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. 2025 Feb 5;10(6):5537–5553. doi: 10.1021/acsomega.4c08152

Valorizing Banana Peel Waste into Mesoporous Biogenic Nanosilica and Novel Nano-biofertilizer Formulation Thereof via Nano-biopriming Inspired Tripartite Interaction Studies

Ajay Kumar †,, Rishabh , Neetu Singh †,*, Yogendra K Gautam §, Priya , Namrata Malik
PMCID: PMC11840586  PMID: 39989758

Abstract

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The present study attempts to valorize banana peel waste (BPW) into high-value precipitated nanosilica-based agri-input. XRD analysis revealed smaller-sized biogenic nanosilica (BNS) with an increase (without heating) or decrease (with heating) in the duration of acid pretreatment during the pre-calcination step. The highest BNS yield was recorded in post-calcinated BPW ash involving simultaneous acid and heat treatment (1 h) (SA-3). FTIR analysis displayed an intense peak at 1078.3 cm–1, indicating “Si–O–Si bond” asymmetric vibrations. FESEM-EDX micrographs revealed high-purity BNS of predominantly spheroid morphology. The BJH plot exhibited mesoporous nanosilica with a median pore diameter of ∼33.82 nm. The bipartite interaction of 0.001 g mL–1 BNS signifies growth-promoting effects on Bacillus subtilis (BS) and Raphanus sativus (RS). The nano-primed RS seeds showed higher germination indices over non-primed seeds at 0.001 g of BNS mL–1. Further, the nano-biopriming studies showed the synergistic response of BNS and BS interaction on RS seeds in terms of higher seedling growth, biomass content, and stress tolerance index. The findings open new avenues for developing nano-biofertilizer formulations that serve multifaceted functions such as waste management and biomass valorization into value-added products and fulfill sustainable development goals.

1. Introduction

Musa paradisiaca L. (M. paradisiaca), commonly known as banana, belongs to the family Musaceae, widely recognized as an edible fruit.1 According to the FAO,2 India is the largest banana producer (27.6 MT), followed by China (12.1 MT), the Philippines, and Brazil (6.9 MT).3 Almost 60% of the banana fruit part, known as the peel, is discarded as waste.4 Nearly 30–40% of banana production is vetoed as a consequence of poor quality standards, and the damage proportion to fruit during transportation is also very high.1

Fruit residues are one of the critical biowaste among different agricultural wastes.3,5 Nearly 3.5 MT/year of banana peel waste (BPW) is produced globally by food industries.3 According to another report, annually, ∼114.08 MMT of banana waste loss is generated worldwide, despite significant hemicellulose, cellulose, and natural fibers contents. The conventional routes for disposing of fruit residues, like composting, burning, and landfilling,6 can attract severe environmental consequences, including GHG emissions that could result in global climate change.7

As per reports, GHG emissions like carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) owing to waste disposal have increased by ∼142, ∼253, and ∼121%, respectively.3 Fruit wastes are the third source of GHG emissions in the USA and China.2,3,8 According to Oelofse and Nahman,9 each ton of fruit/food waste grossly generates ∼4.14 tons of CO2 via direct emissions or indirectly through microbial metabolism.3 N2O, CO2, and CH4 have also been defined as the main contributors to global warming, among which the former have more pronounced effects.3,1012

Banana peel (BP) comprised ∼30–40% of its fruit weight in total, with 60–65, 5–10, and 6–8% of cellulose, lignin, and hemicellulose, respectively.3,11,13 In another study, BP’s hemicellulose, lignin, and cellulose contents were ∼11.87, ∼7.3, and ∼28.57%, respectively.14 Apart from natural biopolymers, it contains proteins, lipids, dietary fibers, secondary metabolites (phenols, carotenoids, flavonoids, amine derivates, phytosterols, etc.),1 and minerals (in mg/100 g) like sodium (∼115.1), magnesium (∼44.5), iron (∼47), calcium (∼59.1), phosphorus (∼211.3), zinc (∼0.033), copper (∼0.51), manganese (∼0.702), and potassium (∼4.39).15

The rich biochemical profile of BPW7 can be valorized into numerous value-added products such as biofuels, biofertilizers, and different nanomaterials.6,1619 Serna-Jiménez et al.16 recorded 182 LCH4/(kg of volatile solids) via mesophilic biomethanation of BPW. Ruangtong et al.20 demonstrated the reducing and capping potential of BPW crude extract in ZnO nanosheet synthesis. Naeem et al.17 utilized BP fibers with bacterial cellulose to develop a reinforced composite with higher tensile strength and thermal stability. Other reported nanomaterials obtained from BPW are carbon quantum dots (∼5 nm),18 palladium NPs,21 CuO/NiO nanocomposites,22 silver NPs,23 and titanium NPs.24 The BPW-derived nanoparticles showed significant antibacterial, anticancer, drug-delivery, and dye degradation potential.20,2527 Therefore, nanotechnology-inspired valorization can be an attractive strategy for constructive mitigation of BPW into high-value products with multifarious utilities.

