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Biological and Medical Applications of Materials and Interfaces
Food-safe modification of stainless steel food processing surfaces to reduce bacterial biofilms Tarek Samir Awad, Dalal Asker, and Benjamin D. Hatton ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03788 • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 11, 2018
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Food-safe modification of stainless steel food processing surfaces to reduce bacterial biofilms
Tarek S. Awad1, Dalal Asker1,2, Benjamin D. Hatton1*
1
2
Department of Materials Science & Engineering, University of Toronto, Toronto, ON, Canada
Food Science and Technology Department, Faculty of Agriculture, Alexandria University, Egypt
KEYWORDS: stainless steel, anti-adhesive, bacterial attachment, food-safe, biofilm, surface roughness, lubricant layer, food processing
*Corresponding author:
[email protected]
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ABSTRACT: Biofilm formation on stainless steel (SS) surfaces of food processing plants, leading to foodborne illness outbreaks, is enabled by the attachment and confinement within microscale cavities of surface roughness (grooves, scratches). We report Foodsafe Oil-based Slippery Coatings (FOSCs) for food processing surfaces that suppress bacterial adherence and biofilm formation by trapping residual oil lubricant within these surface cavities to block microbial growth. SS surfaces were chemically functionalized with alkylphosphonic acid to preferentially wet a layer of food grade oil. FOSCs reduced the effective surface roughness, the adhesion of organic food residue, and bacteria. FOSCs significantly reduced Pseudomonas aeruginosa biofilm formation on standard roughness SS-316 by 5 log CFU cm-2, and by 3 log CFU cm-2 for mirror-finished SS. FOSCs also enhanced surface cleanability, which we measured by bacterial counts after conventional detergent cleaning. Importantly, both SS grades maintained their anti-biofilm activity after erosion of the oil layer by surface wear with glass beads, which suggests there is a residual volume of oil that remains to block surface cavity defects. These results indicate the potential of such low-cost, scalable approaches to enhance the cleanability of SS food processing surfaces and improve food safety by reducing biofilm growth.
1. INTRODUCTION Stainless steel (SS), such as 316 alloy, is common for food processing and handling equipment due to its strength, corrosion resistance, wear resistance, and relatively low cost.1 However, the high surface energy, typical roughness, and hydrophilic nature of SS make it susceptible to biofouling by organic food residue. The standard average surface roughness (Ra) for industrial food processing surfaces (Mill No.4) is a maximum of 0.8 µm, but can increase after wear (>5 µm) to form surface defects such as grooves, scratches and pits.2-3 Organic residue trapped within these concave surface features can enable bacterial cell attachment and biofilm formation, which constitutes a major problem in the food and
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beverage industries.4 Biofilm formation on food processing surfaces causes food product cross contamination and can lead to foodborne disease.5 Pathogens such as Salmonella enterica, Listeria monocytogenes, Escherichia coli, Bacillus cereus, Shigella spp. and Vibrio spp. are among the most common causes of contamination in food processing plants.6-7 Despite intensive efforts to improve hygienic practice, microbial contamination persists as a problem in the food industry, even in the developed world (such as the recent widespread baby formula contamination by Salmonella8 due to the difficulty in reliably disinfecting food processing equipment.9-10 There are significant health and industry costs associated with this microbial contamination. In the United States, the CDC estimates that each year roughly 1 in 6 Americans gets sick, 128,000 are hospitalized, and 3,000 die of foodborne diseases.11 In developing countries, there are an estimated 2 million deaths every year due to foodborne diseases.12 Consequently, foodborne illnesses are a significant cause of morbidity and mortality, and a consistent public health problem. The total cost of foodborne illness in the US was an estimated $93.2 billion a year in 2015.5 In addition to health risks and direct medical costs, there are a number of significant direct and indirect industrial costs associated with product recalls, plant disinfection, down time and lost productivity. Biofouling in food processing plants, such as dairy, also dramatically reduces operating efficiency and increases corrosion rates and maintenance costs.13 Biofilms are surface attached mixed microbial communities encased in a self-produced extracellular polymeric matrix (EPS).14 Bacteria within biofilms are protected from unfavorable environments during cleaning and sanitation and can be 1,000 times more tolerant to antimicrobials than planktonic cells.14-15 Thus, biofilms are notoriously difficult to eradicate and may survive for years in food processing environments (e.g., processing surfaces, filters, pipes and plumbing system). Certain microbes such as
