Biodegradable Films from Phytosynthesized TiO2 Nanoparticles and Nanofungal Chitosan as Probable Nanofertilizers

Titanium dioxide nanoparticles (TiO2-NPs) have great importance for plant nutrition and growth, at little concentrations. The bioactive polymer chitosan and its NPs provide outstanding characteristics for capping and enhancements of nanometals. The phytosynthesis of TiO2-NPswas promisingly achieved using an extract of pomegranate rind, whereas the fungal chitosan (FCt) was produced from Aspergillus brasiliensis biomass and was transformed to nanoform. The phytosynthesis of TiO2-NPs generated homogenous spherical particles with 13 to 64 nm range and 37 nm mean size. The extracted FCt had 92% deacetylation degree and a molecular weight of 28,400Da. The infrared spectral analysis of TiO2-NPs, FCt-NPs, and their nanocomposite indicated their functional groups and biochemical interactions. The released amounts of TiO2-NPs from their nanocomposite with FCt–NPs were 31% and 50% after the first and third hour, respectively. The nanocomposite film had a faster hydrodegradability rate which resulted from TiO2-NP addition. Therefore, the fabricated nanocomposite from FCt/TiO2NPs could have elevated potentiality for application as liquid spray for foliar feeding or as powder for soil amendment.


Introduction
Titanium (Ti) element has important biological consequence for plants, being advantageous at lower concentrations and potentially toxic at higher ones, but with miniature toxicity toward animals or human [1]. Whereas TiO 2 antimicrobial potentiality was reported against many microorganisms [2,3], it has many beneficial consequences for plant physiological attributes, especially at little dosages (the Ti contents in plant dry weight ranged from 1 to 1000 mg/kg with profitable application range of up to 100 mg/L), including their elemental contents, biomass yield, chlorophyll contents, and foliar growth [4][5][6].
Nanofertilizers emerged as promising candidates for enhancing micronutrients' uptake efficiency; their successful practical applications promoted the search for more systems to improve their delivery, e.g., nanocarriers and nanocom-posites [7,8]. Formulated nanofertilizers could have slower release of nutrients, which improves their usage, solubility, bioavailability, and dispersion [7]. Compared to traditional fertilizers, nanofertilizers could be absorbed by crops easily, with sustained nutrient delivery into soil/plant [8].
The practical exogenous applications of TiO 2 -NPs in crop propagation indicated their efficacy for enhancing plant performance, biomass production, photo-reduction activities, and nitrogen assimilation [10,11]. Moreover, these micronutrient NPs could protect chloroplast membranes from rеactivе oxygеn spеcies (ROS) attacking and destruction [4]. TiO 2 -NPs can also improve other native nutrient utilization via support of beneficial microbial activities [12]. The TiO 2 -NPs were effectually phytosynthesized (synthesis enforced by plant derivatives) using extract of various parts of plants [13][14][15], which was advised for increasing the process safety, feasibility, and efficiency.
The biosynthesis (green synthesis) of nanomaterials has recently become a key method for preparation of many bioactive NPs with augmented activity and reduced toxicity during their preparation and applications; the synthesis could be enforced by NPs incorporation with numerous types of biopolymers [16][17][18].
Biosynthesized nanometals and nanocompounds were validated as effectual and powerful antimicrobial agents in biomedical fields and their successful applications were proved [19][20][21].
The extract of pomegranate rinds (PRE), as a byproduct with plenty of valuable phytochemicals and biological activities, was employed for the green phytosynthesis of many metal NPs including silver, iron, and zinc [22][23][24], as the PRE exhibited strong reducing capability for these metals.
The polymer NPs, particularly from Cts and FCt, are well proved for their surplus functionalities and bioactivities as nanocarriers, biochelators, antimicrobial, biosorbent, and plant protectant agents, in individual or composited forms with other bioactive compounds and nanomaterials [28][29][30].
The application of Cts and FCt-NPs was advised for soil and water remediation from heavy metals due to their capabilities for heavy metal chelation/adsorption [28,29].
The incorporation of TiO 2 with Cts was reported to improve the composite structural, mechanical, optical, textural, vapor barring, and thermal properties and its biodegradability [31]. These composites had also augmented antimicrobial, viscoelastic, and biocompatibility attributes [3,32,33].
Accordingly, the intentions from this investigation were to phytosynthesize TiO 2 -NPs using PRE, extract FCt from grown mycelia of Aspergillus brasiliensis and transform it to NP form, then to conjugate both NPs for augmenting their release pattern and hydrodegradability.