Many researchers have advocated nanosilica production from agro-industrial wastes.6,2834 Compared to chemical synthesis, biogenic nanosilica production has various advantages, such as being low-cost, energy-efficient, and eco-friendly.35 Among different forms, the precipitated silica has growing industrial demand.33 Nanosilica has applications in bio-imaging, biomedicine, biosensors, environmental remediation (recovery of heavy metals, nonmetals, radioactive compounds, oil, antibiotics, etc.), agriculture (stimulates the growth of beneficial soil microbes, facilitates toleranceto various abiotic stresses, develops resistance against different fungal and bacterial phytopathogens, regulates activities of antioxidant enzymes, promotes overall plant growth and development, etc.), catalysis, ceramics, optics, thin films, coatings, nanocomposites for construction materials, and use as anticorrosive, antimicrobial, and anticancer agent.6,14,28,3547

The present study uses BPW as a precursor for green-chemistry routed biogenic nanosilica (BNS) extraction. The reaction conditions favoring BNS recovery were optimized. Further, the interaction studies of BNS with agriculturally beneficial bacteria (Bacillus subtilis) (BS) and Raphanus sativus (RS) seeds were performed to develop the novel nanosilica-based biofertilizer formulation with enhanced plant-growth-promoting activities.

2. Experimental Section

2.1. Chemicals and Raw Material

The chemicals employed in the present study, viz., 35–37% hydrochloric acid (maximum impurity levels, 0.0316%), sodium hydroxide (≥97% pure), sodium chloride (99.5% pure), 4% sodium hypochlorite (0.002% maximum impurity as manganese), and ethanol (99.9%), were of analytical grade and used without further purification. The preparation of reagents was performed in triple-distilled water (TDW), and glassware was thoroughly washed and sterilized for ∼2 h at 180 °C.48,49 BPW was procured from the nearby fruit juice corner, sorted, sliced into small pieces, and cleaned with tap water, followed by TDW. Further, the excess water from BPW slices was soaked using blotting sheets, completely dried at 60 °C, ground into fine powder form, and passed through a standard-size sieve to obtain a uniform particle size. The resultant powder was stored in an airtight jar until use.

2.2. Preliminary Analysis of BPW Powder

The BPW powder was subjected to ash, dry matter (DM), moisture, total organic carbon (TOC), and organic matter (OM) analyses.50 Moisture (%) = (PQ/P) × 100; ash (%) = R/P × 100; OM = (QR/Q) × 100; TOC = OM/1.724, where 1.724 is the bemlen factor, and P, Q, and R are fresh weight biomass (FWB), weight obtained after 105 °C heat treatment for 6 h, and that followed by heating for another 6 h at 600 °C, respectively. An FTIR (PerkinElmer, L1600300) spectrum was recorded in the spectral range 4000 to 450 cm–1 to investigate the functional groups present in BPW powder.49,51 Further, the aqueous extract of BPW powder was analyzed for the preliminary phytochemical analysis (i.e., alkaloids, phenols, flavonoids, carbohydrates, cardiac glycosides, tannins, quinones, terpenoids, phytosteroids, proteins, and amino acids),5257 and observations were marked as “+”, “++”, and “+++” for trace, moderate, and high concentrations, respectively.52

2.3. Extraction of BNS

Extraction of biogenic silica from agricultural residues typically involves sol–gel precipitation and alkaline–silica separation.33 The major steps in the methodology adopted for the BNS synthesis from BPW are shown in Figure 1. First, the BPW powder (Section 2.1) was heated for 6 h at 650 °C in a muffle furnace to obtain ash. BPW ash undergoes acid leaching (1:10 (w/v)) for dissolving carbonate components58 and removing metallic impurities.59 Based on acid type, treatment duration, and heating process (80 °C), 7 BPW-ash treatments were designed, viz., SA-1, HCl washing for 1 h; SA-2, HCl washing for 3 h; SA-3, HCl washing for 1 h with heat treatment; SA-4, HCl washing for 3 h with heat treatment; SA-5, H2SO4 washing for 3 h; SA-6, HNO3 washing for 3 h; SC, washing with distilled water for 3 h. The leached ash was left for 12 h in TDW under continuous stirring, followed by centrifuging at 4000 rpm for 30 min. The recovered ash containing silica undergoes alkali solubilization (3 N NaOH in 1:30 (w/v), 80 °C for 6 h in a hot water bath), resulting in sodium silicate formation, which is later precipitated at pH = 7, and heated at 80 °C for 2 h in a water bath. The obtained clear silica gel was aged for 24 h to recover the nanosilica.60 The obtained nanosilica was washed with ethanol and TDW multiple times and calcinated at 100 °C for 6 h to obtain white BNS powder. The general reaction steps involved in HCl/NaOH-inspired extraction of nanosilica are depicted in the following reactions.58

2.3.
2.3.
2.3.

Figure 1.

Figure 1

Schematic view indicating the synthesis and characterization of BNS.