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Pseudomonas spp., can enable biofilms of other species.16 such as Listeria, and Salmonella within the same biofilm community,17-18 and thereby protect other bacteria that do not produce EPS.19 Bacterial attachment to surfaces is a complex process that is affected by many factors including surface roughness.20 High surface roughness and the presence of recessed surface cavities (scratches, grooves and pits) increase surface area and protect bacterial cells from shear forces, environmental disturbances and cleaning/disinfection processes, which enhance cell attachment and colonization rates.2, 21-22
For example, an increase in the average surface roughness (Ra) of SS (type 304) from 0.11 to 0.3
µm made it more difficult to remove adhering organic residues (e.g., milk, soil),23 while an increase in Ra hindered the removal of E. coli cells, which were significantly higher (CFU cm-2 >10) on the rougher surface (1.37µm) than on a smoother (0.14 µm) surface (CFU cm-2 =1).24 In other work, minimal cell adhesion of four pathogenic bacteria on SS-304 with various surface roughness (from 0.03 to 0.89 µm) was found at Ra= 0.16 µm, while adhesion rates increased for both higher and lower roughnesses.25 Surface finishing (e.g., mechanical, electro-polishing and/or passivation) is an important process for removing any surface debris that is formed during the manufacture of SS. According to the 3-A Sanitary Standards and USDA guidelines, the No. 4 (150 grit) finish is a polished (smooth) SS that is recommend for food contact surfaces, and it is necessary to be free of pits, folds, crevices, cracks, and misalignments in the final fabricated form.1 Such surface treatments reduce the surface roughness of SS surfaces to a hygienic standard (Ra ≤ 0.8 µm), reduce topographical sites for bacterial adhesion,26 and also increase their corrosion resistance.27-28 Current sanitation methods in food processing facilities rely on the application of chemical disinfectants, such as hypochlorites, quaternary ammonium compounds (QUATs), hydrogen peroxide and carboxylic acid. The main strategy to prevent biofilm formation is simply to clean and disinfect surfaces regularly 29. Disinfectants can be ineffective due to the protective role of biofilm EPS, and often
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contain chloramines or hypochlorites that damage the passivating oxide of stainless steel, to cause corrosion (further increasing bacteria adherence). Bacteria in microstructured grooves, cracks or scratches are more difficult for disinfectants to reach,22, 30 and ‘hard to clean’ parts of food processing equipment can receive poor or incomplete sanitization. To enhance food safety, the inhibition of initial bacterial attachment is a valuable strategy to prevent biofilm formation on food processing surfaces. There have been two main strategies to improve the hygienic properties and cleanability of SS food processing surfaces; biocidal antimicrobial coatings (e.g., nickel, copper and silver),31-32 and nonbiocidal anti-biofouling coatings or treatments that reduce the fouling of organic materials and microbial colonization. Anti-biofouling treatments include lowering surface energy by ion implantation (H+, F+, and Si+), coating with a hydrophilic polymer brush (such as poly(ethylene glycol), PEG),33 SiOx plasma enhanced chemical vapor deposition, hydrophobic polymer coatings such as polytetrafluoroethylene (PTFE), electroless nickel-PTFE (EN-PTFE)34 and polymeric nano-composite coatings.35 PEGmodification of SS is not well-suited to long-term, large area application. Also, while they have been shown to inhibit protein adsorption (ß-lactoglobulin), PEG-SS does not necessarily reduce biofilm growth.33 PTFE-based surfaces can be more robust. In one example, Ni–PTFE modified SS heat exchanger surfaces in dairy processing plants were found to reduce milk and bacterial fouling by more than 96%.36 In another study, Ni-P-PTFE coatings reduced E. coli attachment by about 90%.37 While these PTFE antiadhesive coatings have shown effectiveness, they are expensive, require sophisticated coating procedures, and may release unsafe contaminants (e.g., perfluorooctanoic acid). Although certain hydrophobic polymers are commercially available, their monomers or oligomers may leach into food products and cause risk of toxicity, which questions their application.38 Recent epidemiology studies have demonstrated the presence of perfluorochemicals (perfluorooctane sulfonate and perfluorooctanoic acid) in human serum (at ppb) that likely originate from perfluoro coatings into
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food.39 Additionally, fluorinated organic compounds have been found to environmentally persistent, bioaccumulative, and potentially harmful.40
Natural non-wetting structures such as the leaves of carnivorours pitcher plants (e.g., Nepenthes) have inspired the design of a new class of ‘slippery’ non-wetting surfaces by trapping a thin lubricating liquid (immiscible with aqueous or other fluids) at a material surface, such as a nano/microsscale porous surface.41 These slippery liquid-infused porous surfaces (SLIPS) can be highly non-wetting, nonadhesive and non-fouling towards a wide range of solvents (alcohols, hydrocarbons),41 complex fluids (food),41-42 solids (ice),41, 43 protein adsorption44-45 and marine biofouling.46 There has been research into SLIPS applications for anti-thrombogenic and anti-biofouling medical devices,44 smart optics/wettability tunable materials45 and anti-biofouling endoscopes.47 Various SLIPS materials have also been shown to significantly