Bio-Inspired Graphene Nanomaterials: Synthesis and Characterization for Ambient Microbial Destruction

doi:10.21203/rs.3.rs-5281348/v1

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Bio-Inspired Graphene Nanomaterials: Synthesis and Characterization for Ambient Microbial Destruction

https://doi.org/10.21203/rs.3.rs-5281348/v1

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This study explores the conversion of carbon-rich coconut waste into bio-inspired graphene oxide (GO) and reduced graphene oxide (rGO) for application as antimicrobial agents, addressing air quality and public health concerns. Both GO and rGO demonstrated significant antimicrobial properties against a variety of airborne microorganisms. GO achieved nearly complete inhibition of bacteria, such as Staphylococcus spp., and fungi, including Aspergillus spp., at low concentrations. Similarly, rGO effectively inhibited Escherichia coli and Brucella spp., highlighting its broad-spectrum antimicrobial activity. The synthesis of GO was performed using an enhanced Hummers' method, producing nanomaterials with distinct surface functionalities, which were characterized by X-ray diffraction (XRD) and Field Emission Scanning Electron Microscopy (FESEM). XRD analysis revealed a notable shift from the sharp graphite peak at 2θ = 26.4°, indicating the (002) plane, to a broadened peak at 2θ = 11.15° in GO, signifying successful oxidation and exfoliation of graphite. For rGO, the disappearance of the 11.15° peak confirmed the partial restoration of graphitic structure. FESEM imaging displayed the nanosheet morphology of GO and rGO, along with microscopic analysis revealing the physical interaction of GO with bacterial and fungal cells, enabling precise identification of microorganisms. Moreover, the GO-coated surfaces exhibited higher surface roughness, promoting bacterial adhesion, which underscores the critical role of surface properties in antimicrobial efficacy. This research contributes to the Sustainable Development Goals (SDG 3: Good Health and Well-being, and SDG 11: Sustainable Cities and Communities) by providing an eco-friendly solution for mitigating microbial pollution in the ambient environment through the use of bio-based nanomaterials.

Biological sciences/Biochemistry

Earth and environmental sciences/Environmental sciences

Physical sciences/Materials science

Physical sciences/Nanoscience and technology

Scientific community and society/Social sciences

Microbial growth in ambient environment aerosols poses a health and environmental risk (Górny, 2020). The ambient aerosols, which typically contain particulate matter, serve as carriers for various microorganisms, including bacteria and fungi, posing a significant health risk. Microbial aerosols can contribute significantly to the overall aerosol mass in the atmosphere, with estimates suggesting they can account for up to 25% of the total (Li et al., 2019; Kumar et al., 2022). Those pathogens can survive for quite a long time in aerosols, thus providing their spread under favorable conditions and giving an increased chance of human exposure. The outbreak of the coronavirus (COVID-19) pandemic in late 2019 highlighted the critical importance of controlling microbial growth and transmission in the ambient environment. The global outbreak of the SARS-CoV-2 virus, marked by an alarming number of cases and deaths (WHO, 2020), served as a stark reminder of the critical role played by airborne transmission in the dissemination of infectious diseases, stressing the importance of adopting stringent control measures to prevent the unchecked growth of microorganisms in the air (Morawska & Cao, 2020; Biswas & Dhawan, 2022). Particulate matter creates an optimal environment for microbial adhesion and survival. These aerosols not only transport harmful microbes, but also promote their stability and dispersion in the atmosphere. These bioaerosols have since been linked to a variety of respiratory and cardiovascular ailments, exacerbating the public health crisis (Kumar et al., 2022). Microbial growth in ambient environment aerosols is influenced by various factors, including temperature, humidity, and the presence of nutrients. In urban areas, the high concentration of pollutants and particulate matter can provide a conducive environment for microbial growth (Joshi et al., 2020). As urban populations and industrial activities continue to grow, the amount of waste produced has also seen a significant rise, providing a fertile ground for microorganisms to grow and multiply (Sharma et al., 2019). Additionally, future pandemics caused by newly developing infections and the misuse of bioweapons underscore the need for novel approaches to reducing airborne microbial threats (Ukuhor, 2020; Hashmi et al., 2022). Traditional methods of microbial control, such as chemical disinfectants and antibiotics, have several limitations, including the development of resistance and environmental toxicity (Zhang et al., 2020; Cesare et al., 2020). Therefore, there is a need for cost-effective and environmentally friendly alternatives.

