A promising antimicrobial bionanocomposite based poly(3-hydroxybutyrate-co-3-hydroxyvalerate) reinforced silver doped zinc oxide nanoparticles

A bionanocomposite based on biosynthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and reinforced with silver@zinc oxide (Ag–ZnO) was synthesized in variable loadings of Ag–ZnO using the in-situ casting dissolution technique. The degradable biopolymer PHBV had been biosynthesized from date waste as a renewable carbon source. The fabricated products were investigated as promising antibacterial materials. The Ag–ZnO nanoparticles were also synthesized using the green method in the presence of Gum Arabic. The Ag–ZnO nanoparticles were loaded within the PHBV biopolymer backbone at concentration of 1%, 3%, 5% and 10%, PHBV/Ag–ZnO(1,3,5,10%). The chemical structure, morphology, physical and thermal properties of the PHBV/Ag–ZnO bionanocomposites were assessed via common characterization tools of FTIR, TGA, XRD, SEM and EDX. One step of the degradation process was observed in the range of 200–220 °C for all the obtained materials. The onset degradation temperature of the bionanocomposites have been noticeably increased with increasing the nanofiller loading percentage. In addition, fabricated products were investigated for their interesting antibacterial performance. A detailed biological screening for the obtained products was confirmed against some selected Gram-positive and Gram-negative strains S. aureus and E. coli, respectively. Overall, the bionanocomposite PHBV/Ag–ZnO(10%) was the most potent against both types of the selected bacteria. The order of bacterial growth inhibition on the surface of the fabricated bionanocomposites was detected as follows: PHBV/Ag–ZnO(10%) > PHBV/Ag–ZnO(5%) > PHBV/Ag–ZnO(3%) > PHBV/Ag–ZnO(1%).


Scientific Reports
| (2022) 12:14299 | https://doi.org/10.1038/s41598-022-17470-y www.nature.com/scientificreports/ synthesized biocomposites-where fillers have enhanced or even totally introduced new features on PHBV-have been investigated 13 . Applications were targeting packaging, biomedical applications, including tissue engineering, drug delivery and others. Most of the published biocomposites had the fillers from bio-origin leading to totally biodegradable composites. PHA applications and their biocomposites were focused in bone scaffolds and tissue engineering 14,15 , therapeutics field 16,17 , their copolymers 18 and many others 1,19,20 . PHBV biocomposites and their applications were reviewed in literature 13,21,22 . PHBV was very much investigated for packaging applications as well [23][24][25] . The antimicrobial properties of silver nanoparticles (AgNPs) and zinc oxide (ZnO) have been incorporated into PHBV for food packaging [18][19][20][21][22][23][24][25][26][27][28] . Stabilized silver nanoparticles 0.04 wt% were used in PHBV to produce a nanocomposite with 56% reduction in oxygen permeability when compared to PHBV, and the films showed a prolonged-up to seven months-antimicrobial activity against the pathogens, Listeria monocytogenes and Salmonella enterica 26 . The use of zinc oxide alone with different morphologies and sizes (micro and nanoparticle sizes) to reinforce PHBV films was reported. Zinc oxide nanoparticles have enhanced the optical and thermal properties of the nanocomposite films in addition to the antimicrobial properties which qualifies it for food packaging 27 . Other examples include the use of zinc oxide with reduced graphene oxide into glycerol-plasticized PHBV to form hybrid nanocomposites that were melt-extruded 29 . PHBV and ZnO nanoparticles were prepared by solvent casting to form composite films where the optimum percentage of ZnO was reported to be 4 wt%. This reported optimum ratio showed the best barrier properties, the maximum Young's and storage moduli and tensile strength in addition to the highest crystallinity. The antibacterial properties were also confirmed 30 . To enhance mechanical and antimicrobial properties, the extract from the leaves of Thymus vulgaris was used for the synthesis of zinc oxide-silver nanocomposites which were incorporated into PHBV-chitosan for the purpose of achieving lowest immigration rate and hence to improve the shelf life of poultry items. Good antimicrobial activity has been reported that qualifies them for food packaging of poultry items 31 . Ternary composites with nanohybrids of silver and cellulose nanocrystals with PHBV via solution casting have been investigated, and higher percentages of silver nanoparticles were associated with better overall migration, mechanical, thermal stability, antibacterial and barrier properties, and suggested as promising materials for food packaging 32 . A nanohybrid of zinc oxide and cellulose nanocrystals with PHBV matrix was synthesized by simple solution casting, the fabricated nanocomposites had antibacterial ratios of 95.2-100% for both bacteria S. aureus and E. coli. They have also degraded with 9-15% after one week. The incorporation of zinc oxide and cellulose nanocrystals has improved barrier properties and hydrophilicity 33 .