2.4. Characterization of BNS

BNS was characterized for structural and optical properties. The phase and size of BNS were determined by X-ray diffractometer (Rigaku, Ultima IV) over the 2θ scanning range up to 80° (X-ray wavelength, i.e., Cu Kα anode = 0.15406 nm; tube current and voltage were 40 mA and 45 kV, respectively; scan speed = 8° min–1).61,62 The crystallite size (D) was determined using the Debye–Scherrer formula: “kλ/βhkl cos θ”, where βhkl = “full width at half-maximum” intensity value in radians; θ = peak position in radians, λ = 0.15406 nm, and k (constant) = 0.9.61 The functional group associated with BNS was analyzed via an FTIR spectrophotometer (PerkinElmer, L1600300) in the transmission mode (4000 to 450 cm–1 spectral range).63 The surface morphology and elemental composition of BNS were determined by “field emission scanning electron microscopy” (FE-SEM; TESCAN, MAGNA LMU), and “energy dispersive X-ray” (EDX) mapping (EDAX AMETEK, “Octane Elite Super EDS system”), respectively.49,61,64 The particle size of nanosilica was determined with TEM (JEOL JEM 2100 PLUS). The absorption spectra of BNS aqueous dispersion were recorded at room temperature via UV–vis spectroscopy (ELICO 150) in the wavelength range from 250 to 400 nm.61 Further, the recorded absorbance data were converted to estimate the band gap energy (Eg, in eV) from Eg = hcmax, where c = 3 × 108 m s–1 and h = 4.135 × 10–6 eV.65 “Brunauer–Emmett–Teller” (BET) analysis (MicrotracBEL Corp., BELSORP-maxII (S/N: 175, Ver 2.0.1.1) was performed on BNS at adsorption temperature 77.355 K under nitrogen atmosphere to study the surface characteristics, i.e., pore diameter and volume through BET and “Barret–Joyner–Halenda” (BJH) plot using data analysis software (BELSORP, BELMaster, Ver 7.2.0.4).66,67

2.5. Maintenance of B. subtilis Culture

Bacillus subtilis (B. subtilis; Gram positive (Gram +ve) bacteria) were maintained in nutrient broth (NB) of the following composition (w/v): peptone (0.5%), yeast extract (0.2%), NaCl (0.5%), and beef extract (0.1%). pH was attuned at ∼7.4. For solid media plates, 2% (w/v) agar was added to the above NB composition. The nutrient medium was autoclaved for 15 min under 15 lbs of pressure and 121°C temperature, followed by cooling at room temperature (RT) before use. For experimental studies, B. subtilis cell suspension maintained at active log phase (inoculated into NB media followed by incubating at 37 °C for overnight) was considered.61,6870

2.6. Disk Diffusion Assay (DDA) and Growth Kinetics Study

The freshly prepared nutrient media were poured into Petri plates and solidified for nearly 30 min. B. subtilis cell culture (BCC) was spread over solidified media plates. The sterile disks of filter paper nearly 6 mm in diameter were dipped in the variable treatment doses of BNS prepared in aqueous solution (until saturation) and equidistantly placed on the inoculated plates. The disks dipped in TDW served as a negative control. The inoculated media plates were incubated in a BOD incubator at 37 °C for a 24 h duration. Serial dilutions for colony count of B. subtilis (cultured in NB with and without BNS) were performed on nutrient agar media, and plates were incubated as mentioned for DDA.71,72 For bacterial growth kinetics studies, the BCC maintained at the logarithmic phase was first diluted to obtain optical density (OD) of about 0.1 at 600 nm (equivalent to ∼108 CFU mL–1) and then grown at 37 °C in NB media (with and without BNS). The turbidity measurements were performed using a UV–vis spectrophotometer (OD at 600 nm).61,7376

2.7. Seed Germination Assay

Raphanus sativus (L.) (R. sativus; radish) was chosen as a model plant in the present study due to its rapid growth and higher germination rate.61 The uniform-sized healthy seeds of radish were surface sterilized using sodium hypochlorite solution (2.5% (w/v))61 for ∼5 min and then washed thrice with sterile TDW. Five seeds were placed equidistantly on the Petri dish equipped with a sterile filter paper soaked with 4 mL of the test sample or TDW (served as a control). The prepared Petri dishes were then incubated at 25 ± 1 °C in the dark77 for ∼7 days, and germination data were recorded upon the emergence of the radicle (∼2 mm)78 beyond the seed coat. The growth indices were measured for germination percentage (G, %), seedling length (SL),79 fresh weight (FWB), and dry weight biomass (DWB),80 vigor index (VI-I and -II),81,82 and stress tolerance index (STI) for seedling parameters such as radicle length (RL), plumule length (PL), SL, FWB, and DWB.83,84

2.7.
2.7.
2.7.
2.7.

The experiments were carried out in triplicates in a “complete randomized design”; the recorded mean values of independent triplicate trials were denoted as mean ± SD.61 We have performed a “Pearson correlation coefficient (r) analysis” to study the association between investigated germination indices under variable treatments, and the r-value closer to +1 or −1 indicated a “strong positive correlation” or “strong negative correlation”, respectively.61,85

2.8. Antioxidant Assay

Total phenol content (TPC) was determined as per Khatiwora et al.86 with slight modifications. Germinated seedlings of radish (GSH) were extracted in 80% (v/v) ethanol, and the volume was marked up to 3 mL with TDW. In the next step, 0.5 mL of FC reagent (1:1 with TDW) was added, followed by 2 mL of 20% sodium carbonate. The test sample was heated for ∼2 min and cooled at RT, and the absorbance was recorded using a UV–vis spectrophotometer at 650 nm wavelength. The curve was plotted against the gallic acid standard, and TPC was expressed as “mg of GAE equivalent/(g of sample)”. Total flavonoid content (TFC) was determined, as mentioned in Baba and Malik,87 with few modifications. A 1 mL aliquot of ethanolic extract of GSH was added to 4 mL of TDW, followed by 0.3 mL of aqueous solution of 5% sodium nitrite and incubated at RT for 5 min. Next, 0.3 mL of aqueous aluminum chloride solution (10%) was added to the test sample and incubated for 6 min at RT, and then 2 mL of 1 M aqueous NaOH solution was added and the volume marked up to 10 mL with TDW. The absorbance was recorded using a UV–vis spectrophotometer at 510 nm wavelength. The curve was plotted against the quercetin standard, and TFC was expressed as “mg of quercetin equivalent per g of sample”.