Graphene and its derivatives, including graphene oxide (GO) and reduced graphene oxide (rGO), have shown remarkable antimicrobial properties against a broad spectrum of pathogens (Liu et al., 2011; Yousefi et al., 2017). The potent antibacterial activity of GO and rGO can be attributed to their extensive surface area, sharp edges, and the ability to generate oxidative stress, which disrupts microbial cells. GO, in particular, stands out as a promising antimicrobial agent due to its distinctive physicochemical characteristics. Graphene itself, composed of a single layer of carbon atoms arranged in a hexagonal lattice, offers exceptional mechanical strength, electrical conductivity, and surface area. These properties enable GO to effectively compromise microbial cell membranes, thereby inhibiting the growth of various pathogens, including both bacteria and fungi (Zhao et al., 2010; Chen et al., 2014; Palmieri et al., 2017; Sengupta et al., 2019; Andrews et al., 2024).

The bio-based technique will utilize biological resources and green chemistry principles, making the synthesis of such nanostructures less expensive and more sustainable than standard approaches. These methods limit the use of hazardous chemicals while increasing the scalability and practicality of manufacturing high-quality graphene nanomaterials. Recent advancements in the synthesis of graphene-based nanomaterials from waste materials offer an environmentally friendly and cost-effective approach to addressing microbial contamination (Morones et al., 2005; Deng et al., 2016; Malik et al., 2022; Balkourani et al., 2022). The use of waste as a raw material offers a dual benefit, reducing production expenses while providing a solution to the mounting issue of waste disposal. This approach aligns with the principles of sustainability and circular economy, making it an attractive option for large-scale implementation.

The primary objective of this research is to develop graphene-based nanomaterials synthesized from waste for the inhibition of microbial activity in ambient environment aerosols. This study will explore the synthesis process, characterize the properties of the resulting nanomaterials, and evaluate their antimicrobial efficacy against various pathogens commonly found in aerosols. By integrating waste-derived graphene nanomaterials into environmental control strategies, we aim to provide a sustainable and effective solution to mitigate microbial contamination and enhance public health protection.

Site Description: Dayalbagh, the experimental study site, is a suburban neighborhood in Uttar Pradesh, India, near the city of Agra. Situated on the southern bank of the Yamuna River, Agra is a city rich in history and culture that is most famous for the UNESCO World Heritage Site that is the Taj Mahal. Situated roughly 10 km northwest of Agra's city center, Dayalbagh is a calm and attractive suburb that offers a distinctive combination of urban and rural features. With a growing population of about 50,000 people, the site is characterized by a mix of residential, commercial, and agricultural land uses. Because of its subtropical plains environment, which features hot summers and mild winters, the area is ideally suited to research the dynamics of microbial aerosols in an ambient environment.

Sampling and Microbial Culturing: Aerosol samples were obtained using two Envirotech air samplers, APM 460 and APM 550, which were set up to gather PM₁₀ and PM_2.5 fractions, respectively. Take note of the starting weight (W₁) of the clean, dry filter. To correctly measure the weight of the filter, where used a precision analytical balance with suitable sensitivity. The samplers were fitted with PTFE filter sheets, and the airflow rate was set to 1.0 m³ min^-1. The sampling time was 24 hr to get a representative sample of aerosols in the ambient environment. After the sampling interval, carefully remove the filter from the sampler and place it in a desiccator to remove any moisture before reweighing. Proceed with the concentration calculation once the filter has reached a constant weight (W₂) after the desiccator process.

Calculation of PM Concentration:

Calculated the PM concentration using the following formula:

PM concentration (μg m^‑³) = ((W₂ - W₁) / V)

Where:

W₂ = Weight of the filter after sampling and deccicator process (μg)

W₁ = Initial weight of the filter before sampling (μg)

V = Volume of air sampled (m³)

t = Sampling time (hours)

After this, one fourth portion of the filter paper was cut into small pieces and transferred to a conical flask containing 25 ml of ultra-pure water. The mixture was then shaken on an electrical shaker to facilitate the extraction of aerosol components. Subsequently, the sample was sonicated using a Sarthak Scientific Ultrasonic Bath (Sonicator) for 15 minutes to ensure complete extraction of biological components. The sonicated sample was then filtered using Whatman 42 filter paper to remove any particulate matter, and the filtered aliquots were used for microbial analysis. The aqueous extracts of aerosols were used to study the biological components of aerosols, including bacteria and fungi. Laminar flow culture techniques were employed to detect and culture these microorganisms. The cultures were incubated in a BOD incubator to facilitate growth.

Culture Media Preparation for bacteria & fungi: The preparation of media is essential for the isolation and cultivation of fungi and bacteria from aerosol samples. For fungal cultivation, Sabouraud Dextrose Agar (SDA) solution was prepared by dissolving 40 grams of dextrose, 0.01 grams of chloramphenicol, and 10 grams of peptone in 1 liter of distilled water. Chloramphenicol inhibits bacterial growth, while the peptone supports fungal growth. In a separate conical flask, 15 grams of agar was added. The dextrose solution (250 ml) was combined with the agar, and the mixture was autoclaved at 121°C for 20 minutes. After cooling to 45°C-50°C, the media was poured into sterile Petri dishes and allowed to solidify.