reduce rates of bacterial biofilm formation.44 As mentioned, certain non-biocidal, anti-biofouling surfaces (generally fluorinated) have been developed to suppress the adhesion of food residue for food contact surfaces,48-49 reduce bacterial attachment and biofilm formation, and limit product cross-contamination. With reduced adhesive forces, such surfaces are also more easily cleaned, which reduces maintenance time and cost.37 But in the published literature there are few examples of superhydrophobic or SLIPS designs for food processing surfaces, which ultimately require the surface coating or modifications to be food-safe, and generally low-cost and scalable to industrial environments. Recently, an antifouling biomimetic liquid-infused stainless steel was developed using a femtosecond laser ablation followed by fluorosilanization and impregnation with perfluorinated oil, which prevented dairy protein adsorption during 90 min of pasteurization.50 However, this surface was not tested for bacterial attachment or biofilm formation. Also, perfluorinated liquids are impractical for food processing surfaces as they are very expensive,
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generally quite volatile, and are not considered food-safe. SLIPS for food packaging (such as LiquiGlide)51 have apparently been developed with non-toxic liquids, but no published results for bacterial or biofilm effects. Finally, a superhydrophobic coating was made using ‘edible’ materials to reduce food waste within containers but also not tested for bacteria.52 For application of SLIPS to food processing environments, shear erosion of the lubricant layer by food particles and liquids is expected to rapidly remove the lubricant layer through fluid and solid contact and shear. Previous studies have tested the durability of SLIPS surfaces using hydrostatic flow conditions, but few have tested physical erosion by solid particles. Howell et al (2015) tested the effects of aqueous flow on perfluorocarbon SLIPS surfaces.53 They found that after the initial loss of excess lubricant, there was no additional loss on both chemically functionalized flat and structured surfaces, and no differences between two different viscosities over multiple flow rates and time points up to 16 h.53 Teseler et al54 tested the change in contact angle for a nanoporous oxide-based SLIPS using ceramic bead grinding media, but did not test the biofouling resistance after this wear. Other researchers tested the decrease in slip velocity of water droplets of various volumes and found poor retention of the lubricating oil (silicone) due to cloaking of the oil around the water drops.55-56 There have no specific studies on the changes in bacterial biofouling for SLIPS surfaces after solid abrasion and wear.
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Figure 1. Suppression of organic residue and bacterial and biofilm formation on untreated and oil-coated SS surfaces. Bare SS (a) was functionalized with C8- and C18-phosphonic acid, then coated with food-safe cooking oil (b). Surface defects enhance the adhesion of organic matters and bacterial cells (top right) while oilcoated surface prevent the adhesion cells (middle right). After exposure to physical wear conditions (c) remaining oil fills the concave surface defects, blocking those sites from organic accumulation and bacterial adhesion.
We have designed inexpensive, practical, scalable and food-safe SLIPS modifications for SS that are suitable for food processing equipment and environments. We have developed these food-safe SLIPS SS to be hydrophobic using food-grade cooking oil lubricant to limit food residue accumulation and bacterial surface attachment between processing batches (Figure 1). This Foodsafe Oil-based Slippery Coating (FOSC) approach has the advantage of not requiring any secondary, topographic coating on the SS, and the lubricant is inherently food-safe. While we expect the oil layer to be physically eroded through shear contact and (likely) miscibility with food, our hypothesis is that residual ‘pockets’ of oil can remain within the concave cavities of the surface roughness. After wear, the retained oil within microgrooves, scratches and voids (10-7 to 10-4 m) will effectively reduce the SS surface roughness and block available sites for bacterial attachment (Fig 1). We have performed
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bacterial biofilm culture experiments on these modified SS surfaces, and have specifically tested the effect of mechanical wear (through wiping and glass bead abrasion) on lubricant depletion and biofilm growth. We have tested the efficacy of this surface modification for two SS surfaces with different surface roughness. Two surface modifiers (alkyl phosphonates), and several types of food-safe oils, were tested to optimize the surface hydrophobicity, durability and repellency to liquid and solid organic materials. The adhesion behavior and kinetics of biofilm formation on SS by anti-adhesive coating was quantified by bacterial enumeration and fluorescence image analysis. The removal kinetics of biofilm formed on native and modified SS after cleaning by both deionized water and alkaline cleaner were quantitatively determined. Ultimately, we believe this simple, low-cost, food-safe SLIPS surface modification (FOSCs) for SS could decrease cleaning cycles, and significantly reduce rates of bacterial adhesion to improve product safety.
