Antibacterial activity of microwave-hydroxyapatite and cellulose blend

Abstract

Introduction

Access to safe drinking water continues to be a global challenge, especially in developing countries [1]. In most rural communities, groundwater remains the main source of drinking water supply [2, 3]. These water sources are easily contaminated by microbial pathogens from sewage and septic system failures, as well as animal waste. Water contaminated with microbial pathogens like Escherichia coli (E. coli), Salmonella species (Salmonella spp.), Staphylococcus aureus (S. aureus), and other faecal coliform bacteria has been linked to the transmission of potentially fatal diseases such as amoebic and bacillary dysentery, cholera, typhoid, and other diarrheal diseases [4, 5].

Various methods have been implemented to decontaminate groundwater for safe human consumption. Common techniques include chlorination, boiling, reverse osmosis, ultraviolet (UV) light treatment, and filtration. While these methods have demonstrated effectiveness against a wide range of pathogens, it is crucial to acknowledge the intrinsic limitations that can hinder their application in water treatment processes. Chlorination, for instance, faces challenges related to antibacterial resistance and the potential health risks associated with the by-products of chemical disinfectants, thereby reducing its long-term effectiveness [6–8]. Similarly, UV light treatment, reverse osmosis, and boiling are associated with high operational costs and energy consumption, which is a constraint on their widespread use [9, 10]. These challenges necessitate alternative options to effectively treat contaminated water.

Biomaterials, with their unique qualities and innate compatibility with biological systems, offer great potential as a promising alternative for antibacterial water treatment [11, 12]. Innovative antibacterial techniques can be developed by utilizing the inherent properties of biomaterials, such as porosity, adsorption capacities, and surface chemistry, to successfully combat bacterial contamination in water sources [11]. Hydroxyapatite (HAp; Ca₁₀(PO₄)₆OH₂), a calcium phosphate mineral with a wide range of biomedical applications, has been widely studied as an effective material for water treatment, specifically in the removal of heavy metals, dyes, and fluorides from contaminated water [13, 14]. The intrinsic properties of HAp have made it an appealing option for pollutant remediation [13, 15]. HAp can be synthesized from both natural and synthetic sources, each offering unique characteristics. Naturally sourced HAp can be obtained from materials such as animal bones, eggshells, seashells, and shells of animals like the giant African snail, presenting a potential low-cost option for sustainable material development [16]. These materials are rich in calcium carbonate (CaCO₃), a precursor in HAp preparation. In contrast, synthetic approaches often involve the use of glassy matrices and polymer networks as templates for HAp formation, allowing for precise control over the material's properties [17, 18].

HAp typically exhibits very minimal antibacterial activity, and this has hindered its use in the antibacterial treatment of contaminated water [19, 20]. Researchers have explored the incorporation of metal and metal oxide nanoparticles into HAp to improve its antibacterial activity. The small size and high surface-to-volume ratio of these nanoparticles facilitate enhanced interaction with bacterial cells, inducing cell wall damage, disrupting key cellular activities, and imposing oxidative stress that ultimately leads to bacterial death [21, 22]. In addition to their antibacterial reinforcement, the nanoparticles in HAp blends enhance mechanical strength, chemical stability, and magnetic and optical functionalities for biomedical applications [23–25]. Similarly, biopolymer blends including cellulose, chitosan, and sericin have been reinforced through the addition of nanoparticles to improve their mechanical strength, antibacterial activity, and chemical stability [26, 27]. While this modification has been proven to be very effective, the potential toxicity, high cost of production, and environmental impact of nanoparticles limit their long-term use [19, 28].

Lamkhao et al. demonstrated in their study that generating radical species in HAp using microwave-assisted combustion synthesis could improve its antibacterial activity [19]. These radical species have the ability to eliminate bacteria by altering the cell wall of bacteria and inhibiting their respiratory activities while remaining non-toxic to human cells [19]. Thus, this study explores the use of cellulose nanocrystals (CNC) as a catalyst for the generation of additional radical species in HAp prepared using microwave-assisted combustion synthesis (HApM). CNCs have the ability to undergo oxidative degradation as a catalyst for the generation of radical species [29]. In the presence of chemical, mechanical, or thermal stimuli, there is a breakdown of the glycosidic bonds between the glucose membranes, releasing very reactive radical species like peroxyl (ROO·) and hydroxyl (·OH) radicals. When CNC is combined with HApM, additional radical species like phosphate (·PO₃) and carbonate (·CO₃) radicals could potentially be generated.