For bacterial cultivation, Nutrient Agar Media (NAM) was prepared with similar sterilization steps, including washing glassware with 70% ethanol. A beef extract solution was prepared by dissolving 3 grams of beef extract, 5 grams of sodium chloride (NaCl), and 5 grams of peptone in 1 liter of distilled water. Separately, 15 grams of agar was added to a conical flask. The beef extract solution (250 ml) was combined with the agar, and the mixture was autoclaved at 121°C for 20 minutes. After cooling to 45°C-50°C, the media was poured into sterile Petri dishes and allowed to solidify. The prepared SDA and NAM media were then ready for cultivating fungi and bacteria from aerosol samples, respectively.

After preparing the Nutrient Agar Media (NAM) for bacteria and Sabouraud Dextrose Agar (SDA) media for fungi, the next step was to inoculate the media with the aerosol sample and incubate it under controlled conditions. To ensure sterility and prevent contamination, all procedures were performed in a laminar flow hood. The laminar flow hood was turned on for at least 30 minutes before use to allow the air to circulate and remove any particles. The hood was then wiped down with 70% ethanol to remove any residual contaminants.

Sterile Petri plates were removed from the autoclave and placed in the laminar flow hood. A small amount of Parafilm tape was used to secure the lid of each plate, ensuring that it was not too tight or too loose. This allowed for gas exchange while preventing contamination. By using a sterile micro pipette, 100 µl aerosol sample was added to the center of each Petri plate. The sample was then spread evenly across the surface of the media using a sterile spreader or inoculation loop. The plates were gently rotated to ensure uniform distribution of the sample. The inoculated Petri plates were then incubated under specific conditions for bacteria and fungi. For bacterial cultures, the plates were incubated at 37°C for a duration of 24 to 48 hours. In contrast, fungal cultures were incubated at 25°C for a longer period, ranging from 5 to 7 days.

Microscopic Analysis of microbial components: For microscopic analysis of microbial components, a sterilized wire loop and needle were used to transfer a small sample of the bacterial or fungal culture onto a clean microscope slide, spreading it evenly. The slide was then stained using a series of dyes, starting with crystal violet (crystaline blue) for 1-2 minutes to stain the bacterial cell walls, followed by iodine for 1-2 minutes to enhance the staining, and then washed with distilled water. The slide was then washed with 95% ethanol to remove excess dye and dehydrate the sample, and finally counterstained with saffranin for 1-2 minutes. A cover slip was then carefully placed over the inoculated area to create a slide. The prepared slide was then examined under a microscope (Motic Microscope) using bright field or phase contrast illumination, with magnification 100x, and the microorganisms were observed and characterized based on their morphology, size, shape, and arrangement.

FESEM Analysis of Microbial growth: For the Field Emission Scanning Electron Microscope (FESEM) analysis of microbial samples using the JEOL JSM-IT800, the sample preparation protocol is as follows:

Initially, samples are fixed in a 2.5% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.2) for duration of 2 to 4 hours. After fixation, samples are thoroughly washed three times with 0.1 M phosphate buffer to remove excess fixative. Subsequently, the samples undergo post-fixation in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 to 2 hours, followed by another series of washes. To prepare the samples for imaging, they are dehydrated through a graduated series of ethanol solutions, gradually increasing in concentration. This step is crucial to ensure that all water is removed. The dehydrated samples are then critical point dried to eliminate any residual ethanol, preventing sample shrinkage during the imaging process. Once dried, the samples are mounted on stubs using a conductive adhesive. To enhance conductivity and minimize charging effects during FESEM analysis, a thin layer of metal with platinum, is sputter coated onto the mounted samples. This meticulous preparation ensures high-quality imaging and accurate analysis of microbial structures.

Extraction of Graphene from Coconut Shell: The methodology (Figure 5) for extracting graphene from coconut shells begins with the collection and preparation of dry coconut shells, which are cleaned thoroughly to remove any residual organic matter or impurities. These shells are then crushed into smaller pieces for increasing the surface area to facilitate subsequent processing steps. The crushed coconut shell pieces are subjected to thermal degradation through pyrolysis in an inert atmosphere, typically nitrogen, at temperatures ranging from 500°C to 800°C (Tsamba et al., 2006). This process converts the organic material into carbon-rich char, also known as activated charcoal. The resulting activated charcoal undergoes sonication in an aqueous medium, utilizing an ultrasonic bath or probe to break down the particles further, ensuring a uniform size distribution. This sonication step enhances the dispersion of the charcoal particles, which is crucial for the oxidation process.

