Microwave assisted starch stabilized green synthesis of zinc oxide nanoparticles for antibacterial and photocatalytic applications

Abstract

Nanostructured particles offer outstanding diversities of applications in the fields of nanotechnology, nano-engineering, nano-biotechnology, etc. Morphological structure, size distribution, electronic behavior including intrinsic characteristics of nanoparticles depend on the source and synthesis methods. Here, an eco-friendly approach using microwave irradiation for the synthesis of zinc oxide (ZnO) nanoparticles has been reported. Zinc nitrate was used as a precursor whereas starch and D-glucose were used as capping and reducing agent, respectively. The synthesized nanoparticles were characterized by different instrumental methods including Ultraviolet-Visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), and field emission scanning electron microscopy (FE-SEM). The characteristic λ_max at 373 nm for ZnO nanoparticles was recorded from UV-Vis absorption spectrum in aqueous system. FT-IR spectrum showed a very sharp peak at 476.62 cm^-1 which confirmed the presence of Zn-O bond. The prepared ZnO was highly crystalline having wurtzite structure and the crystallite size was calculated to be 24.41 nm obtained from XRD analysis. FE-SEM images showed that the synthesized ZnO nanoparticles had near- spherical morphology and particle size was found in the range of 40–90 nm. The antibacterial and anti-biofilm application of ZnO nanoparticles were studied and inhibition zones of Gram negative Salmonella typhi (S. typhi), Klebsiella spp., Escherichia coli (E. coli) and Gram positive Staphylococcus aureus (S. aureus)- 8a were found to be 11 mm, 12 mm, 11.5 mm and 13.5 mm, respectively. Besides, ZnO nanoparticles also showed excellent photocatalytic activity against methylene blue dye solution. The easy and eco-friendly fabrication method would play vital role in other nanoparticles synthesis to meet the demand in textile industry, agriculture and medical sectors.

Introduction

Development of metal and metal oxide nanoparticles with desirable properties hold significant interest to the scientific community in the field of nanotechnology, nanobiotechnology to address electrical, medical and environmental issues^1–4. Zinc oxide (ZnO) is one of the popularly used nanoparticle (NP) that has gained remarkable appeal due to its unique interfacial attraction, catalytic, and outstanding biological properties⁵. Enhanced durability, lower toxicity and high thermal stability make ZnO NPs attractive for transparent electronics, piezoelectric, and catalytic reactions⁶ in the field of biomedical applications, like cancer therapy, tissue engineering, bioimaging, biosensing, drug and gene delivery etc^7–9. ZnO NPs can effectively attach to bacterial surface and demolish them by toxicity or structural damage¹⁰. ZnO-NPs are now considered to have potential to inhibit phytopathogenic fungi because of their ability to produce reactive oxygen species (ROS) in aqueous media^11,12. Crystal facets of ZnO NPs that form because of microscopic surface geometry and capture impurities/states are crucial to efficient photocatalytic performance¹³. ZnO NPs show a distinctive electronic and photonic n-type semiconductor behavior due to high exciton binding energy (60 meV) and broad band gap (3.37 eV) at ambient temperature offering potential catalytic and photochemical activities^6,10.

Many conventional and advanced methods have been used to produce ZnO NPs such as electro-deposition, chemical vapor deposition, sol-gel, microemulsion, spray pyrolysis, ultrasonic, hydrothermal and precipitation^14,15. Chemical synthesis method has been performed extensively because of its easiness, shorter time required, satisfactory product and cost effectiveness¹⁶. Moreover, this method can control the structure and morphology of NPs more precisely with expected properties¹⁷. Various essential factors like capping and reducing agents, solution pH, reactant concentration, solvent type and composition and reaction temperature are usually considered for the chemical synthesis of well-structured NPs^18,19. One of the disadvantages of the conventional methods for the synthesis of nanomaterial is the production of a huge amount of wastes which can be easily prevented in a green approach²⁰. Compared to the conventional methods, the green synthetic approach is well appreciated because it utilizes natural resources, including leaf and fruit extract, organic residue, microorganism etc. as an efficient and easy reagent which are to dispose as they are biodegradable, non- toxic and environment friendly^21,22.