Although the aforementioned studies reported the incorporation of AgNPs and ZnO nanoparticles into PHBV matrices, to the best of our knowledge, no information is available on the incorporation of mixture of Ag doped ZnO nanoparticles in PHBV matrices. Therefore, the main goal of this work was to develop and characterize the antibacterial activity and the physicochemical properties of PHBV bionanocomposites and layer films containing both Ag-ZnO nanoparticles. The Ag-ZnO nanoparticles were synthesized using Gum arabic, and also the PHBV had been biosynthesized by the microorganism Haloferax mediterranei utilizing date waste as carbon source.

Materials and methods
Materials. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was produced by Haloferax mediterranei via biosynthesis utilizing date waste as carbon source and with high 3-hydroxyvalerate (3 HV) content of 18.0 mol%. Date waste had been purchased from local market at Qassim region in Saudi Arabia. The produced PHBV was of high molecular weight (746.0 kDa), narrow polydispersity (PDI = 1.5) and a melting point at 148.1 °C, the details of the biosynthesis were previously reported 34 . Biosynthesis of PHBV polyester. Date waste has been purchased from local market at Qassim region in Saudi Arabia. PHBV had been biosynthesized by halophilic archaeon Haloferax mediterranei from date waste biomass as feed-stock, the complete details of biosynthesis and characterization have been previously described in a published research 34 . The study complies with local and national guidelines in Saudi Arabia.

Synthesis of Ag-doped ZnO nanoparticles using Gum Arabic (GA).
To synthesize Ag-ZnO nanoparticles using Gum Arabic as a green procedure, 50 mL of AgNO 3 (0.001 M) was added to 50 mL of ZnSO 4 .7H 2 O (0.1 M) and the mixture was stirred for 0.5 h. To this mixture, 40 mL of GA solution (1% (w/v)) was added and the stirring was continued for another 0.5 h. The pH of the reaction mixture was adjusted to pH ~ 10 by drop wise addition of 1 M NaOH solution and the mixture was further stirred for 1.5 h at room temperature. The resulting colloidal solution was aged at room temperature for 24 h and was centrifuged. The product thus obtained was washed well with ethanol and distilled water and was dried at 450 °C in an electric oven. The dried product was calcined for 1 h at 400 °C in a muffle furnace.
Synthesis of PHBV/Ag-ZnO bionanocomposites. Different amounts of Ag-ZnO (0.007 g, 0.021 g, 0.035 g, 0.07 g) nanoparticles were weighed and added separately to glass tubes with 5 ml CHCl 3 in each, and the suspensions were sonicated for 15 min. In four 50 ml beakers, 0.7 g of PHBV polymer was dissolved in 18 ml CHCl 3 in each of the four beakers with stirring for 30 min. The nanoparticle suspensions in CHCl 3 were poured separately into the four 50 ml beakers containing the PHBV polymer dissolved in CHCl 3 and the resultant four mixtures was stirred for 5 min. Then the formed suspensions were poured into watch glasses and covered with a glass cover and left to dry. The formed bionanocomposites (1%, 3%, 5% and 10%) were then taken and characterized.
Thermogravimetric analysis (TGA). High resolution dynamic Thermogravimetric analysis (TGA) were performed under a continuous Nitrogen flow and recorded on a TA Instruments Hi-Res TGA Q500 thermogravimetric analyzer with a heating rate of 5 °C per minute.