The conversion of activated charcoal to reduced graphene oxide (rGO) involves several steps. First, the activated charcoal is mixed with concentrated sulfuric acid (H₂SO₄) and potassium permanganate (KMnO₄) in a controlled environment, allowing the KMnO₄ to oxidize the charcoal at a temperature of around 45°C over several hours (Poh et al., 2012). This process introduces oxygen-containing functional groups between the carbon layers, converting the activated charcoal into graphite oxide. The graphite oxide is then exfoliated by adding it to water and sonicating it, resulting in a colloidal suspension of graphene oxide (GO) in water. To convert the GO to rGO, the suspension is treated with hydrogen peroxide (H₂O₂) as a reducing agent at room temperature. The reduction process involves the gradual addition of H₂O₂ to the GO suspension, reducing the oxygen-containing functional groups and restoring the sp² carbon network of graphene.

The resulting rGO is then purified by repeated washing with deionized water to remove residual acids and oxidizing agents. The purified rGO is filtered using a vacuum filtration system to separate the solid rGO from the liquid, and the filtered rGO is collected as a solid residue. Finally, the rGO is dried in a vacuum oven at around 60°C to remove any remaining moisture, resulting in a fine powder.

Characterization of Extracted Graphene Oxide:

FESEM Analysis: The synthesized graphene oxide (GO) in powdered form is initially dried to remove any moisture content by placing it in a vacuum oven at around 60°C for several hours until a consistent weight is achieved, indicating complete dryness. A small amount of the dried GO powder is then carefully placed on a carbon tape affixed to a stub to ensure good electrical conductivity, essential for clear imaging in FESEM (JEOL JSM-IT800). Since GO is a non-conductive material, it is sputter-coated with a thin layer of platinum as a conductive material, by using a platinum coater (JEOL JEC-3000FC Auto Fine Coater), typically for about 30-60 seconds. This coating prevents charging effects and ensures high-resolution images. The prepared sample is then placed into the FESEM chamber, where the instrument is set to the appropriate accelerating voltage, typically between 1-15 kV, depending on the resolution required. High-magnification images are captured to analyze the surface morphology and structure of the GO nanoparticles. The FESEM images provide detailed information about the surface morphology, particle size, and distribution of the GO nanoparticles.

XRD Analysis: The dried GO powder is then ground using an agate mortar and pestle to achieve a fine and homogeneous powder, ensuring the sample is uniformly distributed and free of large agglomerates, which can affect XRD results. The ground GO powder is placed onto a sample holder, typically a flat circular disc, and evenly spread across the surface, gently pressed to form a smooth, flat layer. The prepared sample holder is placed into the XRD instrument (Bruker D-8 Advance), and XRD patterns are recorded over a range of 2θ angles (typically from 10° to 80°) using Cu-Kα radiation (λ = 1.5406 Å). The scan rate and step size are adjusted to obtain clear and well-defined peaks. The XRD patterns are analyzed to identify the crystalline structure and phase composition of the GO nanoparticles.

Antimicrobial Efficacy Testing: The Kirby-Bauer test was performed to evaluate the antimicrobial efficacy of graphene oxide (GO) against Gram-positive and Gram-negative bacteria. The antimicrobial efficacy of graphene oxide (GO) was tested using the Kirby-Bauer method. Clinical strains of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were prepared by touching isolated colony tops with a sterile loop and suspending them in saline. The resulting inoculums were adjusted to 0.5 McFarland units (approximately 1 × 10^8 CFU mL⁻¹) using a densitometer to ensure standardized concentrations. Sterile Mueller-Hinton agar plates were inoculated by spreading 100 µL of each bacterial suspension evenly over the surface with a sterile swab, creating a uniform bacterial lawn. GO samples with high and low oxygen content were sonicated in Millipore water, and 20 µL of each concentration (0.25 mg mL⁻¹, 0.5 mg mL⁻¹, 1 mg mL⁻¹, and 1.5 mg mL⁻¹) was pipetted onto sterile 6 mm Whatman No. 1 filter paper disks. These disks were placed on the agar plates using sterile forceps, and the plates were incubated at 37 ºC for over 24 hours. After incubation, the zones of inhibition around each disk were measured in millimeters, reflecting the antimicrobial activity of the GO against the tested bacteria. This method is based on established protocols (Bauer et al., 1966; Baird-Parker et al., 1970), ensuring reliability and reproducibility in the evaluation of antimicrobial efficacy. The minimum inhibitory concentration (MIC) of GO was determined by identifying the lowest concentration that inhibited bacterial growth. This methodology is based on the standardized protocol described by Bauer et al. (1966) and the Clinical and Laboratory Standards Institute (CLSI, 2019). The results of the Kirby-Bauer test were analyzed according to the guidelines outlined in the Manual of Clinical Microbiology (Jorgensen & Turnidge, 2015).