Materials and Method
2.1. Pomegranate Rinds' Extract (PRE) Preparation. Organic pomegranate fruits (Punica granatum L.), obtained from international markets in KSA, Jeddah, were used in study. Fruits were disinfected (using 5% sodium hypochlorite solution) and peeled and their rinds were dried (for 50 h at 48 ± 2°C) and ground to 60-mesh powder size. The PRE was prepared through powder immersion for 36 h in ethanol (70%) with agitation then filtration to have the extract, which was vacuum dried at 42°C and resuspended in deionized water (DIW) to have 10%(w/v) concentration [34].
For TiO 2 -NP phytosynthesis, 50 mL of TiCl 4 solution in DIW (95 mg/mL) was mixed with equal volume of PRE with strong stirring for 60 min at 25 ± 1°C. The changing solution color to whitish-brown indicated TiO 2 -NP synthesis. Ammonium hydroxide solution (0.2 M) was slowly added to NP solution under stirring at 25 ± 1°C, until reaching alkaline pH of 8.0 and precipitate formation. The precipitated TiO 2 -NPs were collected via centrifugation (at 8600°g), washed with ethanol (95%), recentrifuged, and calcined at 470 ± 5°C for 200 min, then powdered finely.

Preparation of Fungal Chitosan/TiO 2 -NP Composite.
Grown mycelia of Aspergillus brasiliensis (ATCC-16404), after aerobic propagation in broth media of potato dextrose (containing infusion of 200 g potato+20 g dextrose/L), were used as chitosan source by extraction according to Tayel et al. [26]. Briefly, inoculated fungal conidial suspension (10 6 spores/mL), in 500 mL of broth medium, were incubated aerobically under stirring (120 g) for 6 days at 28°C.The FCt extraction involved fungal biomass harvesting and washing with DIW, treatment with 20 folds (v/w) from 1 M of NaOH then HCl and finally deacetylation by treatment with 20 folds from 60% (w/v) NaOH solution for 60 min at 110°C.
Later, 500 mg of FCt-NPs (dissolved in 50 mL of 1.5% acetic acid solution) and 50 mg from TiO 2 -NPs (dispersed in 5 mL of DIW) were combined and then sonicated for 30 min. The uniform composite emulsion was further magnetically stirred for 250 min to enforce NP crosslinkage [3].

Results and Discussion
3.1. TiO 2 -NP Phytosynthesis Using PRE. The TiO 2 -NPs could be successfully synthesized using PRE, as evidenced from their characterization (Figure 1). The phytosynthesized TiO 2 -NPs appeared with spherical and matched shapes with slight NP agglomerations (Figure 1(a)). The TiO 2 -NP size distribution ranged from 13.42 to 63.84 nm, with 34.21 nm median size and 36.71 nm mean size (Figure 1(b)). The recorded Zeta potential average for these phytosynthesized TiO 2 -NPs was -24.8 mV.
Numerous bioactive compounds are contained in PRE, including polyphenols, vitamins, flavonoids, esters, and protein. The polyphenols, vitamin C, and many other phytoconstituents contain extensive hydroxyl groups with strong reducing capability [22]. PRE was acknowledged to contain extraordinarily elevated phenolic compounds as natural antioxidants sources [37]. Thus, these biactive phytoconstituents are assumingly the one responsible for reducing TiO 2 to their NP form.
The PPE was effectively employed for the phytosynthesis of many metal nanoparticles, e.g., silver, gold, and zinc [22,23,38]; this is matching with its capability forTiO 2 -NP synthesis in current study.