Microwave-assisted synthesis is a very efficient way for the synthesis of metal and metal oxide nanomaterials. In this method, instead of conduction or convection heating, the sample is exposed to microwave radiation which accelerates the rate of reaction and produce huge amount of heat within a few minutes. This method is gaining popularity because it offers improved control over NP size and shape, faster synthesis and often higher yields compared to other conventional methods²³.

In our previous article, we reported a new route to synthesize ZnO NPs applying microwave irradiation and using ascorbic acid and polyvinyl alcohol as reducing agent and stabilizer, respectively²⁴. In another article, we reported the synthesis of iron oxide NPs using leaf extract obtained from Carica papaya. The NPs were evaluated for the photocatalytic degradation of Remazol yellow RR dye and antibacterial activity²⁵.

To date, different reaction environments have been selected by researchers for the synthesis of NPs. Numerious types of capping, reducing and dispersing agents such as ascorbic acid, citrates, ethylenediamine, amino acid, polyvinyl alcohol (PVA), poly-acrylamide, polyvinylpyrrolidone (PVP), etc. were reported to prepare ZnO NPs with controlled size and morphology^26–37. It is noteworthy to mention that starch-stabilized silver (Ag) NPs using glucose as a reducing agent by microwave technology with high conversion and below 10 nm particle size was reported by Kumar et al.³⁸. Besides, the synthesis of ZnO NPs by microwave irradiation method using Indian bael (Aegle marmelos) juice was reported by Mallikarjunaswamy et al.³⁹. Moreover, iron chloride and starch aqueous solutions were used for starch-stabilized iron oxide NPs synthesis where trisodium citrate dihydrate was utilized as a reducing agent⁴⁰.

The objective of this study was to prepare ZnO NPs utilizing microwave irradiation in the presence of glucose as a reducing agent and Zn(NO₃)₂ as the precursor in an aqueous solution. Interaction between functional groups of starch and Zn²⁺ and dispersion pattern of cation at the interface of starch provided susceptible environment to generate ZnO NPs. Rapid and instant short period of microwave irradiation helped in controlling particle size, morphology and increasing the yield of NPs⁴¹. Besides, instant heating through microwave reduction method offered the advantages, such as short reaction time, homogeneous distribution of heating, and high rate of reaction as compared with other conventional methods. Herein, starch and D-glucose acted as capping and reducing agents. The ZnO NPs were tested for potential photocatalytic, and anti-microbial applications.

Materials

Zinc nitrate hexahydrate (Zn(NO₃)₂.6H₂O, ACS reagent grade (assay ≥ 96%, MW 297.49 g/mol)) was procured from Merck Life Science Pvt. Ltd., Mumbai, India and used for the synthesis of ZnO NPs. Food grade corn starch ((C₆H₁₀O₅)_n, MW 692.661 g/mol) was collected from local market and used as received. D-Glucose (C₆H₁₂O_6, MW 180.156 g/mol, assay < 100%) was purchased from GSK Bangladesh Ltd. and sodium hydroxide (NaOH, analytical reagent grade, assay > 97%) and ethanol were purchased from Sigma-Aldrich, Mumbai, India. The microbial isolates were cultured in the Department of Microbiology at the University of Dhaka, Bangladesh. The Mueller-Hinton agar medium and tryptic soy broth (TSB) used in anti-bacterial analysis were purchased from Oxoid Limited, UK and Sigma-Aldrich, Mumbai, India, respectively. Distilled and demineralized water were produced in the laboratory from distillation and demineralization units (WDA3000.RW1.5 Fistreem International Ltd, UK) and used throughout the experiments.

Synthesis of ZnO nanoparticles

For preparation of ZnO NPs, 10 gm zinc nitrate hexahydrate (ZNH) was dissolved in 100 mL distilled water and in the same volume of water, 12.11 gm D-glucose was dissolved separately. Before synthesis, 5 gm/L starch solution was also prepared using water as a solvent. Then, 40 mL of starch solution and 24 mL of glucose solution were added to16 mL of Zn(NO₃)₂.6H₂O solution in a beaker successively. The molar ratio of Zn(NO₃)₂ : C₆H₁₂O₆ was kept at 1:2 to increase conversion of Zn²⁺ ions to ZnO and promote the subsequent formation of ZnO NPs. The reaction mixture was then mixed properly using a magnetic stirrer. The resultant homogeneous solution was heated in a microwave oven (Miyako, MD-80D20ATL-DJ, 800 W, 2.45 GHz, Japan) at 400 W for 4.5 min. A turbid homogeneous solution indicated the formation of ZnO NPs, but it did not precipitate even after 12 h due to its tiny size and interaction with starch.