Airborne Microbial Concentration: The culture plates exhibit varying degrees of microbial colony formation, indicating the presence and proliferation of bacteria and fungi on the particulate matter (PM) samples. The bacterial colonies, visible as small, round spots differing in color and size, suggest the presence of different bacterial species. The fungal colonies appear larger, multi-colored, and exhibit various textures and growth patterns, reflecting a more diverse fungal growth compared to bacterial growth. Figure 1 shows particulate matter concentrations in the study varied significantly, with PM₁₀ levels ranging from 190.6 µg m^-3 to 330.4 µg m^-3 and PM_2.5 levels ranging from 139.1 µg m^-3 to 249.2 µg m^-3. Correspondingly, Bacterial concentrations were observed to range from 850 CFU m^-3 to 2285.3 CFU m^-3 in PM₁₀ and from 757.5 CFU m^-3 to 1999.6 CFU m^-3 in PM_2.5. Fungal concentrations exhibited a similar pattern, with values ranging from 100.1 CFU m^-3 to 325 CFU m^-3 in PM₁₀ and from 35.6 CFU m^-3 to 135 CFU m^-3 in PM_2.5. A higher microbial load associated with larger particulate matter (PM₁₀) compared to finer particles (PM_2.5), highlighting the significant role of particulate matter size in influencing microbial proliferation in ambient aerosols. Boreson et al. (2004) and Alghamdi et al. (2014) demonstrated that PM₁₀ had a higher microbial density than PM_2.5. Raghav et al. (2020) later found that the bacterial concentration in PM₁₀ averaged 405.1 CFU m^-3, while in PM_2.5 it averaged 364.9 CFU m^-3. Additionally, the mean fungal concentration in PM₁₀ was 136.5 CFU m^-³, and 20.3 CFU m^-³ in PM_2.5.

Characterization of Microbial Growth: The characterization of microbial growth on particulate matter (PM) involves microscopic and field emission scanning electron microscope (FESEM) analyses to identify and understand the morphology and diversity of microbial communities (Figure 2). The analysis reveals various pathogenic and non-pathogenic microorganisms, indicating significant health implications for humans exposed to these particles.

The microscopic analysis of particulate matter revealed a diverse array of microbial components. Diplo-Coccus pneumonia appears as spherical bacteria occurring in pairs, typically round and about 0.5-1.25 µm in diameter, known to cause respiratory infections such as pneumonia, transmitted through respiratory droplets from infected individuals as showed in fig. 2a and these properties of Diplo-Coccus was also reported by previous studies (Kadioglu et al., 2008; Leo et al., 2016). Diplo-coccus pneumoniae, commonly known as pneumococcus, is a Gram-positive, alpha-hemolytic, aerobic, encapsulated diplococcus. It typically appears as lancet-shaped pairs of spherical bacteria, each measuring approximately 0.5 to 1.25 micrometers in diameter, as illustrated in Fig. 2a. These characteristics of Diplococcus pneumoniae have been previously documented in studies by Kadioglu et al. (2008) and Leo et al. (2016). This bacterium is a significant human pathogen, primarily transmitted through respiratory droplets from infected individuals, and is responsible for various infections, including pneumonia, otitis media, and meningitis. The bacterium Brucella, a tiny, rod-shaped microorganism with dimensions of 0.5-0.7 µm in width and 0.6-1.5 µm in length, is responsible for causing brucellosis, a zoonotic disease that can be transmitted to humans through exposure to infected animals or by consuming contaminated animal-derived food products. This infection manifests with symptoms such as fever, sweats, and muscle pain (Pappas et al., 2006), as shown in Fig. 2b. Aspergillus, a genus of mold distinguished by its conidiophores and spores of varying sizes, is commonly found in decaying organic matter. It can cause respiratory problems, including allergic reactions and lung infections, especially in immunocompromised individuals, as also reported by Shah and Punjabi (2014), as shown in Figure 2c. Aspergillus fumigatus, a specific species within the Aspergillus genus, produces small conidia measuring approximately 2-3 µm in diameter, as shown in Fig. 2d, commonly found in soil and decaying organic material, it can cause invasive aspergillosis, particularly in individuals with weakened immune systems (Latgé, 1999). Bacillus, as shown in Figure 2e, is a rod-shaped, Gram-positive bacterium typically measuring 0.5-2.5 µm in diameter. Ubiquitous in soil and water, some Bacillus species are pathogenic to humans, causing food poisoning and other infections, as also studied by Mayer & Kronstad (2017) and Aljaafari et al. (2020). Lastly, Paramecium, as shown in Figure 2f, is a unicellular ciliate characterized by its oval, elongated shape, typically measuring 50-300 µm in length. Found in freshwater environments, it feeds on bacteria and algae. While Paramecium is not pathogenic to humans, it is widely used in research due to its biological significance (Görtz, 1988). These microorganisms, found on particulate matter, highlight the significant health risks posed by airborne pathogens in ambient environments.