A hydrogen peroxide (H2O2) assay on GSH was performed as per Yahyaoui et al.88 with slight modifications. Briefly, the GSH was homogenized in 5 mL of 0.1% (w/v) chilled trichloroacetic acid (TCA), incubated for ∼30 min at RT, and centrifuged to collect the supernatant. To 1.0 mL of the test sample, equal volumes (2 mL each) of potassium phosphate buffer (10 mM, pH 7) and 1 M potassium iodide were added. The absorbance was recorded at wavelength 390 nm, and H2O2 content was determined from the extinction coefficient value of 0.28 mM–1 cm–1. A lipid peroxidation assay to determine the malondialdehyde (MDA) content was performed according to Iftikhar and Perveen89 with slight modifications. A 1 mL aliquot of supernatant (prepared as mentioned in the H2O2 assay) was added to 4 mL of 0.5% thiobarbituric acid (prepared in 20% TCA) and heated at ∼95 °C for ∼30 min, followed by cooling at RT. The absorbance was recorded at wavelengths 600 and 532 nm. MDA content (nmol/mL) was calculated using [(absorbance532 – absorbance600)/155000] × 106.90

3. Results and Discussion

3.1. Characterization of BPW Powder

BPW showed moisture, DM, OM, and TOC contents (weight %) of 90.82 ± 1.90, 9.18 ± 1.90, 83.60 ± 0.62, and 48.19 ± 0.86, respectively. As per the reports, these parameters can be varied depending on the plant variety, prevailing environmental conditions, etc.91,92 The ash content in BPW was 15.85 ± 0.67%, which is in agreement with the previous findings.93,94 The EDX (Oxford Instruments) analysis of BPW showed “Si” and “O” contents (weight %) of 6.67 and 26.54, respectively. The reported elemental impurities were Mg, P, S, Cl, K, Ca, Mn, Rb, Nb, and Pb95 (Supporting Information Figure S1). Preliminary phytochemical analysis of the aqueous BPW extract showed the presence of alkaloids, phenols, flavonoids, carbohydrates, cardiac glycosides, tannins, quinones, terpenoids, phytosteroids, proteins, and amino acids (Table 1). Previous studies mentioned most of these metabolites in banana peel’s aqueous/organic solvent extracts.9699

Table 1. Preliminary Phytochemical Analysis of the Powdered BPW Aqueous Extract.

Compound Test Resulta Observation
Alkaloids Mayer ++ Whitish/cream colored precipitate
  Wagner +++ Reddish-brown precipitate
Phenols FeCl3 + Intense color
Flavonoids Lead acetate ++ Yellow precipitate
  Alkaline reagent + Intense-yellow color
Carbohydrates Fehling’s + Yellowish/brownish-red precipitate
  Molish Violet color not appearing at the junction of two-liquid layers
Proteins and amino acids Biuret ++ Purplish-violet/pinkish-violet color
  Xanthoprotic ++ Yellow precipitate
  Ninhydrin Blue/Purple color not appeared
Cardiac glycosides Keller-Kiliani + Reddish brown color at junction of two-liquid layers and upper layer appeared bluish green
Tannins Lead acetate +++ Yellow precipitate
Quinones Acid precipitation ++ Red precipitate
Terpenoids Salkowski + Red-brown precipitate
Phytosteroids Hesse’s reaction + Chloroform layer red and acid layer greenish-yellow
a

Concentrations: “+” = trace, “++” = moderate, and “+++” = high.

FTIR spectra of BPW powder show peaks at 2919.47, 2851.2, 1602.8, 1379.2, 1245, 1038.02, and 895.27 cm–1 (Figure 2a). The peaks recorded for BPW ash were at 2988.27, 1985, 1448.3, 1045.7, 880.4, and 702.16 cm–1 (Figure 2b). Similar records were documented in earlier studies. Memon et al.100 in FTIR analysis of banana peel powder, observed peaks at 884.6, 1035.2, 1613.6, 1734, 2850.6, and 2920.3 cm–1 corresponding to NH amine deformation, CO stretching of ester/ether, OH bending, carboxylate stretching, CO, and CH stretching of ester/COOH, respectively. In another work, Udochukwu and Akpoviri101 noted peaks at 2919.47 and 895.27 cm–1 in BPW powder, showing CH-stretching vibrations of CH3/CH2/CH groups, and carbohydrates and water deformation, respectively. The peaks in ash samples at 702.16 cm–1 correspond to C=C bending and C–Cl stretching, 1045.7 cm–1 indicated S=O and C–F stretching, and 1448.3 cm–1 designated calcium oxide, while calcite phases are suggested by 2988.27, and 880.4 cm–1.102104

Figure 2.

Figure 2

FTIR spectra of BPW: (a) homogenized dried powder and (b) ash.

3.2. Synthesis and Recovery of BNS

The key reaction steps involved in the synthesis and recovery of BNS are shown in Figure 3.19 The effect of pretreatment conditions with respect to (wrt) acid type, treatment duration, and temperature on BNS recovery was investigated. The silica yield (in %) was calculated as per the following equation.105

3.2.