The purpose of this study is to develop an HApM/CNC blend that exhibits enhanced antibacterial activity, offering a promising solution for the effective treatment of contaminated water. By leveraging this blend’s potential to eliminate bacterial pathogens, we aim to significantly reduce the incidence of waterborne diseases and improve public health. The addition of CNC to HApM holds the potential for dual applicability in water treatment, making it possible to harness the antibacterial activity of HApM to eliminate waterborne bacteria while leveraging its adsorption capabilities to remove toxic chemicals from the water.

The antibacterial efficacy study of the HApM/CNC blend involved disc diffusion susceptibility testing. bacterial quantification using optical density testing and colony-forming unit (CFU) tests. These analytical tests allowed for a comprehensive investigation and quantification of the antibacterial activity of the blend. Also, the functional groups of the blend were studied using Fourier transform infrared (FTIR) spectroscopy complemented with Raman spectroscopy. The morphological characteristics of the blend were studied using scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDX).

Materials and methods

Materials

All reagents were sourced as analytical-grade substances and used without further purification. CaCO₃ was extracted from the shells of Achatina achatina snails. CNC (92% crystallinity, 10–20 nm width, 300–900 nm length, and 1.49 g/cm³ density) was purchased from PowderNano. Produced via sulphuric acid hydrolysis, this redispersible powder of uniform acyclic nanometric crystals is suitable for high-temperature applications up to 280°C. Diammonium phosphate (CAS 7783-28-0), with a molecular weight of 132.06 g/mol and purity ≥99.0% based on acidimetric analysis, was purchased from Sigma Aldrich.

Synthesis of HAp and HApM

Hydroxyapatite (HAp) was prepared from calcium carbonate (CaCO₃) and diammonium hydrogen phosphate (DHP). Pure CaCO₃ was obtained by calcining aragonite at 600°C. A 1M DHP solution was prepared by dissolving 11.76 g of DHP in distilled water. It was added dropwise under continuous stirring to a 0.6 M CaCO₃ solution prepared by dissolving 3.94 g of CaCO₃ in distilled water. The mixture was stirred at 40°C for 2 h to obtain a white precipitate with a pH of 10.56. The mixture was allowed to age for 24 h at room temperature to improve its chemical structure and stability. The HAp was separated from the solution by filtering, and the filtrate was dried in an oven at 60°C. Microwave-assisted combustion synthesis was done by drying the HAp filtrate using a microwave oven in a cycle of 5 min on and 5 min off to obtain a microwave-assisted hydroxyapatite (HApM). The synthetic procedure for HAp and HApM is outlined in Fig. 1. This figure details the step-by-step process of obtaining the materials, including the reagents and conditions used.

Figure 1.

Schematic illustration of the synthesis of HAp and HApM This illustration details the reagents, conditions, and steps involved in synthesising of HAp and HApM.

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HApM and CNC blending

The HApM/CNC blends were created using a controlled blending process in which precisely measured amounts of microwave-assisted hydroxyapatite (HApM) and cellulose nanocrystals (CNC) were mixed in various ratios. Three blending ratios (80 : 20, 60 : 40, and 40 : 60) were created. For each blending ratio, 1.2, 0.9, and 0.6 g of HApM were mixed with 0.3, 0.6, and 0.9 g of CNC, denoted as 80HAp20CNC, 60HAp40CNC, and 40HAp60CNC, respectively. The dispersal was achieved by continuously mixing for 5 min with a vortex. Figure 2 shows the blending of HAp and CNC to obtain the HApM/CNC blends.

Figure 2.

Schematic illustration of the synthesis of the HApM/CNC blend. This illustration details the blending process for the HApM/CNC blends.

