Evaluation of bactericidal potential and catalytic dye degradation of multiple morphology based chitosan/polyvinylpyrrolidone-doped bismuth oxide nanostructures

In this study, 0.02 and 0.04 wt% of chitosan (CS) were successfully incorporated in a fixed amount of polyvinylpyrrolidone (PVP)-doped Bi2O3 nanostructures (NSs) via a co-precipitation approach. The purpose of this research was to degrade hazardous methylene blue dye and assess antimicrobial potential of the prepared CS/PVP-doped Bi2O3 nanostructures. In addition, optical characteristics, charge recombination rate, elemental composition, phase formation, surface morphology, functional groups, d-spacing, and crystallinity of the obtained nanostructures were investigated. CS/PVP-doped Bi2O3 nanostructures exhibited efficient catalytic activity (measured as 99%) in a neutral medium for dopant-free nanostructures while the inhibition zone was measured using a Vernier caliper against pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) at low and high doses to check antimicrobial activity. Strong bactericidal action was recorded against S. aureus bacteria such that a significant inhibition zone was measured at 3.09 mm.


Introduction
Increased economic activity and fast industrial growth have exacerbated water pollution and health-related concerns globally. 1 World Health Organization (WHO) estimates that each year 2.3 million people die from water-borne (typhoid, cholera, hepatitis, and diarrhea) and carcinogenic diseases. [2][3][4] Around 70% of water pollution is produced due to industrial waste dyes (acidic, basic, and azoic) and heavy metals (cadmium, chromium, nickel, lead, etc.). All such pollutants are highly soluble in nature and pose a serious health risk to humans and wildlife. 5 Dyes are excessively used in paper coloring i.e., temporary hair colorants, cotton dyeing, and paper stock coating. Especially methylene blue (MB), a basic dye, has an aromatic molecular structure that is stable and nonbiodegradable posing strong ecological threat to aquatic life. 6 According to published research, 15% of the most extensively produced dyes are released into water bodies both directly and indirectly. 7 As a result, it is important to employ a method capable of degrading synthetic dye directly into non-toxic molecules such as water and carbon dioxide. Scientists use traditional approaches such as chlorination, aerobic treatment, adsorption, and ion exchange to remove organic contaminants from water. Unfortunately, these techniques have drawbacks and limitations such as high energy consumption, secondary pollution caused by inadequate removal, and transfer of dye. 8,9 Catalysis in the presence of nanomaterial-based semiconductors attracted interest of researchers owing to their minimal toxicity, chemical stability, low cost and naturefriendly characteristics. 10 In order to degrade synthetic dyes such as MB, this research uses a reducing agent and nanocatalyst. [11][12][13] Mastitis has a substantial economic burden on the dairy sector. Infectious agents such as bacteria, viruses, and fungi cause mastitis. Chemical, microbial, and physical changes in milk, and clinical abnormalities in mammary gland tissues, are all associated with this disease. 14 Coliform, Escherichia coli and Staphylococcus aureus are the most prevalent bacterial pathogens linked to mastitis. 15,16 Nanomaterials have attracted researcher's attention due to their unique physiochemical properties and enhanced dyecontaminated wastewater treatment methods. [17][18][19] Small NSs with size ranging from 1 to 100 nm have astonishing surface-tovolume ratios when compared to those of bulk chemical compositions, resulting in signicant increases in chemical (biological, catalytic activity, etc.) and physical properties. Metal oxide nanomaterials have large surface area, and attractive nanostructural, optical, mechanical, and thermodynamic characteristics that are advantageous for catalysis and antibacterial activities. 20 Numerous metal oxide nanomaterials (ZnO, TiO 2 , La 2 O 3 , CeO 2 , and Bi 2 O 3 ) are being used in catalysis and to check antibacterial activity; particularly as an important p-type semiconductor, Bi 2 O 3 has remarkable anode semiconductor properties including a broad band gap, low toxicity, high conductivity, antibacterial activity and degradation capability for organic dyes. [21][22][23][24][25][26][27][28][29] Chemical (co-precipitation, sol gel, and redox reactions) and green synthesis techniques are utilized to synthesize Bi 2 O 3 NSs. 21,22,30,31 Among these, the coprecipitation method is considered as ecofriendly, inexpensive, energy-efficient and easy to use. 23 A number of research studies were conducted on Bi 2 O 3 NSs prepared through various synthesis routes to check the inuence of antimicrobial activity and dye degradation. [32][33][34][35][36][37][38][39][40][41] However, the obtained results were not impressive for bactericidal action and dye degradation performance. The addition of a polymer into metal oxides increases their stability and improves physiochemical properties which results in efficient dye degradation and antibacterial performance.
Polymers can interact with metal ions either through complex or ion-pair formation, which might be an attractive substitute for a stabilizer and thus can be targeted to attain specic physicochemical parameters of NSs. 24 Polymeric materials have received much attention from scientists for usage in biological and environmental applications. 42 Numerous types of polymers (polyvinyl alcohol, polyvinyl chloride, polyvinylpyrrolidone, and chitosan) are used for metal oxide doping to attain signicant outcomes for various applications. [43][44][45] Among them, PVP is a synthetic polymer that is considered an effective capping agent for metal oxide NSs. Its properties are attributed to the presence of both carbonyl groups and functional groups that strengthen metal oxide NSs within its composite. 46,47 As it exhibits excellent physicochemical properties, it is used as an additive in different materials and to stabilize NSs. [48][49][50][51] Coincidently, recent studies have shown that PVP has great water solubility, low toxicity, biocompatibility, and exhibits promising results against antimicrobial activity. [52][53][54][55] Chitosan is an alkaline polymer prepared by partially hydrolyzing chitin, the primary component of crustaceans and fungus cells, and extensively used for pharmaceutical and biomedical purposes. It has superior biodegradability, biocompatibility, low toxicity, and lm-forming characteristics. 56 CS is mainly composed of amino and hydroxyl groups, both signicant in metal ion chemical adsorption, and these groups can bind with metal ions more efficiently than any other polysaccharide, making a strong template for synthesizing metal oxide NSs. [57][58][59][60][61] The motivation of this research is to synthesize PVP/CSdoped Bi 2 O 3 NSs utilizing an ecologically friendly coprecipitation technique for degradation of organic dyes from contaminated water and also to assess material's bactericidal potential. Numerous characterization techniques were employed for detailed analysis of synthesized NSs. Catalytic    Fig. 1(a).