Figure 3.

Figure 3

Key reaction steps involved in synthesis and recovery of BNS (conceptualized from ref (19)).

The BNS yields were recorded from experimental trials carried out in triplicate. The BNS content was highest at SA-3, followed by SA-2, indicating favorable effects of heat treatment and substantial reductions in reaction time. Compared to SA-6, the BNS recovery was greater in the case of SA-5 but lower than SA-2, which preferred pretreatment of ash with HCl for 3 h over HNO3 and H2SO4 (Figure 4). Overall, compared to the control (SC), 59.5, 46.1, 39.9, 38.3, 38, and 22.8% higher BNS yields were noted in treatment samples SA-3, SA-2, SA-6, SA-4, SA-1, and SA-5, respectively. Adebisi et al.106 also reported higher Si contents in maize stalks treated with HCl before (pre-calcination) and after ashing (post-calcination).

Figure 4.

Figure 4

Recovery of BNS under different reaction conditions. (Error bar indicates mean ± SD.)

3.3. Characterization of BNS

3.3.1. XRD

XRD diffractogram showed significant peaks at 2θ angles 27.2–27.3, 31.5–31.7, 45.3–45.4, 53.7–53.8, 56.3–56.4, 66.0–66.2, and 75.1–75.2° (Figure 5a–g). BNS samples SA-2 to SA-5 also demonstrated additional peaks at 28.1–28.5, 40.3–40.7, 50–50.5, 58.5–59, 66.0–66.9, 72.8–72.9, and 83.4–83.8°. Similar peaks were reported in earlier studies by Barma et al.107 (2θ = 27, 31, 45, 56, 75, and 84°), Ali et al.108 (2θ = 40.3, and 53.9°), Silmi et al.109 (2θ = 50.14°), and Periakaruppan et al.110 (2θ = 28°) for SiO2. The HCl pretreated ash (HPA) for 3 h duration (with and without heat treatment) favored smaller-sized BNS than those obtained from HNO3 and H2SO4 treated samples. Further, the short heating duration (SHD) of HPA (SA-3) resulted in reduced-sized BNS compared to the long heating duration (LHD) (SA-4). SA-1 and SA-6 exhibited a higher BNS size than the control (SC) (Figure 5h). Overall, the crystallite size (CS) of BNS obtained at different reaction conditions ranges from ∼51 to 78 nm (except SA-6), which is in concordance with the previous report (Table S1).58 The pretreatment duration and temperature have significant effects on the size of BNS. XRD results demonstrated a decrease in BNS size with an increase (without heating) or a decrease (with heating) in the HCl preatment duration for ash. Dislocation density (δ) was calculated from the CS (D) of BNS using equation δ = 1/D2 to study the defects in SiO2 crystal structure.111 “δ” indicates the degree of crystallization and decreases with an increase in BNS size.61,111 The microstrain (εa) was determined as per equation “εa = β cos θ/(4 × 10–3)” and displayed an increasing trend in proportion with the “δ” values.112

Figure 5.

Figure 5

XRD spectra of BNS: (a) SC, (b) SA-1, (c) SA-2, (d) SA-3, (e) SA-4, (f) SA-5, (g) and SA-6. (h) Size of BNS obtained from Scherrer–Debye equation (nos. 1–7 showing peak positions corresponding to 2θ angles: ∼27.2–27.3, ∼31.5–31.7, ∼45.3–45.4, ∼53.7–53.8, ∼56.3–56.4, ∼66.1–66.2, and 75.1–75.3°, respectively).

3.3.2. UV–Vis Spectroscopy and FTIR

FTIR spectra peaks of BNS (SA-3) at 1078.3 and ∼850 cm–1, corresponding to the “Si–O–Si bond” asymmetric vibrations.63,113 The peak at 586.23 cm–1 also indicates Si–O elements.110 The minor peaks at 1500–2000 and ∼3400 cm–1 are indicative of absorbed water molecules bending and silanol or H–O–H stretching, respectively (Figure 6b).67,114 The optical properties of nanosilica are based on variable defects due to the partial formation of the “Si–O–Si tetrahedral network” (“O” and “Si” vacancies).115 UV–vis spectra of BNS synthesized at different reaction conditions showed maximum absorbance (λmax) at ∼260 nm (Figure 6a).114 The prominent peaks were also noticed at ∼300 nm.116 The band gap corresponding to λmax at 260 nm was ∼4.77 eV.117,118

Figure 6.

Figure 6

(a) UV–vis spectra showing characteristic absorption peaks of BNS synthesized at different reaction conditions. (b) FTIR spectra showing characteristic functional groups in BNS (SA-3).

3.3.3. FE-SEM, TEM, and EDX

FE-SEM micrographs indicated predominantly spheroid morphology of BNS (SA-3), which was further confirmed in TEM investigation (Figure 7a,e).119,120 The particle size calculated from TEM analysis was ∼68–170 nm. EDX mapping revealed elemental composition (weight %) of ∼31 and ∼69 “Si” and “O”, respectively (Figure 7b,c). The atomic percentages of “Si” and “O” were ∼20, and ∼80%, respectively. However, the elemental composition may vary with the choice of template and extraction process adopted.58 Joni et al.121 synthesized SiO2 nanoparticles with elemental “Si” and “O” contents of ∼38 and ∼62%, respectively. Stanley and Nesaraj122 reported 56.63 and 43.37 wt % “O” and “Si”, respectively, in nanosilica (synthesized without surfactant).

