The enrichment of surface passive film on stainless steel during biofilm development in coastal seawater

The surface passive film on UNS S30400 alloy was characterized before and after biofilm development under different regimes of diurnal lighting in quiescent flowing coastal seawater. As exemplified by atomic force microscopy, the passive film grew under all test conditions with conspicuous variations in morphological features. X-ray photon spectroscopy illustrated an enrichment of the outer film by iron oxide and a progressive increase in the iron oxide/chromium oxide ratio with lighting. Mott-Schottky plots reflected the duplex nature of the film, comprising an outer n-type and an inner p-type configuration. The slopes of the plots showed a strong decrease in donor and acceptor densities with biofilm coverage and lighting, thus confirming passive film growth. These results provide new insights that passive film enrichment is an intrinsic process under practical marine conditions, and show that the evolution of the passive film is a key step to sustained passivity and/or its breakdown by microbial mechanisms.     

Strain-specific colonization patterns and serum modulation of multi-species oral biofilm development

Periodontitis results from an ecological shift in the composition of subgingival biofilms. Subgingival community maturation is modulated by inter-organismal interactions and the relationship of communities with the host. In an effort to better understand this process, we evaluated biofilm formation, with oral commensal species, by three strains of the subgingivally prevalent microorganism Fusobacterium nucleatum and four strains of the periodontopathogen Porphyromonas gingivalis. We also tested the effect of serum, which resembles gingival exudates, on subgingival biofilms. Biofilms were allowed to develop in flow cells using salivary medium. We found that although not all strains of F. nucleatum were able to grow in mono-species biofilms, forming a community with health-associated partners Actinomyces oris and Veillonella parvula promoted biofilm growth of all F. nucleatum strains. Strains of P. gingivalis also showed variable ability to form mono-species biofilms. P. gingivalis W50 and W83 did not form biofilms, while ATCC 33277 and 381 formed biofilm structures, but only strain ATCC 33277 grew over time. Unlike the enhanced growth of F. nucleatum with the two health-associated species, no strain of P. gingivalis grew in three-species communities with A. oris and V. parvula. However, addition of F. nucleatum facilitated growth of P. gingivalis ATCC 33277 with health-associated partners. Importantly, serum negatively affected the adhesion of F. nucleatum, while it favored biofilm growth by P. gingivalis. This work highlights strain specificity in subgingival biofilm formation. Environmental factors such as serum alter the colonization patterns of oral microorganisms and could impact subgingival biofilms by selectively promoting pathogenic species.     

The effect of Pseudomonas NCIMB 2021 biofilm on AISI 316 stainless steel

A bioreactor system operating in a continuous mode was designed to generate biofilms on polished and as-received surfaces of AISI 316 stainless steel coupons exposed for 36 d to a pure culture of marine Pseudomonas NCIMB 2021. Scanning electron microscopy (SEM) and atomic force microscopy were employed to determine the degree of surface colonisation and to examine corrosion damage of the steel. X-ray photoelectron spectroscopy analysis was carried out to characterise the chemistry of the passive layers on polished steel stored for a period of time, freshly re-polished coupons, and as-received steel. The effect of biofilms on the composition of layers formed on the steel specimens was evaluated. SEM revealed that the surfaces of polished and stored steel appeared to accumulate more biofilm compared to as-received specimens. Micropitting of steel occurred underneath the biofilm, regardless of surface finish. The concentration of elements in the passive layers differed significantly between freshly re-polished and as-received or polished and stored coupons. In the presence of Pseudomonas NCIMB 2021 biofilm, the composition of the passive layer on the as-received steel surface was considerably altered compared to unexposed steel or steel exposed to abiotic medium.     

Osteopontin reduces biofilm formation in a multi-species model of dental biofilm

Background

Combating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN), a highly phosphorylated whey glycoprotein, on a multispecies in vitro model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms.

Methodology/Principal Findings

Biofilms consisting of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis were grown in a flow cell system that permitted in situ microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence in situ hybridization, changed considerably in the presence of OPN. In particular, colonization of S. mitis, the best biofilm former in the model, was reduced dramatically.

Conclusions/Significance

OPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed in vivo, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.     

Antibacterial isoeugenol coating on stainless steel and polyethylene surfaces prevents biofilm growth

Aims

Pathogenic bacteria can spread between individuals or between food items via the surfaces they share. Limiting the survival of pathogens on surfaces, therefore, presents an opportunity to limit at least one route of how pathogens spread. In this study, we propose that a simple coating with the essential oil isoeugenol can be used to circumvent the problem of bacterial transfer via surfaces.