2. EXPERIMENTAL SECTION
2.1 Materials. Experiments were performed using 316L stainless steel sheet (0.025± 0.01 inch thick, McMaster-Carr), cut in 1 inch squares. Octyl (C8) and Octadecyl (C18) phosphonic acids, (98+%, Sigma-Aldrich) were used as received. Reagents including anhydrous tetrahydrofuran (99.9+%, SigmaAldrich), ethanol (HPLC grade Ultra-Pure, Sigma-Aldrich), isopropyl alcohol (99.9+%, Sigma-Aldrich), methanol (HPLC grade, Ultra-Pure, Biosolve), and dichloromethane (for analysis, Biosolve) were of analytical grade. Mineral oils of different viscosities (light, medium, heavy and extra-heavy) were purchased from Bio Basic Inc. (Markham, Canada). Vegetable oils (olive, corn, and canola) and food sauces were commercial products, as received.
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2.2 Preparation of stainless steel surfaces. Mirror finished SS (SS-MF) was prepared using a polishing wheel and 1µm alumina abrasive. All SS samples were first cleaned using 1% detergent solution, distilled water and pure ethanol in a sonication bath (each 5 min) to remove organic residue. Clean surfaces were then air dried and finally treated for 3 min inside an oxygen plasma chamber (Harrick Scientific Corporation, USA).
2.3 Preparation of self-assembled monolayers (SAM). The basic steps of depositing a selfassembled monolayer (SAM) on SS surfaces are illustrated in Figure 1. In brief, the cleaned and plasmatreated SS was immersed in a 1mM alkylphosphonic acid solution of either short (C8) or long (C18) alkyl chains dissolved in anhydrous tetrahydrofuran (THF) or ethanol, for 5 min at room temperature. To help covalent binding between the alkyl groups and the SS surface, the samples were annealed at 120 o
C overnight. Another set of samples was left to dry at room temperature after SAM deposition (i.e., no
annealing). To remove the physically (weakly) bound alkylphosphonic acid molecules, the samples were rinsed in fresh THF, then in double distilled water under sonication for 1 min (3 times), and finally air dried. A cleaned and plasma-treated SS surface was used as a control. We also varied the surface coating frequency by dipping the surface for 5 min intervals, between which the surface was air-dried at room temperature for 5 min.
2.4 Food-safe oil coating of SAM-functionalized SS surfaces (FOSCs). SAM-functionalized SS surfaces were coated by spreading a drop of oil (mineral, corn, olive). Excess oil was removed by tilting the samples vertically at room temperature for 30 min. To test mechanical erosion of the FOSCs lubricant layer, samples were exposed to silica beads (4 mm, diameter) in a petri dish shaken for 15 min (5 min x 3 cycles).
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2.5 Surface characterization. Surface wettability after chemical attachment of SAM was assessed by measuring the static contact angle (sessile drop method) of constant volume (5 µl) DI water drops using a custom-built goniometer system. Droplet images were analyzed by ImageJ using the contact angle plugin.57 Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR, Spectrum 100 FTIR spectrometer, PerkinElmer) was used to assess the chemical properties of the different SS surfaces after SAM deposition and lubrication. After a background scan, spectra were collected from 4000 to 550 cm−1 using 64 scans at 4 cm−1 resolution. ATR and minimal baseline correction was applied to all spectra. Surface roughness and topography maps were obtained with a 3D Optical profilometer (ContourGT-K 3D, Bruker).
2.6 Bacterial adhesion measurements. Escherichia coli BL21 ATCC 11303 and Pseudomonas aeruginosa PAO1 (Wild-type) cells were cultured in Miller LB Broth (Luria-Bertani) overnight, shaking at 37 °C. Cultured P. aeruginosa cells were then diluted (1:100) in LB without salt (LBNS) supplemented with L-arabinose at a final concentration of 0.5% (w/v), while E. coli cells were diluted (1:100) in LB. 5ml volumes of each diluted culture were then transferred to sterile 6-well polystyrene microtiter plates containing the modified and control SS samples. The plates were incubated statically for specific times (2 or 4 h) at 25 °C to induce biofilm formation. After incubation, biofilm samples were removed, gently rinsed in phosphate-buffered saline (PBS) at pH 7.2, and the adherent bacteria were fixed by 2.5% glutaraldehyde (GDA) in PBS solution for at least 1 h, then rinsed 3 times with PBS to remove unreacted GDA. After fixation, samples were treated with Tween 20 (0.05% in PBS buffer) for 10 min, rinsed with sterile PBS, and stained in the dark with SYTOX green (Life Technologies) for 30 min.58. Samples were imaged by fluorescence microscopy (Olympus BX63) using a 20X air objective, and biofilm fluorescence was observed through a GFP filter (λex/λem 395 and 470 nm). The fluorescence intensity of biofilms was quantified by image analysis (Olympus CellSens software).