After microwave irradiation, an aqueous dilute NaOH solution was added dropwise into the reaction mixture until pH (HANNA instruments) of the reaction solution reached at 13 while the white precipitate became visible. After complete precipitation, the mixture was immediately sonicated (Power Sonic 505, 5.7 L, Hwashin Technology Co., Korea) to prevent agglomeration during nucleation and crystal growth. The reaction mixture was kept rest for four hours and then centrifuged (D-78532, Hettich) to separate the white precipitate. The precipitate was dried in an atmospheric dryer (DO-150, Han Yang Scientific Equipment Co. Ltd. Seoul, Korea) at 105 °C for 180 min and finally calcined in a muffle furnace (NX2-2.5-10G, Henan, China) for 180 min at 500 °C.

Characterization of ZnO nanoparticles

The ZnO NPs were examined for optical, crystallographic, nanostructure and compositional properties by different instrumental techniques. The absorption maxima of ZnO NPs in ethanol (Sigma-Aldrich, Mumbai, India) was recorded using a UV-Visible spectrophotometer (UV-1601, Shimadzu Corporation Ltd., Tokyo, Japan). The chemical bonding between zinc and oxygen atoms with different modes within the particles was explored via recording the FT-IR spectra (FT-IR 8400 S spectrophotometer, Shimadzu Corporation Ltd., Kyoto, Japan) in the range of 4000–400 cm⁻¹ wavenumber (resolution: 2 cm⁻¹; scans: 30). The crystalline properties of ZnO NPs were studied by recording diffractogram in an X-ray diffractometer (Ultima IV, Rigaku Corporation, Japan) with Cu-Ka radiations (λ = 1.5406 Å) operated at a voltage of 40 kV and current 40 mA with scan speed of 3°/min in 2θ range from 10° to 80°. To calculate crystallite size of ZnO NPs, full-width at half-maximum (FWHM) data were utilized. The average crystallite size (D) of the ZnO NPs was calculated using Debye-Scherrer’s formula shown in Eq. (1)⁴².

1

where β is the line broadening at FWHM and k is the shape factor which is equal to 0.9.

SEM (JSM-7600 F, JEOL, Tokyo, Japan) coupled with energy-dispersive X-ray (EDX) spectroscopy was used to study morphology and elemental compositions of NPs. An accelerating voltage of 20 kV in the back scattered electron mode was maintained during operation of the instrument.

Anti-bacterial activity of ZnO NPs

The Mueller-Hinton agar medium was inoculated with 0.1 mL of the bacterial suspensions (1.5 × 10⁻⁶ CFU/mL) using sterile cotton swab followed by creating wells by using a sterile well cutter (4.0 mm diameter) in each agar plate. Next, the three suspensions (5, 20 and 30 mg/mL) of nanoparticles doped in each well individually, were prepared under sterile conditions and then situated on the surface of Mueller-Hinton agar plates. After incubation of the plates at 37 °C for 24 h, the inhibition zone around the discs were measured in millimeter (mm) scale. Here the anti-bacterial activity of ZnO NPs was studied following the well diffusion test using Klebsiella spp. KH15, S. typhi SH7, E. coli EH19 and S. aureus obtained from the Department of Microbiology, University of Dhaka, Bangladesh. The mentioned experimental procedures were similar to our previously published articles^24,25. The bacterial suspensions were adjusted to 0.1 OD using UV–Visible spectrophotometer (Spectrumlab 1200RS, Japan) from 18 h nutrient broth cultures.