Catalysis
The degradation efficiency of synthetic dyes in the presence of sodium borohydride (NaBH 4 ) and the synthesized nano-catalyst was determined through CA measurements. MB is a positively charged thiazine dye frequently used as a reductant in analytical chemistry, and is colorless in the reduced form and blue in the oxidized form. 62 Using a quartz cell, 0.1 M NaBH 4 solution (400 mL) was dissolved in 3 ml MB. Furthermore, 400 mL synthesized NS solution was incorporated in aqueous solution of MB. Absorption reaction progress was spectrophotometrically monitored at room temperature. In the presence of NaBH 4 , MB changed to leucomethylene conrming degradation of dyes. Samples without a nano-catalyst were referred to as blank. % degradation was calculated as: where C 0 represents initial absorbance and C t represents the concentration at specic time.

Catalysis mechanism.
Adding a nano-catalyst and reducing agent to the dye are the major factors considered to be signicant in the catalysis mechanism as demonstrated in Fig. 1(b). The chemical material provides an e À to the ongoing reaction referred to as the reducing agent. MB receives an e À from the diminishing agent in a chemical reaction to act as an oxidizing agent. The redox reaction occurs during CA, and involves the transfer of an e À from the reductant to the acceptor of an oxidant. This leads to electron absorption in MB and causes the breakdown of the synthetic dye. Furthermore, MB was tested in the presence of reducing agent (NaBH 4 ), this oxidation reaction was incredibly slow and time consuming. To overcome these issues, incorporation of nano-catalysts (Bi 2 O 3 and PVP/CS (2%, 4%)-doped Bi 2 O 3 ) into oxidation-reduction reactions serves as electron relay and allows electron transfer from the donor (BH 4 À ) to the acceptor (MB). Adsorption of BH 4 À ions and dye molecules is increased by using NSs while a large number of active sites encourage them to react with each other quicker resulting in efficient dye degradation. 63,64 The presence of a reducing agent and nano-catalyst increases the degradation efficiency. As reported above, a catalytic route was adopted for dye degradation utilizing reducing agents and nano-catalysts in this study. 65

Isolation and identication of Staphylococcus aureus and Escherichia coli
Large quantities of dairy milk specimens were obtained from Pakistani public, private institutions and dairy farms and evaluated for surf-eld mastitis. Furthermore, the acquired samples were incubated in 5% sheep blood agar. On Mannitol salt agar (MSA) and MacConkey agar (MA), colonies were formed in order to isolate Gram-positive (G +ve) S. aureus and Gram-negative (G Àve) E. coli pathogens, respectively. Pharmacological (catalase and coagulase) and morphological (gram staining) methodologies were used to identify distinctive colonies.