Figure 7.

Figure 7

(a) FESEM micrograph at 150k× magnification (100 nm scale) (Inset: enlarged view showing spheroid morphology.) (b) EDX spectra (c) Elemental color mapping from EDX (d) Nitrogen adsorption–desorption isotherm curve (e) TEM image (at 1 μm scale) of BNS (SA-3).

3.3.3.4. BET Analysis

The nitrogen adsorption–desorption curve of BNS (SA-3) displayed type-IV isotherm according to IUPAC classifications (Figure 7d).64 The total pore volume (p/po = 0.990) obtained from the BET plot was 0.01 cm3 g–1. Alhadhrami et al.58 reported a pore volume of 0.062 cm3 g–1 for biogenic silica extracted from RH. In another study, Araichimani et al.123 obtained a pore volume of 0.094 cm3 g–1 from BJH analysis for RH-derived nanosilica. The pore volume can be varied with the substrate material (precursor), operational conditions during the extraction process, and crystallinity of nanosilica.108,124127 Idris et al.124 found lower pore volume for crystalline nanosilica (0.0021a and 0.0045b cm3 g–1) compared to amorphous nanosilica (0.678a and 0.327b cm3 g–1) derived from corncobsa and olive stonesb. Ramasamy et al.125 from wheat straw ash derived amorphous nanosilica with a micropore volume of 0.013 cm3 g–1. The BJH plot of BNS (SA-3) revealed mesoporous nanosilica with average and median pore diameters of ∼19.55 and ∼33.82 nm, respectively,60,128,129 and surface area of ∼2.16 m2g–1.

3.4. Effect of BNS on Bacterial Growth

To explore BNS and BS as a novel combination for plant-growth-promoting (PGP) activities, we first investigated the effect of direct BNS (SA-3) treatment on BS. For this purpose, disc diffusion assay (DDA) and bacterial growth kinetics (BGK) studies were performed (Figure 8). No ZOI was noticed at 0.001 g (T1) and 0.01 g of BNS mL–1 (T2), indicating no apparent inhibitory effect of treatment doses on BS growth. Previously, Ferrusquía-Jiménez et al.40 did not find any toxicity effects of 0.0001 g of nanosilica mL–1 against “Bacillus cereus-Amazcala” (B.c-A.). For cell viability analysis, colony count was performed, and the CFUs recorded for T1 and T2 showed ∼16.8 and ∼12.5% increments compared to the control. The findings of BGK studies allow for determining the bacterial cell density (BCD) and the toxicity of NPs in a liquid medium.61 The absorbance values recorded at 600 nm (OD600) showed higher BCD in T1 than in the control. However, T2 displayed lower OD600 values compared to those of the control. Overall, the findings signify that the BNS treatment up to 0.001 g mL–1 could have growth-promoting effects on BS. Karunakaran et al.45 reported an increase in the growth of four PGP rhizobacteria (PGPRs) (Pseudomonas fluorescens, Bacillus brevis, Azotobacter vinelandii, and Bacillus megaterium) by >20% upon nanosilica treatment. They noted doubling in PGPRs CFUs (×108) counts (g–1 of soil) from 4 to 8.

Figure 8.

Figure 8

Growth kinetics curve of B. subtilis (insets: (a) CFU count; (b) disc diffusion assay results at different treatment doses of BNS) (C = control, T1 = 0.001 g of BNS mL–1, and T2 = 0.01 g of BNS mL–1).

The mechanism of SiO2 adsorption on the cell surface of Gram-positive bacteria is typically governed by the presence of teichoic (TEA) and teichuronic acid (TUA) in the cell wall130 (Figure 9). TEA and TUA contain phosphate and carboxylate groups, respectively, that aid negative charge to the bacterial cell surface (BCS) and facilitate SiO2 binding.130 In some bacteria, proteins (SiP) also played a significant role in SiO2 uptake.45 Studies also mentioned that the hydration property of silica could facilitate its attraction on the bacterial surface.45 Tian et al.131 reported the possibility of SiO2 interaction with BCS-associated proteins in a few bacteria via hydrogen bonding with amino acid residues.

Figure 9.

Figure 9

Probable mechanism of silica adsorption by B. subtilis (conceptualized from refs (45 and 130135)) (BCS, bacterial cell surface; PEM, phosphate enriched media; PDM, phosphate deficit media; SiP, silica induced protein; ABC, ATP-binding cassette; Aa, amino acid).

3.5. Effect of BNS on Seed Germination

Figure 10 shows the general mechanism of silica uptake by the plants. In soil application, silicic acid is the major available form of silica and is taken by roots via passive or active transport. Transport proteins (LSi) play an essential role in the active transport of silica. Silica is deposited in vascular bundles as phytoliths, and in plants such as grasses, it forms a secondary cuticle-Si protective layer. SiNPs, in the case of foliar application, gain entry into leaves via cuticle (through penetration or diffusion) or stomatal pores and translocated to roots via phloem.

Figure 10.

Figure 10

Mechanism of silica uptake by plants (conceptualized from refs (35, 37, and 140143)) (SiNPs, silica nanoparticles; EMC, excess mass infiltration; DF, diffusion; PN, penetration; TPs, transport proteins).