Anti-biofilm activity

Plate method with 96-well microtiter was applied to determine the efficacy of ZnO NPs in biofilm formation. The procedure has already been reported in our recent published articles^24,25. By following the crystal violet staining microtiter biofilm formation assay, anti-biofilm activity was analyzed⁴³. After testing anti-microbial activity of ZnO NPs against selected four pathogens for each were inoculated into 5 mL tryptic soy broth (TSB) of single isolated colonies to produce overnight culture which was suitable for biofilm development. Fabricated ZnO NPs was applied to biofilm to treat on a UV treated glass cover slips incubated in 12-well plate which was washed using 70% ethyl alcohol. In short, the pathway described as the biofilm cover slips was washed with sterile water for three times, followed by placing on 50 mL Falcon tube and adding 15 mL of prepared ZnO NPs. After that, falcon tubes were treated for 5 min at 300 rpm. From falcon tube slips were taken out to remove loosely attached cells by washing with sterile water three times. After ten-fold dilutions bacteria were calculated to determine viable cells by agar plating and incubated at 37 °C for 24 h followed by counting and converting the developed colonies number into colony^44,45. According to the several authors the effectiveness of synthesized nanoparticle anti-biofilm activities were estimated by the following equations respectively^5,7,8.

Photocatalytic performance

To assess photocatalytic performance of ZnO NPs under day light, methylene blue (MB) was taken as a model pollutant. A 400 mL MB  50 ppm stock solution was prepared using distilled water. Initial concentration of MB was measured by UV-Visible spectrophotometer with λ_max absorption at 668 nm. Later, the 100 mL of stock solution of MB was transferred into a suitable beaker where 100 mg of ZnO NPs were added. The solution was then exposed to sunlight for 280 min. A 3 mL solution was withdrawn and ZnO NPs were separated by centrifugation at 6000 rpm for 10 min. The degraded solution was used for absorbance measurement.

The photocatalytic degradation and rate of degradation of MB solution was calculated as below⁴⁶.

where and are the initial and post-irradiation absorbance of MB solution at 666 nm as measured by the UV-Vis spectrophotometer, respectively. k is the rate constant of the reduction reaction. Time required for 50% and 85% degradation of dyes (T₅₀ and T₈₅) were calculated by the following equations.

Statistical analysis

Each experiment was triplicated and data were represented as mean ± standard deviation (SD). Analysis of variance (ANOVA) using IBM SPSS Version 25.0 was used for testing of significant study (p ≤ 0.05).

Results and discussion

The results of the research work have been summarized and critically discussed in terms of synthesis from mechanistic views, spectrometric characterization, microscopic observation and applications including antibacterial, cytotoxic and photocatalytic behaviors.

Synthesis of ZnO NPs

As described earlier, the ZnO NPs were fabricated using glucose as a reducing agent and starch as a dispersing agent from aqueous solution of ZNH using microwave irradiation. ZnO NPs synthesis from ZNH has been reported by many research groups as compiled in the Table 1. Figure 1A shows mechanistic pathway of complex formation between gluconic acid derived from glucose and Zn²⁺. Long chain hydrophilic starch biopolymer facilitates the interfacial interaction with Zn²⁺ for dispersion homogeneously. When ZnO precursor solution with starch was mixed with glucose, a stable complex immediately formed under microwave stimulation as shown in Fig. 1B. This could be due to electrostatic attraction by a self-assembled coordinated chelate complex formation with gluconic acid derived from glucose oxidation under the action of high energy microwave^47–49. Addition of NaOH aqueous solution tuned pH of the reaction mixture and kinetically reduced the time of agglomeration of micelle by accelerating the precipitation of Zn²⁺ core and gluconic acid shell complex. As a result, the smaller size of aggregates bearing fewer Zn²⁺ would interact themselves during nucleation within the micelle. The dried precipitate was then calcined at 500 °C in presence of atmospheric air in a muffle furnace for three hours where adhered starch and glucose derivatives were burned and removed as CO₂ and H₂O leaving nanoscale ZnO NPs in the crucible.

Table 1.

Microwave assisted synthesis of ZnO NPs from ZNH.

Dispersing agent	Reducing agent	Particle size (nm)	References
PVA	Ascorbic acid	70–90	24
	Aegle marmelos/Extract	20	39
--	--	50–150	51
--	Citrullus colocynthis/Extract	27–85	52
--	Pistia Stratiotes/Extract	35	53
Starch	Glucose	65	This study

Fig. 1.

Schematic presentation of ZnO NPs fabrication (A) mechanistic pathway of coordination of Zn²⁺ with glucose derivative and (B) dispersion of Zn²⁺-gluconic acid micelles within starch.