Antimicrobial activity
Antibacterial performance of the prepared NSs was examined through the agar well diffusion approach with germ strain (G +ve and G Àve) swabbed 1. Paper Nanoscale Advances diameter of the inhibition zone that results in the determination of antibacterial performance. One-way variance analysis in SPSS 20 was employed to determine the bacterial efficiency by measuring the inhibitory zone.

Characterization techniques
Structural and crystalline behaviors of obtained powder were determined using powder XRD ranging from 10 to 60 . FTIR spectroscopy was performed between 4000 and 400 cm À1 to identify functional groups present in PVP/CS (2%, 4%)-doped Bi 2 O 3 NSs. The chemical composition, surface study, morphology and d-spacing of PVP/CS (2%, 4%)-doped Bi 2 O 3 NSs were analyzed through EDS, SEM and HR-TEM respectively. Additionally, SAED analysis was performed to check crystallinity of the prepared samples. A Genesys 10S UV-vis spectrophotometer was employed to determine the optical properties while PL spectroscopy was used to investigate electron-hole recombination in the synthesized sample. XRD identies NSs crystallinity, crystal structure, and crystal size ranging from 2q 10-60 ( Fig. 2(a)).  Fig. 2(b)). The Bi-O-Bi stretching vibration, C-C stretching and product vibration mode of NO 3 were assigned to 540 cm À1 , 1076 cm À1 , 1357 cm À1 bands correspondingly. 31 Fig. 2(c-f) suggesting highly crystalline nature of the samples. XRD measurements satisfying Bragg's diffraction conditions were well correlated with various planes of NSs. Fig. 3(a) reveals the band gap energy (E g ) and optical properties of the synthesized samples assessed with a UV-visible spectrophotometer between 250 and 500 nm. It shows a considerable absorption peak at 295 nm for Bi 2 O 3 . 80 The wavelength acquired from UV-visible absorption spectra determined E g of dopant free and PVP/CS-doped Bi 2 O 3 NSs to be 4.18 eV, 4.27 eV, 4.09, and 4.13 eV respectively. 80 Upon doping with PVP, the absorption in higher wavelength (blue shi) was observed, ascribed to an increase in E g and decrease in the crystallite size. Furthermore, addition of CS resulted in absorption toward longer wavelength (red shi) indicating a decrease in E g and increase in the crystallite size. Increasing amount of CS reduces the crystallite size that results in increased E g which is well matched with the XRD results.
PL analysis elucidates the electron-hole pair recombination process in all synthesized samples as shown in Fig. 3(b). The photoluminescence signal is produced when electrons in the VB are excited to the CB at an excitation wavelength and subsequently return to the VB. 81 Bi 2 O 3 NSs emit broad emission peaks in the visible range from 520-542 nm, attributed to Bi 3+ ions, when excited at 300 nm. 82 The luminescence of ions in the green region is produced by the 3 P 1 -1 S 0 transitions, or charge transfer between the bonding oxygen and Bi 3+ ions. [83][84][85] When PVP was incorporated, peak intensity decreased, indicating lower charge recombination while peak intensity increased upon increasing the concentration of CS, which suggests a high photo-generated charge carrier recombination tendency. 86 The chemical composition of PVP/CS (2%, 4%)-doped Bi 2 O 3 NSs determined through EDS is represented in Fig. 4(a-d). Strong peaks of Bi and O were observed that conrm the presence of Bi 2 O 3 NSs in the synthesized samples. The carbon peak is attributed to PVP/CS used in the samples. The sodium (Na) peak was probably generated by the use of NaOH to sustain the pH of samples while Au peaks originate due to the coating sputtered upon the samples to reduce charging effects. Small peaks of Cu and Zn could be attributed to the effect of the brass sample holder utilized during EDS observation and to some contamination. Additionally, EDS mapping of the as-prepared higher doped specimen was carried out to analyze the distribution pattern of its elemental constituents in order to check additional interfacial contact as represented in (e). Five components (Bi, O, Na, Cu, and Zn) were found to spread in the higher doped samples. As already mentioned, Na, Cu, and Zn were assigned to contamination, the sample holder used for EDS analysis.
TEM images conrmed the morphologies of Bi 2 O 3 and doped Bi 2 O 3 as illustrated in Fig. 5(a-d). The image of the control sample showed multiple morphologies including quantum dots while a few nanorods were also observed ( Fig. 5(a)). Addition of PVP showed that quantum dots were covered with PVP ( Fig. 5(b)). Addition of low concentration of CS to PVP/Bi 2 O 3 resulted in agglomeration of nanorods and quantum dots, which led to the formation of nanoclusters (Fig. 5(c)). Upon higher amount of CS addition, agglomeration increased with the signicant nanorod-type structure of CS visible (Fig. 5(d)). Additionally, interlayer d-spacing was calculated from HRTEM images using Gatan soware ( Fig. 5(a 0 -d 0 )). Bi 2 O 3 and PVP/CS (2%, 4%)-doped Bi 2 O 3 NS d-spacing values were found to be 0.271 nm, 0.311 nm, 0.193 nm, and 0.199 nm, which are well compatible with the XRD results.