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3. Results and Discussion
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3.1 Effect of surface roughness on bacterial adherence and biofilm formation. Surface roughness generally increases rates of bacterial adhesion and growth.59 To confirm the effect of SS surface roughness on bacterial adhesion, the roughness of the as-received (SS-316) and mirror finish (SS-MF) surfaces was measured by 3D optical profilometry (Fig 2A-C). The profilometer results showed the average roughness (Ra) of SS-316 (0.18 µm +/- 0.02) was much higher than SS-MF (0.09 µm +/- 0.02) (Fig 2A-C and Table 1). Table 1 also lists the maximum peak height (Rp), valley depth (Rv) and root
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mean square (Rq) values for the two untreated surfaces. The effect of SS surface roughness on the attachment and biofilm formation of P. aeruginosa PAO1 cells was evaluated. In early stages there was a delay in bacterial cell attachment on SS-MF compared with SS-316. After 4 h of incubation, SS-MF showed about one log CFU cm-2 reduction in the number of attached cells compared to SS-316 (Table 1). SEM images also showed a reduction in the cells attached to SS-MF compared to the SS-316 surface after 4 h (Fig 2F,G). Moreover, there is a tendency for bacterial cells to accumulate near groove and
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scratch defects on the SS-316 surface. These results confirm that decreasing the effective surface roughness reduces initial bacterial adherence. After 24 h growth, however, the density of bacterial cells increased on both surfaces to similar levels, independent of the surface roughness (Fig 2F,G), as 9.3 x 106 CFU cm-2 (SS-316) and 1 x 107 CFU cm-2 (SS-MF) (Table 1). 3.2 Characterization of anti-adhesive surfaces. Both SS-316L and SS-MF surfaces were modified by depositing a monolayer of hydrocarbon chains to increase the surface hydrophobicity and affinity to the Figure 2. Effect of SS surface roughness on bacterial cell adherence and biofilm formation. 3D Optical profilometer images of SS-316 (A); SS-MF (B) and example surface profiles (C). SEM micrographs showing attached cell attachment after 4 h (D, E) and biofilm formation after 24 h (F, G) on untreated SS316 (left panel) and SS-MF (right panel) after immersion in P. aeruginosa PAO1 culture at room temperature. The projected area of each profilometry image is 0.35 x 0.45 mm.
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fatty acid chains of the lubricating oils (Fig 1). ATR-FTIR was used to evaluate the deposition and ordering of C18 monolayer. Figure 3A,B shows the IR spectra (C−H stretch region) and phosphate stretching of 1mM C18 SAM on SS-316L and SS-MF. The FTIR stretching mode values are listed in Table S1. The C-H region corresponds to the methylene group of the aliphatic long alkyl chains of the SAM, which have been used to determine the degree of crystallinity and order of monolayers.60 For the C18-functionalized surfaces, there were two main peaks at 2923 cm−1 and at 2853 cm−1, which correspond to the asymmetric and symmetric methylene stretching, respectively (Fig 3A). These values of these peaks were close to those reported on SS-316L and Mg alloy, which suggested the order of the SAMs formed on the two SS surfaces. The peak at 2953 cm−1 is assigned to the out-of-plane asymmetric methyl stretching mode vCH3op.61-62 In addition, the presence of the phosphonate stretching νP-O ( 1088-1095 cm-1), νP=O (1155-1165 cm-1) and νP-O-H (955 cm-1) (Fig 3B) may indicate a tridentate coordination mode of the phosphonate to the SS surface.61
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The uniformity of the SAM functionalization was evaluated by measuring the surface hydrophobicity using static water contact angle (CA) measurements. Untreated native SS-316 has a low CA (∼21 ± 3°) and high surface wettability (Figure 3C) indicative of its hydrophilic character. Coating SS-316 with C8 and C18 SAM decreased wettability and increased CA to 82.5 ± 2° (not shown) and 108.7 ± 3.5° (Figure 3D), respectively, which agrees with previously reported values for well-ordered SAM on SS-316L 63, alkylsilanes on silicon 64, alkanethiols on gold 65 and methyl-terminated SAMs on metals and metal oxides.66 C18 SAM deposition on SS-MF also increased CA from 62 ±1° (untreated) to 108 ±1° (Table 1). Figure 3. Chemical and physical characteristics of SS-316 surface modified by C18-phosphonic acid SAM deposition and mineral oil coating. ATR-FTIR spectra of untreated (SS316), C18-coated SS-316 and C18coated SS-MF of (A) the C–H stretch region showing the methyl (1), the asymmetric (2) and symmetric (3) methylene stretching, and (B) the P-O region showing three peaks for P-O, P=O and P-O-H. Images of water drops on (C) untreated, (D) C18 SAM coated and (E) oil coated SS-316L. Time-lapse optical images showing the mobility of ketchup sauce on tilted (~45°) uncoated (F-H) and oil coated (I-K) surfaces (videos in Supporting Information).