The mechanism of starch stabilized ZnO NPs formation is interesting. During the synthesis, starch works as a stabilizing agent. The mechanism starts with the dissociation of Zn(NO₃)₂ in aqueous solution that generates Zn²⁺ ions which forms coordination complexes with the hydroxyl (-OH) group of starch. These coordination interactions stabilize the Zn²⁺ ions in the matrix of the starch that prevents any agglomeration and confirms particle formation. In mild acidic conditions (~ pH = 5), the Zn²⁺ remains stabilized. However, at higher pH, the formation of Zn(OH)₂ is accelerated. When temperature is increased, at around 80–100 °C, a critical gelatinization process occurs in starch that establish a complex 3D network helping the controlled nucleation and the growth of ZnO NPs⁵⁰.

Spectral characterization

Figure 2A shows the UV–Visible absorption spectrum of the synthesized ZnO NPs. An absorption peak at 373 nm was found for the ZnO NPs in ethanol which is due to π–π* electronic excitation of ZnO^54,55. The remarkably sharp absorption peak of ZnO NPs represents the monodispersity of the synthesized particles’ distribution which is the prerequisite to be an effective catalyst for photocatalytic reaction and biomedical applications⁵⁶. UV–Visible absorption data were utilized to develop the Tauc`s plot where (αhν)² versus photon energy (hν) was plotted and extrapolated to the X-axis according to Eq. 2, yielding a value of 3.33 eV. The color of the sample (white) was in well agreement with the observed band gap value as illustrated in Fig. 2B.

2

Fig. 2.

Fabricated ZnO nanostructures characterization (A) UV–Visible absorption spectrum recorded in ethanol, (B) Tauc`s plot for energy bandgap, (C) functional group study of using FT-IR spectrum and (D) XRD pattern.

FT-IR spectrum of nanostructured ZnO is shown in Fig. 2C. The characteristic metal oxide bond was confirmed from a prominent band at 476 cm⁻¹ which was attributed by the Zn–O stretching vibration mode^57,58. The peaks at 893 and 3437 cm⁻¹ were assigned to the –OH bending and stretching from starch and glucose moieties⁵⁹. In addition, other bands at 1097 and 1610 cm⁻¹ corresponded to the O–C–O and C = O bonds functional groups, respectively⁶⁰.

XRD pattern of ZnO NPs is shown in Fig. 2D. The synthesized ZnO NPs showed a sharp diffraction peak which confirmed its high crystallinity. The recorded XRD peaks of ZnO NPs were in hexagonal wurtzite phase confirmed by JCPDS Card No. 01-089-0510. It is to be emphasized that no impurity phases were noticed in the diffractogram of ZnO NPs. The sharp diffraction peaks confirmed the uniform crystallinity of the particles. In particular, the differences in terms of intensity between the ZnO (101) diffraction peak and the ZnO (102) peak indicates a preferential crystallographic orientation of the ZnO NPs⁶¹. The diffraction peaks on the figure at 31.86°, 34.55°, 36.36°, 47.65°, 56.68°, 62.85°, 66.39°, 68.09, and 69.18° were indexed to the reflection of (100), (002), (101), (102), (110), (103), (200), (112), and (201) planes⁵⁷. The crystallite size for the highest intensity peak in the XRD pattern was calculated to be 24.41 nm whereas, the average crystallite size of ZnO NPs was found to be 21.66 nm.

The elemental analysis of the ZnO NPs was performed using the EDX. Figure 3A shows the EDX spectrum of ZnO NPs. It is revealed that the K and L emission peaks for zinc and oxygen were observed at around 1.1 keV and 0.6 keV respectively. Based on the elemental analysis of ZnO (Zn = 85.85% and O = 14.14% by mass) in Fig. 3B, which is very close to the value (Zn = 84.13% and O = 15.87% by mass) reported by Devraj Singh et al.⁶². Theoretically, ZnO contains 80.34% and 19.66% (by mass) zinc and oxygen respectively. The deviation of experimental value from theoretical value could be occurred due to the limitation of instruments and sample preparation for the test.

Fig. 3.

The elemental analysis of the ZnO NPs was performed using the EDX method. (A) shows intensity (counts) of X-ray emissions vs. keV and (B) mass (%) of elements.

Morphology analysis

The Fig. 4A shows the SEM image of the microwave assisted ZnO NPs. It is clearly observed that most of the particles were in near spherical in shape. The particle size of ZnO nanoparticles was plotted against the population to obtain a trend line for size distribution profile in Fig. 4B. The size distribution pattern confirmed the particle size within the range from 1 to 100 nm with the average particle size of approximately 65 nm.