Antimicrobial activity study of PHBV/Ag-ZnO bionanocomposites. Surface inhibition assay. The fresh bacterial culture was developed one day before the experiment from the stored lab-cultured isolate for the surface inhibition assay on the media plate surface killing. The colony sample was taken from the reserved cultured plate and was inoculated into the 25 ml broth medium, where it was incubated overnight at 35-37 °C. The culture was diluted the next day and grown back to the mid-log phase (4 h incubation) since the most active phase of the log phase was retained and standardized by absorbance reading 0.5 at 600 nm filter and turbidity in culture was maintained according to the McFarland standard with the help of a colorimeter. During cultivation incubation, a stock solution of test compounds (1, 3, 5, 10% Ag-ZnO) was developed in an aqueous solution, and an antibiotic Amoxicillin was used as a positive monitor. Each compound's stock solutions were formulated at a standard concentration of 100 µg/ml. On the media surface, 5 µg/ml of each compound's solution was put on dry-cultured plates. These cultured plates were made from a new culture of the test strain, spread thinly over the plate, and dried for 10 min in the hooded chamber to allow the test strain to settle and the plating liquid to dry out. In addition, these plates were incubated overnight in the incubator without being inverted. Bacterial development around the compounds was found to be disrupted bacterial growth the next day.
MIC and MBC determination.

Results and discussion
Characterization of PHBV/Ag-ZnO bionanocomposites. The pre-synthesized Ag-ZnO nanoparticles with Ag percentage of 1 mol% were added to PHBV pure polymer to form composites with different Ag-ZnO reinforcements (1, 3, 5 and 10%). The final products have been abbreviated as PHBV/Ag-ZnO( x% ) where x is the percentage of the reinforced Ag-ZnO nanofiller. In order to study the synthesized nanoparticles and the new bionanocomposites characteristics, the samples were tested with FTIR, XRD, SEM, EDX and TGA common characterization tools. The FTIR spectrum of the neat PHBV polymer (Fig. 1) shows clearly the characteristic peaks of this polyester; most importantly is the intense peak at 1720 cm -1 that corresponds to the ester carbonyl C=O stretching frequency which also appears as intense peak in all the prepared composites with different percentages 34 .
This most intense peak at 1720 cm -1 is becoming even more intense in the case of the nanofiller percentages above 3%, with the highest intensity in case of 3% filler composition, this finding is in line with previous findings for a similar research 35 , the carbonyl peak-being a main polar part of the polyester-is expected to significantly contribute to the crystallinity nature of the polymer. Also, it has been reported that the stretching frequency position of the C=O is affected by the strength of interaction between polymer chains, namely the interaction C-H-C=O that results from the interaction between a carbonyl group in one chain and an α-methylene group adjacent to another carbonyl in another chain 35 . Frequencies at 1379 cm -1 and 1453 cm -1 corresponds to the amorphous part of the polymer 35 . The peaks at 2874 cm -1 , 2936 cm -1 and 2978 cm -1 corresponds to the different C-H stretching frequencies. Other peaks frequencies assignments are also observed and match with reported 35 . As shown in Fig. 2, the combined spectra of pure PHBV and its composites with different percentages of Ag-ZnO show very similar peaks frequencies at the whole range and this indicates that the PHBV polymer bonds strengths haven't changed any significantly due to the incorporation of the nanofiller.
The XRD crystallographic pattern for the pure PHBV copolymer and Ag-ZnO nanofiller is very well matching the expected diffraction patterns for both samples. The pure PHBV pattern shows peaks (020) 36 , the study has reported these peaks of planes (101) and (002) at slightly higher values for samples with higher percentages of Ag doping, and the peaks positions here in this study matches well with the same previous study reported values for the 1% doping of Ag in ZnO. The authors also claimed that the incorporation of Ag within the lattice of ZnO which occurs through Ag + ions substituting Zn 2+ ions in the ZnO lattice is quite difficult due to the relatively big difference in the ions sizes, (1.22 Å) and (0.74 Å) for Ag + and Zn 2+ , respectively. Another point that is worth-mentioning here is that the existence of the characteristic diffraction peak at 38.0 degrees clearly resembles the existence of crystalline silver clusters in the synthesized nanoparticles, and this is explained by the previous discussion on the size difference between the two ions of Ag + and Zn +2 . Other XRD measurements were conducted on the neat PHVB polymer and for all the bionanocomposites of PHBV with different percentages of Ag-ZnO nanoparticles. As shown in Fig. 3 37 . The intense peaks as 13.7° and 17.1° are connected with the crystalline part of the polymer, while the broad peaks at 2θ = 20° to 22° are corresponding to the amorphous phase in the PHBV 35 . Figure 4 shows the combined XRD spectra for neat PHBV in comparison to the Ag-ZnO nanofiller and the fabricated PHBV/Ag-ZnO( 1, 3, 5, 10% ) bionanocomposites.
