Optimizing Zinc Oxide/Porous Anodic Alumina Composite Films for Superior Antibiofilm Performance

Abstract

This study reports the fabrication of ZnO/porous anodic alumina (PAA) composite films via a two‑step anodization followed by a sol‑gel deposition of ZnO. The films were systematically characterized using XRD, TG/DTA, FT‑IR, SEM, TEM, SAED, and water contact angle (CA) measurements. The antibiofilm activity against Shewanella putrefaciens was quantified by adhesion, colony‑forming unit counts, SEM, and CLSM. Results indicate that ZnO nanoparticles (10–30 nm) nucleate preferentially on PAA pores, and that a 40‑min second anodization yields a highly porous, hydrophobic surface that maximizes ZnO loading. This configuration produced a super‑hydrophobic surface (CA > 150°) and the lowest biofilm adhesion, thereby demonstrating the most effective antibiofilm performance among the tested samples.

Background

Biofilm formation on industrial surfaces leads to corrosion, food spoilage, and significant economic loss. Surface attributes such as roughness, microstructure, hydrophilicity, and antimicrobial additives dictate bacterial adhesion and biofilm maturation. Porous anodic alumina (PAA) offers tunable nanoporosity, making it attractive for biomedical and food‑processing applications. Zinc oxide (ZnO) is a well‑established antibacterial agent; its activity is influenced by crystal structure, particle size, and surface chemistry. Enhancing the hydrophobicity of ZnO films is essential, as hydrophilic surfaces tend to promote bacterial attachment. Shewanella putrefaciens, a psychrotrophic spoilage organism, serves as a relevant model for studying antibiofilm strategies in cold‑chain food preservation.

Materials and Methods

Materials

All reagents were analytical grade; de‑ionized water had conductivity <0.5 mS/cm. Shewanella putrefaciens ATCC 8071 and 99.99 % Al foils (0.3 mm) were sourced from Shengshida Metal Materials Co., Ltd.

Preparation of ZnO/PAA Composite Films

Porous Anodic Alumina (PAA) Films

Al foils (10 × 30 mm²) were polished, degreased, and washed. First anodization: 30 °C, 40 V, 90 min in 0.3 mol/L oxalic acid. Barrier layer removal: 6 wt% H₃PO₄ + 1.8 wt% H₂CrO₄ at 60 °C for 4 h. Second anodization: identical conditions for 0, 40, 60, or 80 min to produce varying pore geometries.

Zinc Oxide Deposition

Equal volumes of 0.02 mol/L zinc acetate and 0.04 mol/L NaOH in ethanol were mixed at 70 °C for 5 min. PAA films were immersed under −0.085 MPa vacuum, heated to boiling to form a blue sol, then rinsed. Samples were dried (−0.085 MPa, 80 °C, 6 h) and calcined at 480 °C for 2 h. Finally, films were modified with 1.0 wt% Si69 in ethanol at 65 °C for 2 h and vacuum‑dried (−0.085 MPa, 40 °C, 12 h).

Characterization

XRD (Rigaku Ultima IV, 40 kV, 50 mA) monitored crystal phases. TG/DTA (Perkin Elmer Diamond) tracked thermal transitions. FT‑IR (Agilent Scimitar 2000) identified surface functional groups. SEM (Hitachi S‑4800) and TEM (JEOL Jem‑2100F) examined morphology; SAED confirmed crystal structure. CA was measured with a sessile‑drop method (3.0 µL de‑ionized water). Biofilm assays included crystal‑violet staining, plate counts, SEM, and CLSM imaging.

Antibiofilm Evaluation

Bacterial suspensions (OD₅₉₅ ≈ 0.5) were diluted 1:200 with 3 % APW and incubated with 0.5 × 0.5 cm film pieces at 28 °C. Biofilm adhesion was quantified by crystal‑violet extraction (OD₅₉₅). Colony counts were obtained via serial dilution and plate counts. SEM visualized biofilm architecture, while CLSM differentiated live (green) and dead (red) cells using acridine orange/propidium iodide staining.

Results and Discussion

ZnO Film Characterization

XRD

Calcination induced a transition from amorphous intermediates to crystalline hexagonal wurtzite ZnO. Peaks at 31.70°, 34.52°, 36.31°, 47.68°, 56.82°, 62.92°, and 67.92° corresponded to (100), (002), (101), (102), (110), (103), and (112) planes (PDF # 36‑1451). Peak sharpening with higher calcination temperatures indicated increasing crystallinity and grain growth.

Thermal Analysis

TG/DTA showed a 68.6 % mass loss up to 100 °C (evaporating ethanol and water) and a minor 3.8 % loss between 100–400 °C (removal of residual hydroxides). No further loss above 400 °C confirmed thermal stability of the wurtzite phase.

FT‑IR

Unmodified ZnO displayed broad O‑H stretching (3600–3300 cm⁻¹) and water‑related peaks. Post‑Si69 modification introduced Si–O vibrations (~895 cm⁻¹) and reduced hydroxyl signals, confirming successful grafting and increased hydrophobicity.

Microstructure

SEM revealed that PAA pore size expanded with second anodization time: 5–10 nm (0 min), 20–40 nm (40 min), 60–70 nm (80 min). ZnO nanoparticles (10–30 nm) preferentially nucleated on smaller pores, forming dense layers on 40‑min PAA. TEM and SAED confirmed the hexagonal wurtzite structure of ZnO particles.

Hydrophobicity

Unmodified ZnO films were hydrophilic (CA < 90°). After Si69 treatment, CA increased dramatically; the 40‑min PAA sample achieved super‑hydrophobicity (CA > 150°), attributable to high ZnO loading and roughness.

Antibiofilm Performance

Biofilm adhesion followed the classic five‑stage model: rapid attachment (0–2 h), growth (2–12 h), maturation (12–24 h), and degeneration (>24 h). The 40‑min ZnO/PAA composite exhibited the lowest adhesion and bacterial counts across all time points. In contrast, the 80‑min sample, despite higher hydrophilicity, showed the highest biofilm accumulation due to lower ZnO density.

SEM images confirmed that the 40‑min sample presented minimal extracellular polymeric substances (EPS) and fewer bacterial colonies, whereas the 80‑min sample displayed dense EPS and mature biofilms. CLSM corroborated these findings, with the 40‑min surface showing a higher proportion of dead cells (red) and fewer live cells (green) at 24–36 h.

Conclusions

By tailoring the second anodization time to 40 min, PAA provides an optimal pore architecture that maximizes ZnO nanoparticle deposition and, after Si69 modification, yields a super‑hydrophobic surface. This configuration effectively inhibits initial bacterial adhesion and disrupts biofilm maturation, establishing the ZnO/PAA composite as a promising antibiofilm material for food‑processing and biomedical applications.

Abbreviations

AO:

Acridine orange

CA:

Water contact angle

EPS:

Exopolysaccharides

FT-IR:

Fourier transform infrared spectrometer

PAA:

Porous anodic alumina

PBS:

Phosphate-buffered saline

PI:

Propidium iodide

SAED:

Selected area electron diffraction

SEM:

Scanning electron microscopy

TEM:

Transmission electron microscopy

TG/DTA:

Thermogravimetric/differential thermal analysis

XRD:

X-ray diffraction