Fig. 4.

(A) SEM image of the microwave assisted ZnO NPs and (B) its size distribution.

Photocatalytic performance evaluation of ZnO NPs

The UV-Visible spectra during degradation of MB dye in the absence and presence of ZnO NPs were recorded at different time interval under sunlight irradiation and were plotted in Fig. 5(A, B). As the time progressed, the absorbance of MB was decreased slowly in the absence of ZnO NPs. In the presence of ZnO NPs, the absorbance decreased drastically. The absorbance and respective percentage degradation of MB was plotted against time in Fig. 5C. It was understood from the figure that the decrease in absorbance rate of MB in the presence of ZnO NPs was higher. Initially, after 40 min, MB degradation with ZnO NPs was about 27%, which was four times higher than the percentage degradation found without ZnO NPs. After 280 min about 2.5 times higher degradation (98.32%) was occurred with ZnO NPs compared to the blank sample. This could be due to the fact that at the half stage (after 45% MB) of degradation, the rate of photocatalysis declined as the ratio of dye molecules to ZnO NPs decreased. Saturation of the catalyst surface, aggregation of dye molecules also might be the reason of this decreased degradation rate⁶³. As shown in Fig. 5D, the MB degradation by ZnO NPs followed the first order reaction kinetics. The rate of reaction with and without ZnO NPs were 0.0089 and 0.0017 min⁻¹ respectively and the reaction was more than five folds faster in the presence of ZnO NPs due to interfacial catalytic degradation reaction with dye molecules. The empirical formulae were followed to calculate time of 50% and 85% MB degradation as mentioned earlier and found to be about 1.3 h and 3 h, respectively. In addition, Table 2 shows a comparative analysis of MB degradation using ZnO NPs at different conditions.

Fig. 5.

UV-Visible spectra during degradation of MB in (A) absence and (B) presence of ZnO NPs were recorded at different time intervals under sunlight irradiation; (C) The absorbance vs. time and percentage of MB degradation vs. time plot and, (D) MB degradation kinetics.

Table 2.

Comparison of photocatalytic activities of ZnO NPs in MB solution among published and present study.

Concentration of photocatalyst	Crystallite size (nm)	Concentration of dye	Dye degradation (%)	Method of synthesis	References
250 mg/L	28–30	20–100 ppm	81-92.5	Precipitation method	64
10 mg/mL	60	100 ppm	90	Precipitation method	65
60 mg	9–38	10 ppm	100	Solution combustion method	66
50–200 mg	20	5–20 ppm	100	Solution combustion method	67
30 mg/100 mL	20	5 ppm	100	Microwave method	39
100 mg/100 mL	24	50 ppm	98.3	Microwave method	Present work

The mechanistic view of photocatalytic degradation of MB by ZnO NPs under sunlight irradiation is schematically depicted in Fig. 6. In the interfacial reaction environment initiated from ZnO NPs, by various ROS species like hydroxyl radical (·OH), superoxide radical (·O₂), and hydroperoxyl radical (·HO₂) were formed and played vital role in MB degradation. After absorbing energy from the sunlight, ZnO NPs liberated electron from the valence band to its conduction band from where superoxide radical originates by capturing it. The oxidation of absorbed water or absorbed OH⁻ in the valence band produces hydroxyl radical and thus prevents the recombination of electron–hole pairs⁵⁷. Labhane et al. reported that plenty of surface oxygen radicals produced by absorbing electron from ZnO NPs could enhance the photocatalytic activity for degradation and discoloration of MB by splitting of the myriad of intermediates produced from initial degradation and finally leaving CO₂ and H₂O molecules⁶⁸.

Fig. 6.

The schematic mechanistic view of photocatalytic degradation of MB by ZnO NPs under sunlight irradiation.