Fungal Chitosan Extraction.
The chitosan was effectually extracted from A. brasiliensis mycelia; the extracted FCt had a molecular weight of 28,400 Da with 92.1% deacetylation degree.
The successfulness of FCt extraction from A. brasiliensis confirmed foregoing investigations that reported the fungi potentialities as sustainable alternative sources for Cts production [26,28,29,39]; these reports applied the extracted FCt, from varied fungal genera, in the environmental and biomedical fields.

International Journal of Polymer Science
NPs), the representative broad peak of Ti-O-Ti stretching was detected around 612 cm -1 wavenumber. The appeared vibrated bands at 1107 and 1120 cm -1 are specified the stretching/bending modes of CH 3 and Ti-OH, respectively, of the NPs surface groups [40].
The sharp peak (at 1638 cm -1 ) and the broad peak (at 3405 cm -1 ) are mainly attributed to the NP adsorbed water and occurrence of hydroxyl groups, respectively [14]. The hydroxyl groups' presence is commonly involving the photocatalytic activity augmentations; with the increased amount of OH− on TiO 2 -NP surface, their higher electron transportability and enhanced photocatalytic activity could be assumed [41].
The appeared peak at 651 cm −1 in TiO 2 -NP spectrum conceivably indicated the involvement of Ti anatase phase in phytosynthesized NPs; this phase could be further confirmed via X-ray diffraction (XRD) analyses. Conversely, the disappearance of any peaks around 2900 cm −1 , which indicates C−H stretching, validated that subtraction of the entire PRE organic components from the TiO 2 -NP sample during their calcination [14].
For FCt-NP spectrum (Figure 2, Cts-NPs), the strong wide peak around 3453 cm -1 could correspond to combined O-H stretching and hydrogen bonding; the peak wideness with increased intensity could indicate hydrogen bonding enhancement after NP synthesis [30]. The FCt-NP spectrum displayed also (at 1171 cm -1 ) a sharp indicating peak for P=O, due to crosslinkage of FCt with TPP. The main characteristic absorption peaks of FCt were detected at 1714 cm -1 (C=O carbonyl stretching within the secondary amide I band), at 1541 and 1322 cm −1 (the bending vibration of N-H in amide II and the amide III absorption, respectively), and at 1083 cm -1 (due to C-O-C stretching).
For FCt/TiO 2 -NPs, the appeared peak at 1391 cm -1 indicates the CH 3 band stretching vibrations in the nanocomposite [32,33], and the TiO 2 -NP incorporation had obvious impact on the intensities of characteristic peaks (Figure 2, Cts/TiO 2 -NPs).
The nanocomposite spectrum displayed many characteristic bands and peaks from both FCt-NPs and TiO 2 -NPs (designated by the vertical lines on the figure). The Ti-O band within 550-700 cm -1 range designated TiO 2 immobilization onto FCt matrix [42]. Compared with pure FCt-NP spectrum, many corresponding bands to amino, hydroxyl, and amide groups were shifted in FCt/TiO 2 -NP composite spectrum; these IR shifts confirmed the interaction between both the conjugated NPs.
The composited FCt/TiO 2 -NP microstructure and morphology were elucidated using SEM imaging ( Figure 3); they appeared with homogenized spherical shapes with some aggregation due to polymer collapse. The TiO 2 -NPs were mostly capped with FCt-NPs and composed uniform mixtures, as was formerly reported [33,42]. The low aggregate size in the nanocomposites is assumingly attributive to the organic nature of FCt that could hinder the aggregation of TiO 2 -NPs [43].