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To optimize the C18 SAM deposition, we studied the effect of immersion time and annealing. As shown in Figure S1, an immersion time of at least 30 min was required to make the surface hydrophobic (CA ~ 105o). In addition, increasing the coating frequency enhanced the surface hydrophobicity after 3 cycles (i.e., 15 min). Annealing was found to not significantly affect the CA, as surfaces that were annealed for 90 min, overnight, or not at all showed CA of ~108º. When the C18 phosphonic acid was solubilized in ethanol, the surface hydrophobicity also increased (CA= 95o). Compared to THF, ethanol is a low cost and low toxicity solvent. Coating the hydrophobic C18-functionalized SS surfaces with a thin layer of mineral or vegetable oil altered the surface chemistry. For lubricated surfaces, ATR-FTIR showed several well-defined peaks (Figure S2), which have been previously reported for mineral oil.67 The bands between about 2952 and 2852 cm−1 correspond to the CH asymmetric and symmetric stretching modes, the peaks at 1458 and 1377 cm−1 correspond to CH bending modes, and the single peak at about 720 cm−1 represents the asymmetric angular deformation of CH2 groups.67 Lubrication also greatly affected wettability of the two surfaces. For example, lubrication increased the surface contact of water droplet with SS-316 surface (Figure 3E), which decreased CA from ~109° to 88° or 100° for SS-316 and SS-MF, respectively (Table 1). In addition, the sliding angles of water droplets decreased from ~95° (untreated) to ~40° and from ~90° (untreated) to ~43° for SS-316 and SS-MF, respectively (Table 1). When surfaces were tilted, liquid droplets (e.g., water and ketchup) were mobile on lubricated C18functionalized surface (Figure 3F-H) while pinned on both lubricated C8-functionalized surface (not shown) and untreated surface (Figure 3I-K). A weak hydrophobic interaction between the alkyl chains of the short (C8) and the oil did not hold the oil layer on rinsing and the surface lost its anti-adherence activity quickly (Video S1). Conversely, the high affinity between mineral oil and the long (C18) terminal chains stabilized the oil layer, which maintained its anti-adherence activity after repeated
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surface cleaning with water (Video S2). When food-grade vegetable oils (canola and olive oils) were used to lubricate these surfaces, similar anti-adherence surface activity for the lubricated C18functionalized surface prevented the adhesion of food liquids compared to untreated or lubricated C8functionalized surface. Figure 4 shows optical profilometer images and surface profiles for the oilcoated surfaces. After lubrication, both SS-316 (Figure 4A) and SS-MF (Figure 4B) surfaces became even (i.e., flat and smooth), and showed significant reductions in roughness compared to untreated surfaces (Figure 2A-C and Table 1). For example, Ra and Rt decreased from 0.18 and 8.66 µm (untreated) to 0.06 and 1.07 µm (oil-coated) for SS-316, and from 0.09 and 7.12 (untreated) to 0.02 and 0.26 µm (oil-coated) for SS-MF. The significant reduction in Ra of both surfaces demonstrates the effectiveness of lubrication to cover surface defects. 3.3 Surface durability. The wear by food particles during mixing is expected to degrade the lubricant layer through physical erosion. To test changes in anti-adhesive (anti-microbial) behavior of modified SS surfaces, we studied the effect of mechanical wear on the depletion of lubricant and surface physical characteristics. Interestingly, the two surfaces showed an increase in CA (more hydrophobic) after wear which may be due to the exposure of more hydrophobic C18- functionalized surface (superhydrophobic Wenzel effect). The sliding angles values increased slightly for both the 316L and mirror finish, due to more pinning points in the underlying surface roughness. ATR-FTIR of the two surfaces after wear indicated the existence of weak mineral oil signals (Figure S2). Optical profilometry measurements showed that Ra increased substantially to 0.38 µm after exposure to wear cycles (Figure 4C) compared with the oil immobilized SS-316 surface (Figure 4A), due to decreased oil layer thickness. As shown from the 3D profiler images and surface profiles (Figure 4A,C,E), the surface topography changed from smooth (excess oil) to a roughness similar to that before treatment (Figure 2A). However, the surface 2D image (Figure S3A) showed that the surface grooves (blue lines) are interrupted by regions of
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shallower surfaces (green), which suggests the presence of oil trapped inside the grooves. As for the smoother (no visual grooves) SS-MF, the Ra value also increased after wear from 75 nm (Figure 4B) to 0.25 µm (Figure 4D,F), which is higher than for the untreated surface (Figure 2C). In addition, the appearance of circular patterns in the 2D surface image (Figure S3B) was evident, which also supports the presence of oil on the surface. This evidence may indirectly suggest that the worn surfaces can still hold an amount of residual oil inside the concave grooves after wear conditions (15 min). The slight increase in Ra after wear compared to the surfaces before lubrication maybe due to uneven distribution of oil. To confirm these assumptions, we tested the bacterial anti-adherence activity for the two surfaces after the same wear conditions, to mimic the conditions of food processing (mixing) operations.