In order to study the crystallization behavior of the bionanocomposites compared to the neat PHBV, the XRD characteristics peaks of both the neat polymer and its bionanocomposites have been investigated. The investigated diffraction peaks positions of the planes (020), (110) and (040) have shown no significant changes in their positions with a slight increase in peak widths which implies that the formed bionanocomposites are slightly less crystalline than the neat PHBV but are still highly crystalline in nature, and without any noticeable phase changes. On the other hand, a significant decrease in the sharp peaks intensities at 13.7°, 17.1° and 26.9° suggests a considerable decrease in the crystallinity of the bionanocomposites compared to the neat PHBV. Also, as expected, the peaks at 31.7°, 34.45°, 36.27° and 38.0° corresponding to the nanofiller are increasing in intensity Additionally, samples of neat PHBV, Ag-ZnO nanoparticles and their bionanocomposites PHBV/ Ag-ZnO (1, 5, 10%) have been all analyzed by thermogravimetric analyzer in the temperature region of RT to 800 °C. As can be seen in Fig. 5, it was depicted from the analyses that peak degradation temperatures have been shifted towards higher values for the composites of 1% and 10%, indicating a better thermal stability for the polymer by introducing these percentages of the filler. One step degradation step has been observed in the range of 200-220 °C for all the tested products. Also, the onset degradation temperature has been noticeably increased in case of the composite with 10% filler. Noticeable increase in the thermal stability has been investigated in comparable to the pure copolymer. This general increases in the thermal stability of the pure biosynthesized   www.nature.com/scientificreports/ copolymer can be attributed to the incorporation of the metal oxide Ag-ZnO between the chains with possible bonding between metal part from the filler and the carbonyl groups in the neat copolymer. The SEM images of the Ag-ZnO nanoparticles and PHBV/Ag-ZnO (1,5,10%) bionanocomposites, are illustrated in Fig. 6. The typical Ag-ZnO nanoparticles images are given in Fig. 6a,b. Micro forest structures of flower like porous ZnO were observed. Whereas, the different compositions of Ag-ZnO reinforced to pure PHBV copolymer chains are displayed in Fig. 6c-h. These images confirm the homogenous and uniform distribution of Ag-ZnO nanoparticles within the copolymer matrix. In addition, the Ag-ZnO nanoparticles size in the bionanocomposites ranging from 40 to 70 nm in a rod-like shapes that are unevenly distributed within the PHBV  www.nature.com/scientificreports/ cavities. Also, the existence of Zn has been confirmed by EDX analysis of specific spots during SEM imaging of PHBV/Ag-ZnO (5%) bionanocomposite as a selected example as illustrated in Fig. 7. Ag peaks are not visible as its doping percentage is below the detection limit. The Au peaks are due to the gold coating used for SEM sample preparation.

Antimicrobial activity of PHBV/Ag-ZnO bionanocomposites
Bacterial surface inhibition. The surface inhibition assay is a critical parameter for the determination of bacterial resistance to compounds added to the media layer. They are monitored to interact with increasing microbes and visible patterns are observed. The Kirby and Bauer disk diffusion assay has been updated for this process. Without automatic tests, it is a feasible alternative to the broth dilution method of substance testing. Without using a filter paper disk impregnated compound, a known concentration of test compound (5 µg) was directly added to the surface of the cultured plate, where it absorbed the water from the agar and diffused the surrounding compounds. During the incubation period, the compound interacted with the culture bacteria.