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Material characterization

The crystallographic structure and phase composition of the HAp, HApM, CNC, and 40HApM/60CNC blends were analysed using XRD patterns obtained from a benchtop X-ray diffractometer (MiniFlex) at 40 kV and 15 mA. A Fourier transform infrared (Perkin-Elmer) spectrometer was used to determine the functional groups present in the materials. The scan was in the range of 400–4000 cm⁻¹. The morphology of the materials was analysed using the Phenom World ProX desktop scanning electron microscope.

Antibacterial activity tests

Antibacterial activity testing of the materials was performed on E. coli (gram-negative) and S. aureus (gram-positive). The density of the bacterial inoculum was standardized using the 0.5 McFarland standard (1.5 × 10⁸ CFU). Sterile conditions were observed by autoclaving all glassware, culture media, and other tools at 121°C for 15 min.

Kirby-Bauer disc susceptibility tests

Bacterial quantification tests

The growth and concentration of bacteria in the materials were assessed using optical density tests and colony-forming unit (CFU) tests. Optical density testing involves the measurement of the turbidity of liquid culture to provide an assessment of bacterial growth, while CFU tests involve plating diluted liquid culture onto agar media and counting the visible colonies to provide an assessment of individual bacterial cell viability and ability to form colonies. Sterile conditions for both tests were ensured by autoclaving all tools and equipment used.

Optical density tests

1 ml of E. coli and S. aureus inocula was pipetted into individual test tubes containing 5 ml of peptone water as the growth medium. 0.2 g of the materials were dissolved in 5 ml of distilled water and added to their respective inocula in the liquid medium. The mixture was thoroughly vortexed and incubated at 37°C for 24 h. After the incubation period, the transmittance of each material was measured using the spectrophotometer (Spectronic 21D) at a wavelength of 640 nm.

Colony forming unit (CFU) tests

A 4-fold serial dilution (dilution factor of 10⁻⁴) was performed on each material after recording their respective transmittances. A volume of 1 ml of each diluted material was pipetted into empty petri dishes. Eosin-Methylene Blue (EMB) agar and Baird-Parker Agar (BPA) solutions were prepared by dissolving 19 g of the EMB agar powder and 32.5 g of BPA, respectively, into 500 ml of distilled water separately. EMB agar is a medium for the quantification of gram-negative bacteria like E. coli, while BPA is used for the quantification of gram-positive bacteria like S. aureus. The media was autoclaved at 121°C for 15 min, allowed to cool, and poured into the corresponding petri dishes. The plates were gently swirled to mix the agar and left to solidify for 10 min. The plates were then incubated at 42°C for 48 h. Visible bacterial colonies on each plate were counted using a colony counter.

Statistical analysis

The results are presented as the means of all data with their standard deviations as errors using one-way analysis of variance (ANOVA) and Tukey’s post hoc test. The statistical significance was recognized at a confidence level of P < 0.05.

Results and discussion

Material characterization

X-ray diffraction (XRD) analysis

Figure 3 shows the XRD patterns of HAp, HApM, CNC, and the 40HAp60CNC blend. In the XRD pattern of HAp, prominent phases of HAp manifest at 2θ angles of approximately 22.8, 26.4, 31.8, 39.2, 43.1, 48.5, and 57.3°, corresponding to the lattice planes of (002), (210), (211), (300), (202), (310), and (220) [32]. Concurrently, minor phases of calcite are observed at 29.3°, 35.8°, and 47.6°, attributed to (211), (300), and (310), respectively. The presence of both HAp and calcite phases signifies an incomplete conversion of calcium precursors to the desired HAp phase. In the XRD pattern of HApM, new diffraction peaks occur at the 2θ angles, which correspond to different lattice planes [33, 34]. The diffraction peaks are attributed to the introduction of a pyrophosphate (P₂O₇₂-) phase, resulting from microwave-induced conversion of HAp [34]. The loss of water molecules (condensation) during the combustion reaction causes the breakdown of the Ca-P bond, leading to the formation of diester bonds between the phosphate groups and oxygen [35]. This results in the formation of pyrophosphate groups, which consist of phosphate groups linked to each other through a P–O–P linkage. The XRD pattern of CNC is characterized by broad peaks at 15.6°, 20.5°, 22.5°, and 34.6°, signifying lattice planes (1 1 0), (1 0 2), (2 0 0), and (0 0 4). The broadness of these peaks implies the presence of relatively small crystallite sizes within the CNC particles. Examination of the 40HAp60CNC blend reveals the combination of the individual spectra of CNC and HApM, showing the distinct phases of HAp, calcite, pyrophosphate, and CNC in the blend. This suggests that the different crystalline structures and lattice planes characteristic of each material are retained, and their diffraction peaks contribute to the overall XRD pattern of the blend.