FESEM Analysis

The characterization of microbial components using Field Emission Scanning Electron Microscopy (FESEM) revealed a variety of bacteria and fungi present in particulate matter samples, highlighting the potential health risks posed by these airborne pathogens. The images include a range of microorganisms, each with distinct morphological characteristics and environmental origins.

The Died Bacteria images (Fig. 2I) depict bacterial cells that have lost viability, often due to environmental stress or antimicrobial treatment. Although these bacteria are non-viable, they can still elicit immune responses and cause inflammation in humans. Aspergillus flavus (Fig. 2 II), known for producing aflatoxins, is a potent carcinogen. This species is characterized by conidiophores and conidia typically 2-4 µm in diameter and is commonly found in soil, decaying vegetation, and stored grains (Klich, 2002). Coccus bacteria (Fig. 2 III), which are spherical and about 0.5-1.25 µm in diameter, are prevalent in various environments, including human skin and the respiratory tract. Pathogenic species such as Staphylococcus and Streptococcus can cause infections in humans (Zondervan et al., 2021). In the fungal category, Aspergillus Germ Tubes are observed as early-stage fungal growth characterized by tube-like structures (Fig. 2 V). These germ tubes, varying in length, can be found in soil and decaying vegetation and can cause aspergillosis in immunocompromised individuals (Latgé, 1999). Rod-shaped Bacteria images (Fig. 2 VI) show bacteria typically 0.5-2.5 µm in diameter, found in soil, water, and human microbiota. These include both pathogenic and non-pathogenic species (Madigan et al., 2006). Vaccinia virus (Fig. 2 VII), used in the smallpox vaccine, is characterized by its complex, brick-shaped structure approximately 300 nm in diameter, providing immunity against smallpox through vaccination (Fenner, 1993). The Bacterial Type images display a variety of bacteria, including both pathogenic and non-pathogenic species commonly found in environmental samples such as air, water, and soil. The presence of Smut Spores (Fig. 2 VIII), which are fungal spores from smut fungi, is notable. These spores are typically spherical to oval and around 5-10 µm in size, and they can cause respiratory issues in humans as well as significant damage to agricultural crops like corn and wheat (Agrios, 2005).

Slime Spores (Fig. 2 IX) produced by slime molds are generally spherical and about 10-20 µm in size. These spores are found in moist, decaying organic matter and are not pathogenic to humans (Stephenson & Stempen, 1994). Curvularia lunata Spores (Fig. 2 XI), crescent-shaped and about 10-20 µm in size, are found in soil and decaying plant material. This fungus is associated with plant diseases and can cause mycoses in immunocompromised individuals (Ellis, 1971). Finally, the Fluffy Surface of Curvularia lunata (Fig. 2 XII) is indicative of its spores. Found in soil and decaying plant material, this fungus is associated with plant diseases and opportunistic infections in humans (Ellis, 1971).

Characterization of Extracted Graphene Oxide:

The extracted graphene oxide (GO) was subjected to a thorough characterization process to elucidate its physical and structural characteristics. High-resolution imaging of the material's surface features was achieved using Field Emission Scanning Electron Microscopy (FESEM), which revealed the surface morphology and particle size of the GO. Furthermore, X-Ray Diffraction (XRD) analysis was performed to examine the crystal structure and phase composition of the GO, providing valuable information about its atomic arrangement and crystalline properties.

FESEM Analysis of Extracted Graphene Oxide:

The FESEM images provided showcase the morphological characteristics of graphene oxide (GO) extracted from coconut waste (Figure 3). These high-resolution images, taken at magnifications of 15,000x and 25,000x, reveal significant details that confirm the successful extraction of GO.

The left image in Figure 3, magnified 15,000 times, displays a layered, flake-like structure typical of graphene oxide. The crumpled and wrinkled sheets are indicative of the two-dimensional nature of GO, consistent with the literature on graphene oxide morphology (Park & Ruoff, 2009). The oxidation process introduces oxygen-containing functional groups, such as hydroxyl, carboxyl, and epoxy groups, onto the graphene oxide (GO) sheets, which significantly influences their structural arrangement. As a result, the material's physical and chemical properties are altered. The oxygen-to-carbon ratio in GO determines the extent of disorder in the sheet stacking, leading to the characteristic overlapping and disordered layers. This distinctive feature of GO is a direct consequence of the presence of these functional groups on the basal planes and edges of the graphene sheets (Dreyer et al., 2010). This increases the interlayer spacing, resulting in the observed expanded and crumpled morphology. The right image, taken at a higher magnification of 25,000 times, provides a more detailed view of the surface texture of the GO sheets. It shows a more pronounced wrinkling and folding, with edges that appear thin and sharp, reinforcing the presence of few-layered graphene oxide. The high magnification reveals the fine, flaky nature of the GO, which further corroborates the successful exfoliation of graphite into graphene oxide. The presence of these wrinkled sheets is essential, as it increases the surface area, a critical factor for applications in sensors, catalysis, and as a reinforcement material in composites (Stankovich et al., 2006). Additionally, the images confirm that the extraction process from coconut waste did not introduce significant impurities or structural defects, which can be inferred from the uniformity and consistency of the GO flakes. The absence of large, irregular particles or non-layered structures suggests a high purity of the extracted GO, which is crucial for maintaining its desirable electrical, thermal, and mechanical properties (Pei & Cheng, 2012).