Anti-bacterial activity

Gram negative (S. typhi, Klebsiella spp. and E. coli) and Gram positive (S. aureus) bacteria were studied using ZnO NPs suspensions of different concentrations to evaluate the anti-bacterial activity in aqueous nutrient broth^69–78 as shown in Fig. 7. Table 3 shows that the inhibition zone was increased with increasing ZnO NPs concentration in the suspension. It is revealed that all the bacteria were resistant against 5 mg/mL concentration. ZnO NPs having concentration 20 mg/mL and 30 mg/mL showed inhibition zones 13 ± 0.03 mm and 13.5 ± 0.01 mm, respectively for S. aureus. The lowest inhibition zones 10 ± 0.06 mm were observed for 20 mg/mL and 11.5 ± 0.04 mm for 30 mg/mL of ZnO NPs (p < 0.05) in case of E. coli.

Fig. 7.

Images of zone of inhibition of ZnO NPs suspensions against S. typhi, Klebsiella spp., E. coli and S. aureus.

Table 3.

Antibacterial activity with variable concentrations of ZnO NPs against tested bacteria.

Nanoparticle (ZnO) concentration (mg/mL)	Diameter of inhibition zone (mm)
	S. typhi	Klebsiella spp	E. coli	S. aureus
5	R	R	R	R
20	10.5 ± 0.04a	11 ± 0.01c	10 ± 0.06e	13 ± 0.03g
30	12 ± 0.05b	12.4 ± 0.02d	11.5 ± 0.04f	13.5 ± 0.01h

Different letters represent statistical significance among different treatments (p<0.05). R-Resistant (no zone).

Relatively similar inhibition was observed for S. typhi (10.5 ± 0.04 and 12 ± 0.05), Klebsiella spp. (11 ± 0.01 and 12.4 ± 0.02) in both 20 mg/mL and 30 mg/mL concentration. The slightly higher inhibition against Gram-positive S. aureus—is likely influenced by differences in bacterial cell wall structure. Gram-positive bacteria possess a thicker peptidoglycan layer and numerous pores which may interact more readily with ZnO nanoparticles, leading to enhanced anti-bacterial effects. On the other hand, Gram-negative cell wall structure composed of lipopolysaccharides, phospholipids, and lipoproteins which together form a barrier allowing only micro-molecules to enter the cell⁷⁹. A comparative anti-bacterial activity including crystallite size and concentration of green synthesized ZnO NPs among reported articles and the current study has been furnished in Table 4.

Table 4.

Comparison of antibacterial activities of green synthesized ZnO NPs among the published and present study.

Bacterial species	Crystallite size (nm)	Concentration of ZnO	Zone of inhibition (mm)	References
S. aureus	60	10 mg/mL	16.01	80
S. aureus	27–85	0.1 mg/mL	6.8 ± 0.36	52
S. aureus	47.27	75 µg/mL	11.4	81
E. coli	7	1 mg/disc	19.36	82
S. aureus			22.20
S. typhi	24	20 mg/mL 30 mg/mL	10.5 ± 0.04 12.0 ± 0.05	Present work
Klebsiella spp		20 mg/mL 30 mg/mL	11 ± 0.01 12.4 ± 0.02
E. coli		20 mg/mL 30 mg/mL	10.0 ± 0.06 11.5 ± 0.04
S. aureus		20 mg/mL 30 mg/mL	13.0 ± 0.03 13.5 ± 0.04

Anti-biofilm activity

In this study, likewise anti-bacterial performance, ZnO NPs was examined against S. typhi, Klebsiella spp., E. coli, and S. aureus for evaluation of the anti-biofilm activity. The results of anti-biofilm activity are shown in the Table 5. It is observed that ZnO NPs in 20 mg/mL and 30 mg/mL shows anti-biofilm activity against all four studied bacteria and these concentrations regulate the effect. For instance, in the absence of ZnO NPs, the highest amount of bacterial count was observed for E. coli (6.77 × 10⁸ CFU/mL) whereas when treated with 20 mg/mL and 30 mg/mL of ZnO NPs, the bacterial count was reduced to 4.92 × 10⁸ CFU/mL and 4.05 × 10⁸ CFU/mL respectively. The highest anti-biofilm activities were observed against S. aureus (2.89 × 10⁸ CFU/mL) whereas the count was reduced to 3.19 × 10⁸ CFU/mL for 20 mg/mL of ZnO NPs. The Table 5 shows that, 30 mg/mL of ZnO NPs killed 41%, 38%, 37%, 53% of E. coli, Klebsiella spp., S. typhi and S. aureus bacterial strains with an LR value of 0.23, 0.19, 0.21, 0.33 respectively. Moreover, ZnO NPs killed 72.67%, 67.92%, 73.55%, 52.46% of E. coli, Klebsiella spp., S. typhi and S. aureus bacterial strains with and PK value of 28%, 33%, 27%, 48%, respectively at 20 mg/mL concentration. It is revealed that, all the Gram-negative bacteria strains were survived significantly for both treatment and the lowest survival fractions (52.46% and 47.53%) were observed for Gram-positive bacteria (S. aureus) after the treatment with 30 mg/mL and 20 mg/mL, respectively. This more tolerant biofilm formation occurs because the Gram-negative bacterium strains secret more component for biofilm generation^64,83.