Figure 3.

XRD spectra of HAp, HApM, CNC, and the 40HAp60CNC blend. The symbols •, *, ¨, and ° are pyrophosphate, HAp, calcite, and cellulose phases, respectively.

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Fourier transform infrared (FTIR) analysis

Figure 4 displays the FTIR spectra of the materials, showcasing two regions: the main region and the fingerprint region. The main region covers a wide range of wavenumbers and exhibits important peaks that correspond to the various functional groups present. The fingerprint region, located on the right side of the spectra, provides more detailed information about the unique molecular vibrations and specific functional groups present [14]. The FTIR spectrum of HAp, serving as the reference spectrum, displays six characteristic peaks within the range of 4000 to 400 cm⁻¹. A notable broad band is observed at 3356 cm⁻¹, corresponding to the stretching vibrations of the hydroxyl (OH–) groups [36]. The peak at 1431 cm⁻¹ corresponds to the symmetric stretching of the carbonate (⁠$CO32−$⁠) groups associated with the CaCO₃ component of HAp [36, 37]. The characteristic peak is observed at 1021 cm⁻¹, which is identified as the stretching vibration of the phosphate (⁠$PO43−$⁠) groups, while those at 869 and 598 cm⁻¹ correspond to the bending vibrations of the $PO43−$ groups [14, 38]. The peak at 561 cm⁻¹ corresponds to the bending vibrations of the OH– groups. In the spectrum of HApM, new peaks emerge in addition to the characteristic HAp peaks. These additional peaks are attributed to the complex chemical transformations that occur during the energy-intensive combustion synthesis method. The combustion process can induce the breakdown of certain chemical bonds, the rearrangement of atoms, and the generation of new species [39]. These changes can manifest as distinctive vibrational modes that are detected as new peaks in the FTIR spectrum. These new peaks are identified as pyrophosphate groups formed from the reaction between phosphate groups and water, observed in the spectrum as the vibrational modes of the P-O-P bonds [40, 41]. Additionally, the shift of the CO₃₂ peak from 1431 to 1423 cm⁻¹ in HApM suggests structural changes induced by combustion synthesis. After the combustion reaction, the HApM and CNC (green) spectra appear to be very similar in terms of the positions of their peaks. This indicates similar bonding patterns between the two materials [42]. This claim is further supported by 40HAp60CNC's (red) spectrum, which also coincides with the characteristic functional groups of HAp. The CNC spectrum includes peaks related to OH– groups, aliphatic C–H bonds, and C–C bonds, while the 40HAp60CNC spectrum shows peaks characteristic of hydroxyapatite [43]. In the CNC spectrum, the peak identified at 3356 cm⁻¹ is the stretching vibrations of the OH- groups [43, 44]. The 2856 cm⁻¹ peak corresponds to the stretching vibrations of the aliphatic C-H bonds, while that at 1645 cm⁻¹ is identified as the bending vibrations of the C-C bonds [32]. The peaks at 1423 and 1369 cm⁻¹ correspond to the bending vibrations of CH₂ and CH₃ groups, respectively. The peak at 1163 cm⁻¹ reveals the presence of a sulphate (SO₂) group, which could be caused by the sulphonation of cellulose during synthesis [45]. The peaks identified at 1058 and 1021 cm⁻¹ represent the stretching vibrations of C-OH bonds [46]. The peak at 869 cm⁻¹ is identified as the stretching vibration of the C-C groups, while those at 664, 564, and 460 cm⁻¹ correspond to the bending vibrations of the O–H, C–H, and C–O–C groups. The peak identified at 3356 cm⁻¹ in the 40HAp60CNC spectra is attributed to the stretching vibrations of the OH– group. The characteristic $CO32−$ group is observed at 1423 cm⁻¹, while the $PO43−$ groups are identified at 1021 cm⁻¹ [14]. The peaks at 2856, 1645, 1369, 1163, 1058, 896, 664, 564, and 460 cm⁻¹, which coincide with the characteristic peaks of CNC, could be attributed to the presence of functional groups of CNC like OH–, C–H, and C–C, as well as the pyrophosphate groups.