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Figure 4. Reduced effective surface roughness after coating with mineral oil, before and after wear. 3D Optical profilometer images of (A) oil-coated SS-316; (B) oil-coated SS-MF and after wear (C, D). The surface profiles are combined before and after wear for (E) SS-316 and (F) SSMF. Oil-coated surfaces were subjected to wear conditions for 15 min. The projected area of each profilometry image is 0.35 x 0.45 mm.
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3.4 Lubrication suppresses bacterial cells attachment and biofilm formation. Initially, C8- and C18-functionalized SS-316L surfaces were coated with canola oil then exposed to P. aeruginosa PAO1 under static culture conditions for 4 h at room temperature. Bacterial cell attachment was reduced to just a few individual cells on the C8-functionalized and oil-coated surface (Figure S4c) compared to those untreated (Figure S4a) or functionalized with C18-SAM (Figure S4b) surfaces. In addition, a complete elimination of bacterial attachment was observed on the C18-functionalized and oil-coated surface (Figure S4d). Therefore, the prevention of E. coli adherence on the C18-functionalized SS after lubrication with three mineral oils with different viscosities (light, heavy and extra heavy) were studied by exposing the
Table 1. Effect of treatment on surface hydrophobicity (contact and tilt angles), surface roughness and bacterial cell attachment on native (SS-316) and mirror finished (SS-MF) SS 316-L before and after treatment, and after wear. Surfaces were first treated by octadecyl (C18) phosphonic acid SAM deposition then lubricated with mineral oil. Values are reported as averages.
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oil-coated surfaces to diluted cultures of E. coli (Fig S5). Densely attached bacterial cells on both control (Fig S5a) and C18-functionalized (Fig S5b) SS-316 surfaces were observed. After surface coating with oil, bacterial attachment decreased with increasing the viscosity of mineral oil. While light mineral oil coated surface inhibited bacterial adherence to some extent (Fig S5c), there were less cells on the surfaces coated with the heavy and extra heavy mineral oils and olive oil (Fig S5d-f). Image analysis of the number of attached cells and percentage of cells reduction (Fig S5g) confirmed the same results. SEM, fluorescence microscopy and CFU cm-2 were used to characterize P. aeruginosa adhesion on the extra heavy mineral oil coated SS 316 and SS-MF compared with uncoated surfaces for 4 and 24h. While the untreated SS-316 surfaces were completely covered with adherent bacterial cells after 4 h (Fig 5A) and 24 h (Fig 5B), oil coated surfaces reduced bacterial cells attachment after 4 h (Figure 5A) by > 3 log CFU cm-2 (Fig 5C) and biofilm formation after 24h (Fig 5B) by >5 log CFU cm-2 (Fig 5D). In comparison, the oil-coated MF-SS was also anti-adhesive (Fig S6) as it reduced bacterial attachment by ~2 log CFU cm-2 after 4 h (Figs S6A,C) and ~3 log CFU cm-2 after 24h (Figs S6B,D). From these results, it is reasonable to conclude that the oil layer immobilized on the surface fills the grooves, which prevents the contact between bacterial cells and surface and colonization. Increasing the surface roughness (i.e., SS-316) increases the surface area and the immobilized (trapped) oil amount compared with the smooth SS-MF surface, which does not significantly trap oil.
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Furthermore, the antibiofouling activity of the oil-coated surfaces was tested after wear with glass beads for 15 min. After wear, the oil coated SS-316 maintained its antibiofouling activity for 4 and 24 h corresponding to 3 and 2 log reductions compared to the uncoated surfaces, as shown in Figs 5A,C and 5B,D, respectively. Similar results were obtained after wear of the oil coated SS-MF, which showed 4 and 3 log reductions after 4 and 24 h, as shown in Figs S6C and S6D, respectively. SEM and fluorescence images showed the durability of the anti-adherence (Figure S6A) and antibiofilm (Figs S6B) of the coated SS-MF surfaces. These results strongly suggest that, despite the depletion of the surface excess oil layer due to wear, the residual oil trapped within microscale defects can still provide the surface with remarkable antibiofouling activity. Early tribology studies showed that surface lubricant is retained on metal surfaces to a greater extent as a function of microstructural roughness.68 Also, tribology measurements on ‘lubricant starved’ surfaces, where the lubricant layer is less than the Ra roughness, show that trapped volumes of liquid partially fill recessed areas of surface topography.69
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3.5 Cleanability of oil-coated surfaces. We studied the effect of oil coating on the cleanability of SS surfaces after soaking in diluted detergent (2%) and rising with copious amounts of water. After washing, the number of attached cells (CFU cm-2) on uncoated SS-316 surfaces decreased by 2 and 3 log CFU cm-2 on the surfaces with grown biofilm for 4 and 24 h, respectively (Figure 6A,B). However, a complete elimination of bacterial cells was observed on the oil coated SS-316 surface with 4 h grown biofilm (Figure 6A). For the 24 h grown biofilm (Figure 6B), although bacterial attachment increased by 3 log CFU cm-2 for the oil coated SS-316, no bacterial cells were observed after washing demonstrating a 4 log CFU cm-2 reduction compared with the uncoated surface. Similar results were obtained for SSMF surfaces before and after washing (Figure S7). These results demonstrate a significant increase in the cleanability of oil-coated surfaces. In summary, functionalization of SS surfaces with long chain
Figure 6. Reductions in attached P. aeruginosa cell counts (log CFU cm−2) of (A) 4 h and (B) 24 h grown biofilms on uncoated; oil coated; and oil coated (after wear) SS-316 surfaces before and after washing with alkaline cleaner and DI water.