Table 5.

Bacterial biofilm development on ZnO NPs treated glass coverslips at 37 °C.

Bacterial strain	No. of surviving cells before treatment×108 (CFU/mL)	No. of surviving cells after treatment×108 (CFU/mL)	Survival Factor (%)	Kill (%)	Log Reduction
ZnO NPs (20 mg/mL)
E. coli	6.77 ± 0.01a	4.92 ± 0.03e	72.67	28	0.14
Klebsiella spp.	6.39 ± 0.02b	4.34 ± 0.01f	67.92	33	0.17
S. typhi	5.52 ± 0.02c	4.06 ± 0.01g	73.55	27	0.14
S. aureus	6.08 ± 0.05d	3.19 ± 0.04h	52.46	48	0.30
ZnO NPs (30 mg/mL)
E. coli	6.77 ± 0.01a	4.05 ± 0.02e	59.82	41	0.23
Klebsiella spp.	6.39 ± 0.02b	4.01 ± 0.03f	62.75	38	0.19
S. typhi	5.52 ± 0.02c	3.52 ± 0.01g	63.77	37	0.21
S. aureus	6.08 ± 0.05d	2.89 ± 0.02h	47.53	53	0.33

Different letters represent statistical significance among different treatments (p<0.05).

Conclusion

In summary, we have demonstrated a simple convenient, efficient and environment friendly microwave-assisted pathway to fabricate ZnO NPs. UV-Visible, FT-IR spectroscopy and XRD analysis showed maximum absorbance at 373 nm, 476 cm⁻¹ and the average crystallite size of 21.66 nm, respectively, which proved the formation of ZnO NPs. The band gap was found to be 3.33 eV and the average ZnO NPs particle size was calculated to be 65 nm. The rate of photocatalytic reaction for MB degradation with and without ZnO NPs in aqueous system was 0.0089 and 0.0017 min⁻¹, respectively. It’s revealed that the reaction rate was more than five folds faster in the presence of ZnO NPs. The maximum inhibition zones of ZnO NPs having concentrations 20 mg/mL and 30 mg/mL were found to be 13 ± 0.03 mm and 13.5 ± 0.01 mm, respectively for S. aureus. Besides, 30 mg/mL of ZnO NPs killed 41%, 38%, 37%, 53% of E. coli, Klebsiella spp., S. typhi and S. aureus bacterial strains with LR values of 0.23, 0.19, 0.21, 0.33 respectively. Moreover, ZnO NPs killed 72.67%, 67.92%, 73.55%, 52.46% of E. coli, Klebsiella spp., S. typhi and S. aureus bacterial strains with PK values of 28%, 33%, 27%, 48% respectively at 20 mg/mL concentration. The facile pathway reported herein offers a promising approach for the synthesis of ZnO NPs for photocatalytic and anti-bacterial applications. The future research should explore the stability of the ZnO NPs in bio-environment and testing scalability of the process through pilot-scale experiments.

Author contributions

Md. Ashaduzzaman: Conceived and designed the experiments; supervision, analyzed and interpreted the data; fund awarded and contributed reagents, materials, analysis tools or data; review and editing the manuscript; Md Abdullah Al Muhit: Performed the experiments; drafting the manuscript; Shaikat Chandra Dey, Malay Kumar Das: Data curation, analyzed and interpreted the data; HN Mahmudul Hasan, Md Kaium Hossain: Prepared and analyze graphical presentation; Md Mizanur Rahaman, Nusrat Mustary: Performed the biological experiments; analyzed and interpreted the data, review and editing the manuscript.

Funding

The authors received fund from University Grants Commission of Bangladesh, Agargaon, Sher-e-Bangla Nagar, Dhaka-1207.

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.