Figure 4.

FTIR spectra of HApM, HAp, CNC, and 40HAp60CNC.

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Scanning electron microscope (SEM) analysis

Figure 5 reveals the distinctive morphology of HAp, characterized by irregularly shaped particles of varying sizes and a granular texture. The particles exhibit a closely packed arrangement, forming an intricate network with a number of pores, which is an inherent property of HAp [47]. Figure 6 shows the SEM images of the HApM sample, which reveal structural changes as compared to the HAp sample. The images show the nanoscale particles of the material arranged in agglomerates with a textured surface and porous regions. The particles appear to have undergone a process of sintering or fusion, where adjacent particles have melted together due to the intense heat generated during the synthesis. The particles exhibit a range of sizes, indicating the presence of both individual nanocrystals and larger aggregates. Figure 7 displays the microstructure of CNC as a smooth nanocrystal surface with interconnected clusters of intricate patterns and varying degrees of porosity. The surface texture remains consistent, displaying smooth regions with minimal irregularities, reflecting the inherent characteristics of CNC [48]. The agglomerates of the nanocrystals are well-dispersed but form denser patches, indicating their tendency to self-organize into microscale structures. Figure 8 shows the images of the 40HAp60CNC blend, revealing the interfacial interaction between the HApM and CNC particles. The micrograph shows the irregularly shaped HApM particles dispersed in a matrix of CNC particles. The high-resolution image highlights the intimate contact between the CNC and HApM components, suggesting strong interlocking between them. The surface of the blend appears to have a rough texture, possibly due to the uneven distribution of HApM and CNC particles. Some areas exhibit agglomerations of particles, potentially indicating localized regions of higher particle concentration. The particles of the blend appear to be more closely packed as compared to the pure HApM and CNC samples, resulting in reduced pores between them.

Figure 5.

SEM image of HAp and its interactive 3D surface plot.

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Figure 6.

SEM image of HApM and its interactive 3D surface plot.

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Figure 7.

SEM image of CNC and its interactive 3D surface plot.

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Figure 8.

SEM image of 40HApM60CNC and its interactive 3D surface plot.

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Antibacterial activity testing

Kirby-Bauer disc susceptibility tests

The Kirby-Bauer disc diffusion method, a widely accepted technique for evaluating bacterial susceptibility to antimicrobial agents, was employed to assess the antibacterial activity of the test materials against two bacterial strains, E. coli and S. aureus. The size of the inhibition zones recorded for each material provides an indication of the effectiveness of the material in inhibiting bacterial growth. The results showed that all the materials, HAp, HApM, CNC, and the HApM/CNC blends, exhibited varying degrees of antibacterial activity against both E. coli and S. aureus. Notably, the HApM/CNC blends showed superior antibacterial activity, characterized by larger inhibition zones compared to the pure materials. Through statistical analysis, 60HAp40CNC and 40HAp60CNC were identified to show significant antibacterial activity (P < 0.05) with inhibition zones of 12.5 ± 2.12 and 12.5 ± 0.7 mm (for E. coli and S. aureus) and 13.5 ± 0.7 mm and 17 ± 1.41 mm (for E. coli and S. aureus), respectively. These blends, containing higher CNC content, exhibited the best antibacterial activity. The CNC generated additional radical species (·CO₃, ·PO₄, ·OH, and ROO· radicals) in the blend materials, and 40HAp60CNC (made up of 40% HApM and 60% CNC) had the highest CNC content. The 40HAp60CNC blend generated the most radical species and exhibited the most pronounced antibacterial effect. Moreover, our results align with the well-established trend of gram-positive bacteria, such as S. aureus, generally showing higher susceptibility to antibacterial agents than gram-negative bacteria like E. coli [49, 50]. Unlike gram-positive bacteria, gram-negative bacteria are surrounded by a double membrane made up of lipopolysaccharides. The enhanced susceptibility of S. aureus can be attributed to its distinct composition and structure of the cell wall and the absence of some specific resistance mechanisms, such as efflux pumps or antibiotic-modifying enzymes in S. aureus [50]. Consequently, S. aureus displayed higher susceptibility to most of the tested samples compared to E. coli. Figure 9 shows the relative inhibition zones achieved by each material against E. coli and S. aureus.

