Prevention Of Microbial Adhesion In Food Processing Environment Using Multifunctional Nanopillared Surfaces

<p>Task 1. Study nanoscale patterning and treatment techniques for aluminum/stainless steel substrates and develop a prototype anti-microbial adhesion surface with controllable surface wettability and slipperiness. The PI group will use the developed protocols and recipes for processing different pore sizes and pillar heights during test trials. Based on the PI's input, Dr. Choi's research group will further optimize the wettability and slipperiness of the specimens controlled by precision spin coating with hydrophobic polymer i.e., Teflon and infusing the prepared surfaces with low-surface tension lubricant i.e., Krytox. Surface roughness measurement: Surface roughness characterization will be performed by the use of an atomic force microscopy (AFM) equipped with carbon nanotube tip (10-30 nm in diameter and < 1 µm in length, N-Tracer Scanning Probe Microscope, NanoFocus, Inc.).Scanning electron microscopy: Bare substrates will visualized using FESEM (Hitachi S-4800, 50,000X, Biological Electron Microscope Facility, UH). Substrates exposed to bacterial cells will be immersed in nitrogen slush for 10 s, freeze-dried using a Labconco FreeZone freeze dryer (Labconco Corp., Kansas City, MO) for 24 h, and then mounted onto stubs. Images obtained by SEM will be visually analyzed to determine the size and number of bacterial appendages that were visible on the attached cells. The number of appendages was categorized into the following groups: 0 to 5, 6 to 10, 11 to 15, 16 to 25, and >> 25. Cells categorized as >> 25 have too many appendages to count. The average roughness (Ra), root-mean-square roughness (Rrms), and 10-point height (Rz) will be extracted from the AFM images by using the free and open source software Gwyddion. Wear test: The wear and friction tests for evaluation of tribological properties of the developed nanopillared surfaces will be carried out using a Nanovea tribometer (Microphotonics, Irvine, CA). </p><p>Task 2. Evaluate the effect of the developed surface on microbial adhesion and furthermore biofilm formation using various types of pathogenic microorganisms. The PI and Co-PI (Dr. Li) at UH will test the anti-microbial attributes of the developed nano-engineered surfaces using pathogens including E. coli, Salmonella Typhimurium., S. aureus, and L. monocytogenes at various wall shear rates. The detailed bacterial adhesion tests are in the following.1. E. coli, Salmonella Typhimurium, S. aureus, and L. monocytogenes cells will be separately cultured in tryptic soy broth (TSB) at 37°C for 24 h before used. Bacterial adhesion tests will be carried out on hydrophilic, hydrophobic, and slippery nano-engineered surfaces, all placed together side-by-side inside a parallel-plate flow channel. To perform the stagnant adhesion tests, the prepared bacterial suspensions will be held inside a pre-cleaned test chamber for 1, 5, 10, 18, 24, and 96 hrs. On the other hand, bacterial suspensions will individually pumped through the test chamber at various flow rates for dynamic adhesion test.3. The growth of biofilms on the proposed nanopillared surfaces can be monitored using an optical laser scanning microscope at the Biology Department. Biofilm culture will be prepared by incubation at 28°C with bacterial suspension and culture medium on the surfaces. Randomly selected biofilm samples from all replicates are thoroughly washed three times with 0.05 M phosphate buffer at pH 7.4. Each sample will be subsequently immersed in 1 mL solution of PBS for 5 min twice. For staining, samples will be added to 5 µL of 0.5% (w/v) fluorescein isothiocyanate (FITC) stock solution in 1 mL of PBS in the dark. Finally, samples will be washed with PBS and then fixed with 4% glutaraldehyde at room temperature for 1 hr. The scanning microscope will be used for the image analysis of samples.4. FITC and green fluorescent protein (GFP) with clear hydrophobic markers will be used to inspect their adhesion with the proposed hybrid surfaces and the control for a comparative study. A fluorescence microscopy (Nikon Ti-U inverted microscopy, Available in Dr. Li's lab) will be used to inspect the samples after they are incubated with FITC and GFP. ImageJ software, (NIH, Bethesda, Maryland) is used for image processing and analysis.</p><p>Task 3. Study and develop the comprehensive model to elucidate the mechanics of microbial adhesion to the nanopillared surfaces with control factors such as surface wettability and slipperiness e.g. kinetics, cell-surface interaction theories (UH and SIT)1. A mathematical model to simulate the three-dimensional single bacterial attachment to nano-engineered surfaces (i.e. nanopillared) will be developed and tested with bacteria i.e. E. coli, Salmonella spp., S. aureus, and L. monocytogenes in different wall shear rates. The model combines accurate diffusion-reaction equations to simulate substrate diffusion with cellular automation-like criteria to model initial bacterial adhesion process as well as biofilm growth and colonization on solid substratum. Therefore, the cell-surface interactions e.g., attachment kinetics, effects of surface nano-topography, hydrophobicity, and slipperiness can be determined.2. The concentration of substrates in the bulk liquid simulates environmental conditions where bacterial biofilms can develop. Chemical species used in the model can be lactate, hydrogen, methane, acetate, bicarbonate, sulfate, and hydrogen sulfide, and the mass balance equation of a chemical species m in a biofilm element.3. The numerical optimization of the 3D model is based on the separation of the biological, physical, and chemical phenomena in three distinct steps: (a) diffusion of chemical species, (b) growth of biomass within each biofilm element, and (c) biofilm expansion and colonization. Commercial computational software i.e., COMSOL multiphysics will be used to calculate the transient concentration of chemical species and multiplication of cells within each biofilm element for a given time step, and distribute excess cells from overpopulated biofilm elements to neighboring elements.</p><p>Task 4: Design pilot-scale washing equipment for fresh-produce processing and test anti-adhesion property of nano-engineered surfaces1. Designing of the pilot-scale washing station is necessitated for the use of less water and samples as well as to simulate a real-time situation when the proposed nano-engineered surface would be applied. The dimension of the developed unit will be approximately 2.5'´1'´1' and consist of air bubble jets, water recirculator, and sample loading area. The bottom surface will be tiled with two different surfaces (nanopillared and control surfaces). Flow characteristics, treatment time, and system schematics will be key parameters for effective adhesion testing of microbial contaminants on the surfaces. A typical versatile water system in fresh produce industries is a flood washer that has underwater jets with or without the use of chlorine. The proposed washing station will consist of holes on the sidewall where washing water will be sprayed through. High spray water injection through side holes will create water bubble effects to release microbial contaminants from fresh produce.2. Microbial testing: Experimental conditions can be defined as follows: </p><p><ol><li> Only cabbages will be contaminated; </li><li> Only washing water (spiked with bacterial solution) will be contaminated; and </li><li> Both cabbages and washing water will be contaminated. Fresh produce washing in the pilot-scale equipment will be allowed to run for 1, 5, 10, 18, 24, 96 hrs. Thereafter, the same procedure as in Approach 2 will be repeated for bacterial adhesion testing. After draining washing water, individual pieces of nanopillared and control surfaces will be separated from the station base and subject for microbial counts and anti-biofilm tets.</li></ol></p>