Catalytic activities of pure and PVP/CS (2%, 4%)-doped Bi 2 O 3 NSs with NaBH 4 for MB degradation under acidic, neutral, and basic conditions were investigated using a UV-vis spectrophotometer. Dye sludge is frequently released at various pH levels; the rate of degradation is inuenced by the pH solution and affects nano-catalysts that have been synthesized. Undoped and PVP/CS (2%, 4%)-doped Bi 2 O 3 nanomaterials showed the maximum degradation of 99.48%, 77.90, 97.95%, and 75.54% in neutral (pH ¼ 7), 98.35%, 98.93%, 98.27% and 96.68% in basic (pH ¼ 12), and 89.54%, 68.07%, 94.51% and 93.84% in acidic (pH ¼ 4) media ( Fig. 6(a-c)). In all media, PVP/CS (2%)doped Bi 2 O 3 demonstrated the highest catalytic activity. The surface area crystallite size and shape of the nano-catalyst substantially inuence CA. On doping with CS, variation in  Paper Nanoscale Advances the dye degradation was observed, which is attributed to the presence of more active sites provided by catalyst's large surface area which results in high catalytic efficiency. In addition, the surface area is generally large, but the inuence of the nanocatalyst is limited due to micro-porosity, which inhibits the reactants from diffusing to the catalyst surface. 87 Furthermore, a slight difference between an acidic and basic medium is ascribed to increased electrostatic attraction between MB + , a positively charged dye and the catalyst which is negatively charged. The nanocatalyst surface in the basic medium tends to   Fig. 7(a). The rate constants (k) have been calculated for catalytic degradation kinetics by measuring slopes of ln(C 0 /C t ) against time. Degradation rate constant k for undoped and doped Bi 2 O 3 NSs was calculated to be 0.03095, 0.24174, 0.06604 and 0.66335 min À1 , respectively Fig. 7(b).
Investigating the stability of the nano-catalyst is of economic importance. As mentioned earlier, catalytic activity in the basic medium exhibits excellent dye degradation results. Therefore, the stability of the catalyst in the basic medium was investigated by allowing the experiment to stay for at least 72 hour in order to examine whether the reduction of dye as observed in the presence of the nanocatalyst is stable or not. In this case, the degraded dye was kept in the dark and the degradation was monitored using absorption spectra obtained through a UV-vis spectrophotometer every 24 hours, as shown in Fig. 7(c). The obtained results indicate that no loss of degradation occurred under stable conditions for 72 h. Degradation was observed to be in its fairly original form which affirms the stability of the catalyst. Fig. 8 Fig. 9(a and b). All concentrations of E. coli at low dose exhibited zero efficacies as shown in Fig. 9(d). A negligible efficiency was shown by Bi 2 O 3 for E. coli and S. aureus at low and high doses respectively Fig. 9(c and d). Furthermore, inhibition zone 4.25 mm against S. aureus and E. coli for ciprooxacin (positive control) parallel to 0 mm DIW (negative control) was recorded. Apart from this, doped Bi 2 O 3 NSs showed substantial (P < 0.05) antibacterial efficacy against S. aureus as compared with E. coli. In general, cell walls of Gram negative bacteria are thicker and have a more complicated structure than Gram positive bacteria. The comparison of the present work with the literature is presented in Table 2.
Nanomaterials produce oxidative stress that is directly proportional to their concentration, shape and size. The particle size and concentration affect antibacterial activity. The size of the material has an inverse relationship with the antimicrobial efficacy. 90 Small sized particles produce more reactive oxygen species (ROS) causing cytoplasmic components to extrude and kill bacteria by harmful microorganism membrane implant. 91,92 Sufficient distribution of Bi 3+ inside bacterial cells increases its antimicrobial activity as it destroys bacterial membrane stability and inhibits biolm formation as shown in Fig. 10. 91

Conclusion
In the present work, Bi 2 O 3 and PVP/CS-doped-Bi 2 O 3 NSs were successfully synthesized to achieve an improved bactericidal and catalytic activity. Among all the prepared samples, CS doping in PVP-Bi 2 O 3 with 2% and 4% concentrations showed effective catalytic and antimicrobial activities, respectively. In view of the experimental results, Bi 2 O 3 exhibited a monoclinic

Data availability
Data is available on suitable demand.

Conflicts of interest
The authors declare no conict of interest.