In the present study, RS seeds exposed to filter papers soaked with 0.001 (Te) and 0.01 (Tf) g BNS mL–1 (nonpriming experiments) showed lower germination indices compared to the control. However, RL and RLTI values were higher in “Te” than “Tf” and control. In the case of seeds nanoprimed with 0.001 g mL–1 BNS (Tc), higher PL, RL, SL, FWB, VI, and stress tolerance indices were obtained than those of control, “Te”, “Tf”, and "Td". Also, the marginal increment in G (%) and DWB was recorded in control over “Te”. Studies reported growth-prompting effects of nanosilica on several crops like wheat, tomato, sugar cane, maize, rice, soybean, potato, etc., which help to alleviate abiotic stress such as salinity, heavy metal, heat, drought, etc.37,38,136,137

The comparative assessment of nanopriming and nonpriming experiments indicated growth-promoting effects of seed nanoprimed with 0.001 g of BNS mL–1 (Tc), which concords with previous reports.138 Sun et al.138 recorded an improvement in phyto-biomass, seed germination, chlorophyll, and protein contents in wheat and lupin at mesoporous nanosilica concentrations of 0.0005 and 0.001 g mL–1. Zaheer et al.139 also found an increase in plant height and biomass weight in Vigna radiata (L.) after application of nanosilica (0.002, 0.0002, and 0.000002 mg L–1) on 6 days old plants, compared to control. Karunakaran et al.45 noted 100% seed germination in maize in the case of nanosilica treatment, as against 97, and 95% in microsilica and control experiments, respectively. Elamawi et al.42 performed a foliar application of 0.00005 mg L–1 nanosilica on Fusarium fujikuroi infested rice seedlings and witnessed an increase in grain yield and a reduction in bakanae disease symptoms. We observed that “Tf” exhibited lower germination indices than the control, "Te", "Td", and "Te", indicating phytotoxicity of direct BNS exposure at elevated doses on RS seeds.

3.6. Tripartite Interaction Studies

The tripartite interaction of RS seeds with BS (biopriming) followed by BNS (nano-biopriming) showed a synergistic response in terms of seedling growth, biomass content, vigor indices, and stress tolerance potential. The nano-biopriming using 0.001 g of BNS mL–1 (Ta) indicated ∼21, ∼27.5, ∼23.9, ∼17, ∼12.4, ∼16.5, and ∼35% higher PL, RL, SL, FWB, DWB, VI-2, and VI-2, respectively, compared to control (Figure 11a,b). PL, SL, FWB, DWB, and VI-2 also showed increments in “Ta” seedlings with respect to “Td” (nanopriming using 0.001 g of BNS mL–1). These values, coupled with higher tolerance indices in “Ta”, favored the growth-promoting effects of combined BS–BNS treatment over solitary treatment of BNS (Figure 11e). Ferrusquía-Jiménez et al.40 performed co-application of B.c-A. and 0.0001 g mL–1 of nanosilica on chili pepper plants and observed increments in leaves number, G (%) of seeds, plant height, yield, and number of fruits.40 Except for RL, we found that all of the germination indices values were lower in treatment "Tb" (nano-biopriming using 0.01 g of BNS mL–1) compared to control (Figure 11a,b). The data recorded for “Tb” and “Tf” when compared to control, “Ta”, and “Td” demonstrated phytotoxicity at a higher dose of BNS (0.001 g mL–1) irrespective of treatment mode. An integration of biopriming along with BNS treatment displayed positive interaction with RS seeds and supported PGP activities together with stress tolerance potential. In this way, the effective dose of BNS has also been reduced substantially, hence promoting judicious use of agri-inputs, keeping sustainable agricultural practices in view. Table 2 shows strong positive correlation between SL-PL, SL-PL, FWB-RL, and FWB-DWB (R2 ≥ 0.95); DWB-RL, DWB-SL, and SL-FWB (0.94 ≥ R2 ≥ 0.90); and PL-RL, PL-FWB, and PL-DWB (R2 ≤ 0.89).

Figure 11.

Figure 11

Effect of BNS and BS treatments on (a) seedling length and (b) biomass content. Antioxidant activity: (c) total phenol and flavonoid contents and (d) hydrogen peroxide and malonaldehyde content. (e) Stress tolerance indexes for different growth parameters (Ta = BS/0.001 g of BNS mL–1 (priming), Tb = BS/0.01 g of BNS mL–1 (priming), Tc = 0.001 g of BNS mL–1 (priming), Td = 0.01 g of BNS mL–1 (priming), Te = 0.001 g of BNS mL–1 (nonpriming), and Tf = 0.01 g of BNS mL–1 (nonpriming); error bars indicate mean ± SD).

Table 2. Correlation Analyses between the Recorded Germination Indices of R. sativusa.

3.6.

a

Note: Growth parameters were recorded for experimental trials “C”, and “Ta” to “Tf”.