The extraction method employed likely involved chemical oxidation followed by exfoliation, common steps in the preparation of GO from various carbonaceous precursors (Hummers & Offeman, 1958). Utilizing coconut waste as a precursor offers a dual benefit. On one hand, it provides a cost-effective and sustainable source of raw material. On the other hand, it supports environmental sustainability by repurposing agricultural by-products that would otherwise be discarded, thereby aligning with eco-friendly objectives. This approach highlights the potential for large-scale production of GO with reduced environmental impact compared to traditional methods that use more hazardous chemicals and expensive graphite sources (Dreyer et al., 2010).

XRD Analysis: X-ray diffraction (XRD) analysis is a valuable tool for understanding the structural transformations that occur during the conversion of graphite to graphene oxide (GO) and reduced graphene oxide (rGO), as illustrated in Figure 4. The XRD pattern of graphite is characterized by a sharp peak at 2θ = 26.4°, which corresponds to the (002) plane and indicates a highly ordered crystalline structure, as previously reported by Liang et al. (2009) and Hansora et al. (2015). The XRD pattern of graphite undergoes a significant transformation upon oxidation to graphene oxide (GO). The main peak shifts to a lower angle, from 2θ = 26.4° to 2θ = 11.15°, corresponding to the (001) plane of GO, as reported by Rahmawati et al. (2018). This shift is caused by the introduction of oxygen-containing functional groups, such as hydroxyl, carboxyl, and epoxy groups, which increase the distance between the graphene layers (Hummers & Offeman, 1958; Luo & Zhang, 2018; Yu et al., 2020). The resulting broad peak and shift to a lower angle confirm that the graphite has been successfully exfoliated and oxidized to form GO. The reduction of graphene oxide (GO) to reduced graphene oxide (rGO) leads to a distinct change in the XRD pattern, characterized by the emergence of a peak at 2θ = 26.6°, which is similar to, but not identical with, the peak of the original graphite. This shift is consistent with previous findings by Setiadji et al. (2018) and Rochman et al. (2019), who reported that GO typically exhibits a peak between 2θ = 10-12°, whereas rGO shows a peak in the range of 2θ = 24-28°. The shift in the XRD pattern towards the original graphite peak suggests that the graphitic structure is partially restored during the reduction of GO to rGO. This is because some oxygen-containing groups are removed, decreasing the interlayer spacing. However, the structure does not completely return to its pristine graphite state (Stankovich et al., 2007). The reduction process, which can be achieved through chemical or thermal means, removes a significant amount of oxygen functionalities, thereby recovering electrical conductivity and some crystalline order. Nevertheless, defects and residual oxygen groups remain, indicating that the structure is not fully restored.

These structural changes, confirmed by the XRD analysis, are essential for validating the successful synthesis of GO and rGO. The presence of a peak at 2θ = 11.15° for GO confirms the effective oxidation and exfoliation, while the shift to 2θ = 26.6° for rGO indicates successful reduction. The partially restored structure of rGO, as evidenced by the XRD peak, is particularly advantageous for applications in antimicrobial agents. The recovery of electrical conductivity and the presence of residual functional groups in rGO enhance its ability to interact with microbial cells, potentially leading to the disruption of cellular membranes and oxidative stress. These mechanisms are effective in exerting antimicrobial action, as suggested by Elbasuney et al. (2021).

Therefore, the XRD analysis not only confirms the phase transformations from graphite to GO and rGO but also underscores the potential utility of rGO as an antimicrobial agent. The XRD peaks provide valuable insights into the structural properties of rGO, such as interlayer spacing and the degree of reduction, which are crucial in determining its effectiveness in inhibiting microbial activity. The potential of graphene-based materials, including rGO, in addressing microbial contamination in ambient aerosols is well-supported by previous studies (Perreault et al., 2015; Tadyszak et al., 2018; Lekshmi et al., 2021; Liao et al., 2018), highlighting their promising applications in environmental and biomedical fields.

Assessment of Antimicrobial test: The antimicrobial efficacy of graphene oxide (GO) and reduced graphene oxide (rGO) was systematically evaluated using Kirby-Bauer tests, a well-established qualitative method for assessing microbial inhibition (Boyle et al., 1973; Vj et al., 1973). The results summarized in Table 1 demonstrate the potent antimicrobial activity of both GO and rGO against a range of bacterial and fungal pathogens, highlighting their potential as effective agents for controlling microbial growth.