Figure 9.

A graph of the relative inhibition zone sizes of the samples on E. coli and S. aureus. The statistical significance is denoted by *, **, and *** and is set at P < 0.05.

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Bacterial quantification tests

The amount of viable bacteria in each test material after incubation was determined using optical density measurements and colony-forming unit (CFU) tests. The Kirby-Bauer disc susceptibility test relies on the diffusion of antibacterial agents into the agar medium to generate inhibition zones that are measured to evaluate the susceptibility of the bacteria. The size of the inhibition zones produced is influenced by the concentration of ions (radical species) impregnated on the paper disc, which interact with bacterial cells and induce cell death [51]. However, additional bacterial quantification methods like optical density measurements and CFU tests are necessary to ensure the reliability of the Kirby-Bauer test results [52]. Optical density measurements and CFU tests were performed to provide insights into bacterial viability and growth.

Optical density (OD) tests

Where T is the transmittance value and A is the absorbance value. The relative optical density values obtained by each sample against E. coli and S. aureus are shown in Fig. 10.

Figure 10.

Relative optical density measurements for the test samples against E. coli and S. aureus. Statistical significance was is established at P < 0.05 (*, **, ***, ****).

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The optical density measurements revealed significant differences in absorbance values among the test materials. Higher absorbance values indicate higher bacterial concentrations, and vice versa [53]. The control, consisting of growth media and respective bacterial cells without any test material, exhibited the highest OD values of 0.303 ± 0.01 for E. coli and 0.325 ± 0.007 for S. aureus, indicating substantial bacterial growth in the absence of the test materials. Conversely, the remaining materials containing the test materials displayed lower absorbance values for both bacteria strains, and this was confirmed by statistical analysis. While the absorbance values were seen to decrease in the order of HAp, HApM, CNC, 80HAp20CNC, 60HAp40CNC, and 40HAp60CNC, statistical analysis revealed significant differences in absorbance values for only 40HAp60CNC (for E. coli) and 60HAp40CNC and 40HAp60CNC (for S. aureus).

The observed decrease in absorbance values indicates a reduction in bacterial density or growth inhibition for the 60HAp40CNC and 40HAp60CNC samples for both bacteria strains. These findings align with the results from the disc diffusion tests and support the presence of radical species contributing to the antibacterial activity of the 40HAp60CNC blend.

Colony forming unit (CFU) tests

After 48 h of incubation, the colonies on each plate were counted and recorded. For the CFU tests, a dilution factor of 10⁻⁴ and a volume of 1 ml were used for spreading on the agar plate. Figure 11 shows the relative CFU/ml values obtained by each sample against E. coli and S. aureus. The observed variations in CFU/ml values among the different materials provide valuable insights into their respective antibacterial activities and growth inhibitory effects. The control exhibited the highest CFU/ml value of 0.012 ± 0.0004 for E. coli and 0.029 ± 0.0003 for S. aureus. The HAp, HApM, and CNC materials had statistically similar values for both bacteria strains, indicating that these materials promoted greater bacterial growth and demonstrated relatively weaker antibacterial effects. For E. coli, the 80HAp20CNC, 60HAp40CNC, and 40HAp60CNC samples had significant CFU/ml values. For S. aureus, the CNC, 80HAp20CNC, 60HAp40CNC, and 40HAp60CNC materials had significant CFU/ml values. These materials displayed lower CFU/ml values for both E. coli and S. aureus. The reduction in CFU/ml values indicates that the blends exerted a more pronounced antibacterial effect, inhibiting bacterial growth to a greater extent.

