metals and the high cost of production of many others have made them ill-suited for widespread use by humans. On the other hand, HWT Al does not present these limitations as the process is inexpensive and non-toxic [10].
The antibacterial properties of several metals are primarily due to the release of metal ions from the surface of the metal. These ions can puncture holes in the bacterial cell membrane, exposing its genome to dangerous conditions outside the membrane [11]. Studies also show metal oxide nanostructures can kill bacteria through oxidative stress, protein dysfunction, and membrane damage [12]. Two mechanisms are commonly proposed for nanostructures' ability to kill bacteria: Contact killing and deactivation via reactive oxygen species (ROS). Contact killing proceeds by a mechanism whereby bacteria attach to the nanostructures causing the puncturing of the cell membrane; this causes the cell to rupture, leading to cell death [13]. Reactive oxygen species (ROS) can kill bacteria directly by inducing oxidative stress to the bacterial cell, which inhibits bacterial growth by disabling the DNAs' ability to replicate. This process causes the bacteria to self-destruct due to the bacterial cell's stress as it engages with the reactive oxygen species [14,15].
Food packaging and containers have become necessary in food purchases over the past century. The food is packaged with the aim of food preservation and storage. Metal is a common material of choice in the food industry because it preserves food and beverages [16]. The preferred metal in the food industry is Al due to its lightweight, low cost, and capacity to be recycled. Al foil is highly flexible and can preserve and protect food in most environments [17]. Previous studies have shown the capability of Al foil to reduce pathogens and foodborne illnesses [18,19].
In this study, hot water treatment was used to enhance the antibacterial properties of Al foil by creating nanostructures on the surface of the foil, which kills the bacteria faster than untreated Al foil. Considering the need and usefulness of antibacterial metals, the present study was taken to analyze the antibacterial properties of nanostructured Al foil and the time dependency of the inactivation [8,12,19]. Our HWT nanostructures were tested against two types of bacteria, Gram-negative E. coli and Gram-positive S. epidermidis, as examples.

Materials and methods
Fabrication of nanostructured aluminum foil For HWT, 2 × 2 cm 2 Al foil (Reynolds Wrap Heavy Duty Aluminum Foil) coupons were placed in a beaker containing DI water with a resistivity of 18.2 MΩ cm and temperature of 75°C [13,18]. The treatment was carried out for 5 min After the hot water treatment, the Al foil was dried for 75 min in ambient air and tested for bacterial growth immediately after. Similar HWT experiments were repeated for water temperatures of 85°C and 95°C. The morphology of the samples was characterized utilizing the JEOL JSM-7000F scanning electron microscopy (SEM) at 50 K magnification [19].
Bacterial strains and growth conditions K-12 Escherichia coli cells and Staphylococcus epidermidis were inoculated into the LB broth and incubated for twenty-four hours at 37°C. The spectrophotometer was used to determine the initial bacteria count. 50 μl of bacterial culture was placed on each HWT 2 × 2 in 2 Al foil coupon. The bacteria were left on the Al foil for different durations ranging from 30 seconds to 60 min to test the effect of contact time on antibacterial performance. The Al foil containing the bacteria was placed in 10 ml sterile tubes containing phosphate buffer solution (PBS). The tubes were shaken using a vortex for 2 min at medium speed. The Al foil was removed from each tube, then disposed of properly. The serial dilution was done with the bacteria culture. 100 μl of bacteria was added in 900 μl of PBS. Then, 100 μl of each dilution was plated onto a nutrient agar plate brought to room temperature to prevent temperature shock to the bacteria. Bacteria were spread on the agar plate evenly using a sterile metal spreader. Agar plates were incubated, lid down, at 37°C for 18 h. Colonies were then counted and recorded using ImageJ analysis. This experiment was repeated three times on different days, using three replicates for each experiment. The calculation of bacterial counts was averaged using these data points [8,19].

Results and discussion
Characterization of surface morphology The SEM image of figure 1(a) shows that the surface of the untreated Al was smooth with minimal bumps and grooves, which could have caused due to the foil processing during manufacturing. Results of the Al foil treated at 75°C for 5 min ( figure 1(b)) show interconnected nanostructures, which are called 'nanograss' [17]. As the HWT temperature is increased from 75°C to 95°C, the nanostructure growth also increases, forming more mature structures (figure 1). The nanograss network extends more with sharper features as the HWT temperature increases. This finding is consistent with our earlier work that reported a similar nanograss formation made of aluminum oxide (Al 2 O 3 ) on Al surfaces after HWT [8].