3.7. Antioxidant Activities

TPC and TFC were estimated for “treated” and “control” RS seedlings. “Ta”, and “Tc” showed ∼20.3 and ∼28.2% higher TPC and ∼24.1 and ∼34.6% higher TFC, w.r.t. control, respectively (Figure 11c). The results promoted nanopriming and nano-biopriming of seeds using BNS (0.001 g of BNS mL–1) over other treatments and control. Sun et al.138 observed no oxidative stress up to 0.002 g mL–1 of nanosilica and recommended for applications in plants over the tested range. Even Ferrusquía-Jiménez et al.40 noted defense-associated response of nanosilica (0.0001 mg L–1)/B.c-A. co-application in chili pepper plant in terms of higher superoxide dismutase and catalase activities. The H2O2 content analysis demonstrated higher values in the following treatments (compared to control): Td (∼22.6%) > Tc (∼18.2%) > Tf (∼17.7%) > Tb (∼12.5%) (Figure 11d). MDA contents showed ∼43.6, ∼35.1, ∼31, and ∼20.4% increments in “Td”, “Tc”, “Tb”, and “Tf”, respectively, w.r.t. control, indicating significant stress at elevated doses of BNS, precisely at direct seed treatment (Td) (nanopriming).

3.8. Credit of BNS for Long-Term Agricultural Applications

To ascertain the suitability of developed BNS for long-term agricultural applications, we have performed interaction studies of BNS with BS and RS. The bipartite interaction of BNS with B. subtilis (BS) showed no obvious toxicity at both of the tested doses (i.e., 0.001 and 0.01 g mL–1). The increase in CFU counts at 0.001 g of BNS mL–1 was in agreement with the previous reports. Karunakaran et al.45 observed the growth-promoting effects of nanosilica against four PGP rhizobacteria. Their seed germination studies also revealed no phytotoxicity symptoms and oxidative stress in RS at 0.001 g of BNS mL–1. Despite this, we have witnessed the synergistic response of BNS and BS interaction on germination indices, tolerance index, and antioxidant levels of RS.40 However, to better understand the credit of nanosilica for long-term applications, we recommend additional investigations exploring their effect of introduction on the environment during agriculture applications via foliar spraying or soil fertigation.

3.9. Economic Analysis

As per the “Nano Silica Market Research” report, the nanosilica market from USD ($) 4.6 billion (valued in 2021) is expected to reach $8.6 billion by the year 2031, rising at a 6.5% CAGR from 2022 to 2031.144 We have encountered few reports on the cost–benefit analysis of biogenic nanosilica production. Maroušek et al.115 extracted nanosilica from coir-pith via an acid-based sol–gel method and projected cost valued at 1.3 Euros (€)/g (expenses breakup owing to energy, reactants, feedstock and processing, labor, equipment depreciation, and others were 0.2, 0.3, 0.1, 0.2, 0.4, and 0.1 €, respectively) against the wholesale market price of about 2.4–3.3 €/g. Singh et al.67 estimated Sapindus mukorossi seed extract stabilized nanosilica production cost of $24.19 per kg (expenses breakup include the cost of paddy straw, S. mukorossi seeds, chemicals, labor, energy, and capital) from paddy straw ash against the market price of ∼$198 per kg, with net profit of about $173.81 per kg. These figures suggest the tremendous feasibility of commercial nanosilica production by exploring various low-cost precursor substrates.

4. Conclusions

Banana peel waste has been successfully utilized as a low-cost precursor substrate for developing biogenic nanosilica (BNS). The highest BNS yield at SA-3 directed toward favorable effects of heat treatment and a significant reduction in reaction time. The obtained BNS was of comparable grade as those per previous reports on silica extraction from rice straw, sugar cane bagasse, etc. XRD crystallite and TEM particle sizes were ∼66.52 and ∼68–170 nm, respectively, and FTIR analysis revealed silanol functional groups. The average pore diameter calculated from the BJH plot was ∼19.55 nm. Overall, BNS (SA-3) characterization studies confirmed the nanosized, mesoporous structure with a predominantly spheroid morphology. The findings instate positive tripartite interaction of seeds with B. subtilis/BNS (SA-3) that can be productively translated into the growth-promoting novel nano-biofertilizer formulation. The study potentially served as a stepping stone toward green chemistry routed facile, cost-effective, eco-benign, and energy-efficient biogenic silica production exploiting various phytobiomass-derived agro-industrial waste. It is anticipated that the recorded observations could facilitate the concerned stakeholders engaged in the area of biomass valorization and rural development toward the productive materialization of plenteous agricultural residues into a comprehensive platform catering to waste management, generating high-value products, and promoting a circular bioeconomy in the purview of sustainable development goals.

Acknowledgments

The authors are grateful to the CRF, IIT Delhi, for FE-SEM, EDX mapping, and BET; CIF, Jamia Millia Islamia (New Delhi), for XRD; Professor Shailendra S. Gaurav, Department of Genetics & Plant Breeding, CCSU (Meerut) for FTIR; and Sprint testing solutions, Nagpur for TEM and EDX facilities. The support given by all during the study is acknowledged, and the authors are thankful to all who dedicated themselves by all means. Errors, if any, are purely unintentional. We attempted to acknowledge the copyright holders of all matters reproduced in this work.

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c08152.

  • (Figure S1) EDX analysis of BPW; (Table S1) XRD crystallite size of biogenic nanosilica obtained at different reaction conditions (PDF)

Author Contributions

A.K.: Conceptualization, investigation, writing—original draft, review and editing, and supervision. R.: Experimental, writing—original draft, and literature survey. N.S.: Overall supervision and writing–review and editing. Y.K.G.: Writing—review and data interpretation; P.: Experiment. N.M.: Supervision.

No funding was received to assist with the preparation of this manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao4c08152_si_001.pdf (170.8KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao4c08152_si_001.pdf (170.8KB, pdf)

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.


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