Figure 11.

Relative CFU/ml values for the test samples against E. coli and S. aureus. The statistical significance is set at P < 0.05 and indicated by *, **, and ***.

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However, just like the results from the disc diffusion test and the optical density test, the 40HAp60CNC blend demonstrated the best antibacterial activity, consistent with the findings from the Kirby-Bauer disc diffusion test and optical density tests.

The observed antibacterial activities of the HApM/CNC blends, particularly the 40HAp60CNC, hold significant practical implications for water treatment applications and other biomedical engineering applications. The superior antibacterial activity of the 40HAp60CNC blend, as indicated by the large inhibition zones, lower absorbance values, and lower CFU/ml values for both E. coli and S. aureus, suggests that this blend has the potential to effectively combat waterborne pathogens, demonstrating its suitability as a promising antibacterial agent for water treatment processes. Additionally, the pronounced antibacterial activity of the 40HAp60CNC sample highlights the importance of selecting the appropriate blending ratio to achieve optimal antibacterial activity.

Antibacterial mechanism of the blend

The antibacterial activity of HApM is observed to be higher than that of HAp due to the presence of pyrophosphate groups, which have more negative charges and the potential to generate more radical species such as hydroxyl (·OH), peroxyl (ROO·), and phosphate (·PO₃) radicals [54, 55]. The generated radical species interact with the bacterial cells and cause oxidative stress on them, which ultimately leads to their death [56]. The combination of HApM and CNC showed higher antibacterial activity relative to pure HApM and CNC due to two probable reasons. Firstly, during the production of pyrophosphate ions, the microwave radiation may crack the residual calcite, producing Ca²⁺ and $CO32-$ ions. These free Ca²⁺ ions have the potential to recombine with the negatively charged pyrophosphate ions to form calcium pyrophosphate, thereby reducing the potential to produce adequate radical species to eliminate the bacterial cells. Upon adding CNC to form the blend, the CNC has the potential to oxidize and remove the Ca²⁺ ions, preventing the recombination of pyrophosphate ions and Ca²⁺ ions. As a result, the HApM/CNC blend exhibits enhanced antibacterial activity as compared to the pristine HAp and CNC materials.

Secondly, there is the possibility of a nanoparticle of sulphonated CNC hydrogen bonded with a pyrophosphate nanoparticle. The interaction might have conferred a negative charge on the pyrophosphate nanoparticle, preventing it from recombining with another pyrophosphate nanoparticle through the counter-ion effect, which prevents the agglomeration of the pyrophosphate nanoparticles. The combined pyrophosphate-CNC particle will therefore continue to exist as nanoparticles with a large surface area-to-volume ratio. Particles with a large surface area-to-volume ratio are highly reactive and therefore penetrate the membranes of the microbes through a well-established mechanism called endocytosis (direct entry), eliminating them as a result.

The interaction dynamics differ when the HAp and CNC materials are combined. Conventional HAp synthesis methods like wet chemical synthesis typically result in a material with fewer inherent radical-generating capabilities compared to microwave-synthesized HAp [19]. When incorporated with CNC, HAp retains its structure and serves as a reinforcing phase within the composite [57]. The surface functionalities of HAp, including exposed calcium and phosphate groups, interact primarily with the carboxyl and hydroxyl groups of the CNC, leading to a more stable composite. This interaction is predominantly through hydrogen bonding and van der Waals forces rather than involving free radicals [58]. Consequently, there is minimal generation of free radicals, which reduces the potential for interactions with bacterial cells, thus limiting any inherent bactericidal effect of the material. This synergy, however, can potentially offer enhanced mechanical properties, which could be used in drug delivery and tissue engineering applications [57].