Antibacterial activity
In this study, Escherichia coli and Staphylococcus epidermidis were used as model Gram-negative and Grampositive bacteria, respectively. The initial bacterial count of Escherichia coli cells was 3.1 × 10 9 cells ml −1 . As shown in figure 2, when comparing the untreated Al foil (control), as the contact time increased, more bacteria were killed, perhaps due to the antibacterial properties of the elemental Al to a certain extent [8]. However, antibacterial efficacy was significantly improved when the Al foil was treated with hot water. At 60 min, the 95°C hot water-treated Al foil demonstrated 97% more antibacterial effectiveness than the control. HWT Al foil at 75°C was 58% more effective in killing the bacteria after 30 seconds of contact time than the untreated foil. At 1 min contact time, the 75°C HWT was 79% more effective when compared to the control. When the HWT temperature was increased to 85°C, for 30 seconds and 1 min contact times, the nanostructured HWT Al foils antibacterial properties were enhanced by 63% and 84%, respectively. For 95°C HWT, at 30 seconds and 1 min contact times, the nanostructured Al foil killed 73% and 88% more bacteria, respectively, compared to the control.
As shown in figure 3, the initial bacterial count for Staphylococcus epidermidis was 2.72 × 10 9 cells ml −1 . The 75°C HWT at 30 seconds was 88% more effective when compared to the control. When the time increased to 1 min at 75°C and 85°C, it was more effective than the control by 96% and 97%, respectively. As the temperature increased to 95°C for 30 seconds, it was 93% more effective than the control. And, at 1 min, the HWT was more effective in inactivating S.epidermidis than the control by 98%. When comparing the control to the 95°C HWT Al foil at 60 min. The HWT Al foil was 100% more effective (shown in figure 2). As with the Escherichia coli, the increase in the antibacterial efficacy of the HWT Al foil was directly proportional to the contact time with the S.epidermidis; as the contact time increased, the antibacterial effect of the HWT also increased.
As can be seen clearly from the results summarized above, HWT Al foil was able to kill more bacteria than the untreated foil as the duration of contact time increased, demonstrating the enhanced antibacterial properties of the nanostructured Al foil produced by HWT. The results also show that Gram-positive S. epidermidis had a more significant antibacterial response than Gram-negative E. coli, which may have been due to the nanostructures interacting more with the thicker peptidoglycan layer of S. epidermidis [20]. Additionally, since Gram-negative bacteria have a lipid bilayer outer membrane, they are not likely to be as permeant to any ionic species as Gram-positive bacteria, although there is no experimental evidence to support this notion.
Once the aluminum oxide nanostructures are in contact with the bacteria, they can potentially cause the bacteria to rupture via irreparable mechanical damage to the bacterial membrane, precipitated by the sharp end points of the nanostructures. Also, forming reactive oxygen species (ROS), initiated by the nanostructures, can increase oxidative stress within the bacterial cell [14,15]. The increased levels of free radicals can result in irreparable damage to nucleic acids, proteins, membranes, and organelles, which eventually leads to the  activation of cell death. Either or both of these processes might have contributed to killing the bacteria in our study [19].
This research demonstrates the advantages of using HWT to synthesize a nanostructure surface in a simple, low-cost, scalable, and environmentally friendly manner and to introduce a robust antibacterial property to metallic materials. Utilizing this novel technology has the potential, in the healthcare sector, to decrease the amount of bacterial growth on medical equipment and instruments, leading to a decrease in nosocomial infections and bacterial resistance. This process could impact the years of life lost and positively benefit the healthcare and health insurance sectors by decreasing the financial burden caused by preventable illnesses and deaths. This process also has the potential to benefit the food packaging industry by effectively eliminating up to 100% of foodborne pathogens in a cost-effective manner. This process can decrease illness and death due to foodborne bacteria and economic loss due to spoilage.
In summary, we demonstrated the ability of aluminum oxide nanostructures, produced on the surface of Al foil by a simple hot water treatment method, to kill bacteria rapidly after a short contact time of seconds to minutes. HWT Al foil could deactivate Gram-positive bacteria faster than Gram-negative bacteria. The efficacy of the HWT Al foil was further enhanced by increasing the contact time and temperature of the hot treatment. HWT Al foil with an extended nanograss layer after 5 min of HWT at 95°C resulted in ∼97% antibacterial efficacy for E. coli and 100% antibacterial efficacy for S.epidermidis after 60 min of contact time. Our results indicate that the morphological enhancements achieved by the nanostructured Al foil led to superior antibacterial properties. Once the nanostructures are in contact with the bacteria, they can cause irreparable mechanical damage to the bacterial membrane, precipitated by the jagged end points of the nanostructures. This contact inevitably can lead to the loss of viability in E. coli and S. epidermidis. The high surface area of the HWT nanostructures is also believed to be facilitating the generation of reactive oxygen species (ROS), another potential mechanism leading to the inactivation of bacteria. ROS can kill bacteria via oxidative damage to the bacteria cell membrane and nucleic material, preventing the ongoing replication of bacteria [14,15].

Conclusion
The increased number of bacterial infections and deaths, along with the bacteria's growing resistance to antibiotics and biocides, is a major public health concern with a need for immediate alternative solutions. This study presents an aluminum oxide nanograss layer produced on the surface of Al foil by a simple hot water treatment method to kill bacteria. This novel, environmentally safe, low-cost, and practical method of producing nanostructures via hot water treatment for the inhibition of bacteria provides an alternative mode of action to antibiotic-resistant bacteria. Al foil antibacterial activity was enhanced via the HWT method. Our results reveal HWT Al foil could deactivate Gram-positive bacteria faster than Gram-negative bacteria, which is highly significant due to the Gram-positive thicker cellular wall. The efficacy of the HWT Al foil was further enhanced by increasing the contact time and temperature of the hot treatment. HWT Al foil with an extended nanograss layer after 5 min of HWT at 95°C resulted in ∼97% antibacterial efficacy for E. coli and 100% antibacterial efficacy for S.epidermidis after 60 min of contact time. This is to be expected from a combination of both physical and chemical mechanisms. Our hot water treatment method can decrease bacterial infections and the formation of biofilm formation on the Al surface, which in turn can improve the quality of life.
Future research is being considered in medical supplies, such as wheelchairs and walkers. The immunocompromised group could greatly benefit from our HWT process's added protection. Another potential research area is using our HWT method to produce nanostructures on food preparation surfaces and food packaging material to create a layer of protection for the consumer. This procedure also presents the added benefit of revenues gained by producers by decreasing the quantity of product lost to bacteria during the packaging and delivery phase. The HWT approach represents one vehicle that can be effectively utilized in the ongoing battle against bacteria and antibacterial resistance [32][33][34][35][36][37][38][39][40][41][42].