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alkyl (C18) phosphonic acid monolayer (SAM) enhanced the surface hydrophobicity, surface interactions with oil molecules and antiadhesive activity, which formed a physical barrier (FOSC) that hindered the ability of bacterial cells to adhere and consequently form a mature biofilm that is difficult to eradicate or disinfect. This anti-adhesive character of FOSC also facilitates the removal of all the bacterial cells from the surface or within the surface grooves upon rinsing in a detergent solution. We suggest these FOSC treatments are a low-cost and scalable solution to improving the cleanability and resistance to bacterial contamination of large industrial food processing facilities. A major advantage of FOSC treatment is that the oil lubricant can be replaced between food processing batches (such as mixing). Also, while alkylphosphonates are not currently classified Generally Recognized as Safe (GRAS) by the FDA (to be food-safe, various phosphonates are used in water treatment and FDAapproved drugs. Further, there are several alternative approaches (and products) available in the food industry to modify SS surfaces with hydrophobic wettability. The fluid modelling and experimental work of the Stone group at Princeton have studied the viscosity ratio of the lubricant liquid to the external contacting fluid, to predict when shear flow removal of lubricant (within surface topography) should occur.70 We intend to further optimize our choice of lubricant oil against bacterial adhesion under a range of wear/erosion conditions with these viscosity ratios in mind, since there is a vast range of hydrocarbon and complex fats, oils and waxes which could be designed for this application.
3. CONCLUSIONS Adherence of food debris to SS surfaces during food processing encourages bacteria to attach, colonize and form an ineradicable biofilm. Prevention, rather than treatment, of biofilm formation is the ideal solution to avoid food contamination and risks of food-borne infections as cleaning and disinfection of surface-bound biofilms can be very challenging. We have developed a simple, low cost and food-safe
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treatment (FOSCs) for stainless steel surfaces as the first example of food-safe SLIPS for food processing surfaces. This approach appears to block concave surface defects (scratches and grooves) as significant sites for initial bacterial adhesion and biofilm growth. The deposition of monolayers (SAM) of long chains (C18) linear alkyl phosphonic acids enhances the surface hydrophobicity to aid the wetting of the oil layer. The optical profilometry results showed that the oil layer remaining after wear still covers areas of the surface microstructure to fills in the defects and reduce the surface area. Even after wear, this residual oil acts as a physical barrier to reduce P. aeruginosa surface adherence and biofilm formation by a factor of log ~ 4 and 5 CFU cm-2 after 4 and 24 h, respectively. This surface treatment (i.e., FOSCs) is relatively simple and non-toxic (food grade/FDA approved oils), and could be scalable to large industrial equipment, unlike other more complex SLIPS surface treatments such as microstructural patterning, coatings or surface oxidation. Also, our FOSC approach allows for a wide variety of edible oils and waxes that can be tuned compositionally to achieve the optimum viscosity and durability against wear.
Supporting Information. Effect of octadecylphosphonic acid immersion time on surface hydrophobicity (Figure S1), effect of wear on lubricant stability (Figures S2 and S3), suppression of biofilm formation of P. aeruginosa (Figure S4), Effect of oil type and viscosity on suppression of E. coli adherence (Figure S5), suppression of P. aeruginosa cells attachment and biofilm formation on mineral oil coated SS-MF (Figure S6) and its cleanability (Figure S7) are supplied as Supporting Information. Funding: The authors wish to acknowledge the support of a Natural Sciences and Engineering Research Council of Canada (NSERC) Engage Program grant (#EGP 488391-15), and a Canada Foundation for Innovation (CFI) John Evans Fund grant (#31799).
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