When an antimicrobial compound is present at the critical point of diffusion, bacteria's growth at critical biomass is blocked, and a clear zone forms around the applied bionanocomposite. As expected, and shown in Fig. 8, the bionanocomposite with 10 wt% Ag-ZnO (PHBV/Ag-ZnO (10%) ) created a maximal zone of 13 and 14 mm inhibition against gram-positive S. aureus and Gram-negative E. coli bacterial stains, respectively. The order of antimicrobial action against bacterial strains in the sequence of composites according to the increasing percentage of the added filler of Ag-ZnO against both bacterial test strain S. aureus and E. coli, the order of bacterial growth inhibition on the surface PHBV/Ag-ZnO (10%) > PHBV/Ag-ZnO (5%) > PHBV/Ag-ZnO (3%) > PHBV/Ag-ZnO (1%) . Overall, the bionanocomposite with PHBV/Ag-ZnO (10%) was the most potent against both types of the bacterial strains.

MIC and MBC determination
The minimum inhibitory concentration was calculated using the broth dilution process, which involved subculturing compound-treated bacteria onto agar plates using the spread plate technique without changing the assay agent in the plate. However, MIC is the lowest antibacterial compound concentration that decreases the viability of the original bacterial inoculum by at least 99.9%. The MBC is the minimum bactericidal concentration which is the lowest concentration of test compound that destroys a bacterium in a fixed-dose or comparatively long period, such as 24 h, under optimal conditions. Overall, the MBC is a supplement to or expansion of the MIC test; moreover, the MIC test determines the lowest level/concentration/dose of test compound that significantly inhibits bacterial development. The tested composite is considered bactericidal if its MBC value does not surpass four times the MIC value. The MBC test can be used to assess the potency of a drug and to rule out issues during formulation, such as the active ingredient being bound to other compounds and thus unable to kill bacteria in the media. Gram-positive bacteria (S. aureus) were found to be more susceptible than Gram-negative bacteria (Escherichia coli) in this research and this was in line with previously reported research which showed that E. coli is more resistant to ZnO than S. aureus 38 . The bionanocomposite of PHBV/Ag-ZnO (10%) was the most effective antibacterial composite against all types of bacteria when synthesized composites were used as antimicrobial agents. The bionanocomposite with PHBV/Ag-ZnO (10%) had MIC and MBC levels of 15 and 30 µg/ml against E. coli and 10 and 20 g/ml against S. aureus, respectively. The bionanocomposites x% Ag-ZnO antimicrobial property in MIC and MBC against S. Aureus and E. coli are depicted in Table 1. As MIC and MBC findings were compared, PHBV/Ag-ZnO (1%) was the least toxic against both bacterial strains, and hence the conclusion that

Conclusion
Pre-synthesized Ag@ZnO nanoparticles with Ag percentage of 1 mol% had been successfully composited with biosynthesized PHBV copolymer to form novel bionanocomposites with different percentages of Ag-ZnO reinforcement (1, 3, 5 and 10%) and were used for the fabrication process with given abbreviations of PHBV/ Ag-ZnO (1,3,5,10%) . The synthesized bionanocomposites had been characterized by FTIR, XRD, SEM and EDX which confirmed the chemical structures for the end products. The thermal behavior had been studied by TGA thermal analysis. All the products gave the same degradation profile. A fast one step of degradation thermogram had been seen for the fabricated PHBV/Ag-ZnO (1,3,5,10%) . The antibacterial performance of the casted bionanocomposites was confirmed by the study against selected Gram positive and Gram negative strains (S. aureus and E. coli), respectively. The order of antimicrobial action against bacterial strains was found in the sequence of composites according to the increasing percentage of the added filler of Ag-ZnO against both bacterial test strains S. aureus and E. coli. The maximal observed inhibition zone against gram-positive S. aureus and Gram-negative E. coli bacterial strains was associated with PHBV/Ag-ZnO (10%) which gave 13 mm and 14 mm inhibition diameter, respectively. The synthesized bionanocomposites are considered of great interest in the development and design of biodegradable active packaging materials for food industry.