Release Pattern of TiO 2 -NPs from FCt-NP Nanocomposite.
The release pattern of TiO 2 -NPs from their nanocomposite with FCt-NPs is performed throughout 180 min releasing time ( Figure 4); the released TiO 2 amounts from the nanocomposite were 31.2% after the first hour and reached 50.2% at the experiment end. The release pattern TiO 2 -NPs and its influence with stirring time was indicated from other colloids, cream, and sunscreen [44,45]; the TiO 2 release percentages in these studies were higher than the obtained values in a current study, which indicate the high capability of FCt-NPs for entrapping and capping the TiO 2 -NPs and preventing them from disintegration. The main suggested factor for TiO 2 -NP    International Journal of Polymer Science releasing could be the degradation of FCt-based film that assists the liberation of capped metal NPs. The controlled release of TiO 2 -NPs, via their conjugation with FCt-NPs, is highly important for sustained fortification of plants with Ti ions for longer times and required concentrations [12,46].
Many patents and inventions advocated TiO 2 -NP applications in plant fertilization purposes, either as liquid or colloidal compositions, which could promote foliar and root growth [6,12]; this supports the potential application of current fabricated FCt/TiO 2 -NP composites as sustained and controlled source of Ti ions for plant fertilization. In addition, the controlled release of Ti, via incorporation in FCt/TiO 2 -NP composites, could be highly beneficial for providing the plant with this essential element without its potential toxicity at higher doses [6]. The sustained release of TiO 2 -NPs could, additionally, provide advantageous long-lasting antimicrobial potentiality against the pathogenic microbial communities in treated soils [2,3], whereas the application of TiO 2 -NP fertilizers was statеd to havе no еffеct on thе community structurе of еithеr rhizobia or arbuscular mycorrhizal fungi that colonized plant roots, at any concеntration [47]. This expected antimicrobial power from FCt/ TiO 2 -NPs could have influential consequences to protect soils and plants from pathogenic microbes.

Hydrolytic Degradation Patterns of Nanocomposed Films.
The hydrolytic degradation patterns of composed films from FCt-NPs and FCt/TiO 2 -NPs are illustrated in Figure 5. Both NPs-based films were gradually degraded and lost their weights with prolongation of experiments; the FCt/TiO 2 -NPs-based film showed faster degradation than FCt-NPsbased film. The FCt/TiO 2 -NP film completely degraded after 7 h of treatment, whereas the FCt-NPs film lost 67.4% of its weight at this time and its degradation percentage was 93.2% after 10 h of treatment ( Figure 5).
The degradation rate of FCt/TiO 2 -NPs-based film could be correlated with TiO 2 -NP release from this nanocomposite, as the degradation rate was 42.8% after 3 h and the liberated TiO 2 -NPs was slightly higher than this percentage, at the same time. The excess liberated amounts from the nanopolymer composite are assumingly due to decreased electrostatic bonds between them and electron exchanges within the nanocomposite after its hydrolytic destabilization [36,48].
Theoretically, TiO 2 -containing films are assumed to have a slower rate of degradation because of the antimicrobial action of TiO 2 -NPs that can retard microbial degradation of composited films [48], but the composed films from the PLA/TiO 2 composite exhibited greatly higher hydrolytic degradation rate than films from PLA resin [36]; this increased degradability of TiO 2 -incorporated films was attributed to the photodegradation properties of TiO 2 -NPs, which is activated by NP exposure to UV and visible lights and lead to faster degradation of their composited films [36,49]. These harmonized results with ours could advocate the incorporation of TiO 2 -NPs into nanocomposites to control their hydrodegradability [42]. Additionally, the improvement in the biodegradability of chitosan/TiO 2 hybrid composite was reported as a TiO 2 dose dependent [31,48], which advocates further experiments to specify the exact optimum TiO 2 -NPs for controlling films' degradability.
However, the fabricated nanocomposite here from FCt/ TiO 2 -NPs could have elevated potentiality for application as liquid spray for foliar feeding or as powder for soil amendment [50].

Conclusion
The phytosynthesis of TiO 2 -NPs was innovatively achieved using PRE and their nanocomposites with FCt-NPs had homogenous organization and miniature sizes. The nanocomposite had a faster hydrodegradability which resulted from TiO 2 -NP addition, which advocates its application as liquid spray for foliar feeding or as powder for soil amendment. These formulated nanocomposites could be possible candidates for application as nanofertilizers to deliver TiO 2 -NPs into plants in a controlled manner. However, more investigations are required to judge the practical application of FCt/TiO 2 -NP nanocomposite as a fertilizer.

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
This article does not contain any studies with human or animal subjects.

Conflicts of Interest
The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.