The results of this work are consistent with findings reported in literature. Lamkhao et al. used microwave-assisted combustion synthesis to enhance the antibacterial activity of HAp. They attributed the enhanced antibacterial activity to the presence of phosphate, hydroxyl, peroxyl, and carbon dioxide radical anions, which were induced by the combustion synthesis. Similarly, Yusoff et al. utilized microwave-assisted combustion for HAp synthesis and enhanced the antibacterial activity of their material by incorporating silver (Ag) nanoparticles. Their Ag-HA composites demonstrated significant inhibition zones against S. aureus (15.12 mm) and Streptococcus mitis (14.75 mm) [59]. The antibacterial effects were attributed to the leaching of Ag ions, which interacted with microbial cellular components and disrupted their functionality. Kalaiselvi et al. used a similar microwave-assisted synthesis for HAp and enhanced its antibacterial activity by adding Moringa oleifera flower extract (MOFE). Their pure HAp material exhibited inhibition zones of 11 mm against S. aureus. They attributed the antimicrobial effect of HAp nanorods to the rough surface structure due to surface defects and aggregates, which mechanically damaged bacterial cell membranes [38]. The incorporation of MOFE improved the antibacterial efficiency against S. aureus (13 mm) through enhanced interaction with the electronegative microbial surface.

In our study, we also employed microwave-assisted combustion for HAp synthesis; however, unlike the referenced studies, we identified a pyrophosphate phase in the HApM material. Pyrophosphate ions, as previously explained, are known for their superior ability to generate radical species, leading to enhanced antibacterial activity in our HApM material [54, 55]. Furthermore, the addition of CNC to our synthesized HApM material facilitated the generation of additional radical species, such as alkoxyl, peroxyl, hydroxyl, phosphate, and carbonate radicals, through cellulose oxidation [60]. Consequently, our HApM/CNC blend achieved enhanced antibacterial activity against E. coli (13.6 mm) and S. aureus (17 mm), highlighting the efficacy of the microwave-assisted combustion synthesis method and the addition of CNC in improving antibacterial activity. Our findings indicate an improvement because we used pyrophosphate and CNC to improve the surface roughness and antibacterial efficacy of HApM and CNC composites.

Conclusion

This study developed a microwave-assisted hydroxyapatite and cellulose nanocrystal (HApM/CNC) blend and investigated its potential as a strong antibacterial agent for water treatment applications. Compared to pure HApM and CNC, the HApM/CNC blends exhibited significantly enhanced antibacterial activity, with inhibition zones measuring up to 13.6 mm for E. coli and 17 mm for S. aureus. XRD, FTIR, and SEM were used to fully characterize the materials' structural and functional properties, providing valuable insights into the material composition and functional groups. The microwave-assisted combustion method facilitated the formation of a calcium pyrophosphate phase in HApM, contributing to the generation of radical species that enhanced its antibacterial activity. The incorporation of CNC into HApM promoted the release of additional radical species through cellulose oxidation, further enhancing the antibacterial efficacy of the blend. This research highlights the importance of synthesis methods in tailoring material properties for specific applications. It opens up new avenues for further research and practical application, potentially revolutionizing water treatment techniques with a more effective and sustainable approach.

Data availability

The paper contains all relevant data. Any additional data related to this study are available from the corresponding author upon reasonable request.

Authors’ contributions

Sheila Priscilla Kyeremeh (Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Writing—original draft [equal], Writing—review & editing [equal]), Bernard Owusu Asimeng (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Funding acquisition [equal], Investigation [equal], Supervision [equal], Writing—review & editing [equal]), Lily Paemka (Formal analysis [equal], Resources [equal], Writing—review & editing [equal]), Michael Ainooson Kojo (Investigation [equal], Project administration [equal], Writing—review & editing [equal]), Ebenezer Annan (Supervision [equal], Validation [equal], Writing—review & editing [equal]), and Elvis K. Tiburu (Supervision [equal], Validation [equal], Writing—review & editing [equal])

Funding

The project was made possible with financial support from the Building a New Generation of Academics in Africa (Banga-Africa) Project III, which is funded by the Carnegie Cooperation of New York.

Conflict of interest statement: The authors declare no conflict of interests.

References