The inhibition results reveal that GO exhibits significant antimicrobial properties. Specifically, at a concentration of 300 µg mL⁻¹, GO inhibited Coccus bacteria by 48%. This inhibition can be attributed to the unique properties of GO, particularly its high surface area and the presence of oxygen-containing functional groups that can interact with bacterial cell membranes, disrupting their integrity. These interactions likely induce oxidative stress within the bacteria, leading to cell death. Moreover, GO demonstrated complete inhibition (100%) against Aspergillus at a lower concentration of 175 µg mL⁻¹. The fungicidal activity of GO may be linked to its ability to penetrate fungal cell walls, thereby disrupting cellular processes and leading to cell lysis. This finding is particularly relevant in the context of fungal infections, which can pose significant health risks, particularly in immunocompromised individuals.

In comparison, rGO exhibited even more pronounced antimicrobial effects, particularly at lower concentrations. For instance, E. coli showed an impressive inhibition rate of 88% at just 100 µg mL⁻¹ of rGO. This enhanced efficacy can be attributed to the restoration of electrical conductivity in rGO, which may facilitate stronger interactions with bacterial cell membranes, enhancing membrane permeability and promoting cellular uptake of rGO. The disruption of membrane integrity could lead to leakage of intracellular components and ultimately result in bacterial cell death. Additionally, rGO completely inhibited Brucella at a concentration of 175 µg mL⁻¹. Brucella spp. is known for their pathogenicity and ability to evade the immune system, making effective treatment strategies essential. The complete inhibition observed here underscores the potential of rGO as a viable antimicrobial agent against such resilient pathogens.

These findings are consistent with previous studies on the antimicrobial properties of graphene-based materials. For example, Akhavan and Ghaderi (2010) reported significant antibacterial activity of both GO and rGO against E. coli and Staphylococcus aureus, attributing GO's superior efficacy to the oxidative stress induced by its functional groups. Similarly, Liu et al. (2011) found that rGO exhibited enhanced antibacterial effects due to its increased electrical conductivity, which supports our observations regarding the improved inhibitory effects at lower concentrations. This research highlight the considerable antimicrobial potential of graphene-based materials, particularly GO and rGO. Their ability to effectively inhibit a variety of microbial pathogens not only emphasizes their relevance in biomedical applications but also paves the way for their use in developing novel antimicrobial agents. Given the growing concerns surrounding antibiotic resistance, the integration of graphene-based materials into therapeutic strategies could provide a promising alternative for combating microbial infections.

Table 1. GO & rGO activity in Inhibition of microbial growth

Graphene Materials	Bacteria Type	Evaluation Method	Concentration	Inhibition
GO	Coccus	Plate count	300 µg mL^-1	48%
GO	Aspergillus	Plate count	150 µg mL^-1	100%
rGO	E. coli	Plate count	100 µg mL^-1	88%
rGO	Brucella	Plate count	175 µg mL^-1	100%

This study successfully synthesizes and characterizes graphene oxide (GO) and reduced graphene oxide (rGO) from carbon-rich coconut shell waste, presenting an eco-friendly and sustainable approach to nanomaterial production. XRD analysis confirms the effective structural transformation from graphite to GO and rGO, demonstrating successful oxidation and reduction processes. FESEM imaging provides further validation, displaying the characteristic wrinkled and layered morphology of the synthesized nanomaterials. The antimicrobial efficacy of both GO and rGO was demonstrated against a wide range of airborne microbial strains, with rGO showing particularly strong inhibitory effects at lower concentrations. The surface properties of the nanomaterials, including the increased surface roughness of GO-coated surfaces, contributed to enhanced bacterial adhesion and subsequent inhibition. These results highlight the significant potential of bio-based graphene nanomaterials as novel, cost-effective solutions for controlling airborne microbial contamination. By utilizing waste materials and employing green synthesis methods, this research aligns with global efforts toward sustainability, addressing the pressing need for eco-friendly technologies in air quality management.

ACKNOWLEDGEMENT

We are grateful to Prof. Rohit Shrivastava, Head of the Department of Chemistry, Faculty of Science, DEI for his kind support and encouragement along with Prof. Shyama Prasad and Dr. Manju Srivastava for their assistance with the FESEM analysis.

Author contribution statement: All authors listed have significantly contributed to the development and the writing of this article.

Consent to participate: The authors declare their consent for the submission.

Consent to publish: The authors give their consent for the publication.

Data availability statement: Data will be made available on request.

Declaration of competing interest: The authors declare no conflict of interest.

Ethics statement: No potential conflict of interest was reported by the authors.

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