Methods and Results

Two commonly used materials, stainless steel and polyethylene, were coated by physical adsorption, and the coatings were characterized by Raman spectroscopy, atomic force microscopy and water contact angle measurements. We quantified and visualized the colonization of coated and uncoated surfaces by three bacteria: Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens. No viable cells were detected on surfaces coated with isoeugenol.

Conclusions

The isoeugenol coating prepared with simple adsorption proved effective in preventing biofilm formation on stainless steel and polyethylene surfaces. The result was caused by the antibacterial effect of isoeugenol, as the coating did not diminish the adhesive properties of the surface.

Significance and Impact of the Study

Our study demonstrates that a simple isoeugenol coating can prevent biofilm formation of S. aureus, L. monocytogenes and P. fluorescens on two commonly used surfaces.     

Variation in sessile microflora during biofilm formation on AISI-304 stainless steel coupons

Coupons of stainless steel type AISI-304 were exposed to the industrial cooling system of a petrochemical plant fed by seawater from the Guanabara Bay, Rio de Janeiro, Brazil, in order to study thein situ formation of biofilms. Bacteria, microalgae and fungi were detected on the coupons as soon as 48 h after exposure. Their respective numbers were determined at times 48, 96 and 192 h and over the following 8 weeks. Aerobic, anaerobic and sulfate-reducing bacteria were quantified according to the technique of the most probable number, and fungi by the pour plate technique. The number of microorganisms present in the forming biofilm varied over the experimental period, reaching maximal levels of 14×10¹¹ cells cm^–2, 30×10¹³ cells cm^–2, 38×10¹¹ cells cm^–2 and 63×10⁵ cells cm^–2, respectively, for aerobic bacteria, anaerobic bacteria, sulfate-reducing bacteria and fungi, and the dynamics of this variation depended on the group of microorganisms.Bacillus sp,Escherichia coli, Serratia sp andPseudomonas putrefaciens were identified among the aerobic bacteria isolated. Additionally, microalgae and bacteria of the genusGallionella were also detected. Nonetheless, no evidence of corrosion was found on the stainless steel type AISI-304 coupons over the experimental period.     

Demonstration of biofilm removal from type 304 stainless steel using pulsed-waveform electropolishing

Abstract

This article describes an electrochemical method to remove bacterial biofilm from a stainless steel (SS) surface using a potential pulse/reverse pulse technique. This technique employs a periodic waveform that consists of anodic and cathodic pulses. The pulses can effectively strip a thin layer of metal off the SS surface, along with the adherent biofilm, in a saline solution. Not only can the pulses effectively remove biofilm from the SS surface, but they also regenerate the original mirror-like shiny surface. The importance of this electrochemical biofilm removal method is its wide applicability for any types of biofilms. That is, instead of directly removing the biofilm, it removes a very thin layer of the metal under the biofilm. Thus, the removal process is independent to the nature of the biofilms. Furthermore, this electrochemical biofilm removal method is rapid (less than 30?s of potential pulse time) and does not require hazardous chemicals.     

Terpinen-4-ol and carvacrol affect multi-species biofilm composition

The aim of this study was to investigate the cytotoxic activity and inhibitory effect of terpinen-4-ol (T4ol) and carvacrol against single- and multi-species biofilms. The toxicity of each compound was tested on oral keratinocytes and evaluated by XTT assay. Inhibition and eradication of single-species biofilms were analyzed by crystal violet assay and the effect on multi-species biofilm composition was evaluated by qPCR. T4ol and carvacrol did not affect the epithelial cell viability, in contrast to chlorhexidine, which showed a high cytotoxic effect. Inhibition and eradication of single-species biofilms treated with T4ol and carvacrol were observed. The same inhibitory effect was observed for multi-species biofilms, especially on periodontal pathogens. In conclusion, specific concentrations of T4ol and carvacrol without toxicity towards the epithelial cells reduced the numbers of periodontal pathogens in single- and multi-species biofilms.     

First air-tolerant effective stainless steel microbial anode obtained from a natural marine biofilm

Microbial anodes were constructed with stainless steel electrodes under constant polarisation. The seawater medium was inoculated with a natural biofilm scraped from harbour equipment. This procedure led to efficient microbial anodes providing up to 4 A/m² for 10 mM acetate oxidation at −0.1 V/SCE. The whole current was due to the presence of biofilm on the electrode surface, without any significant involvement of the abiotic oxidation of sulphide or soluble metabolites. Using a natural biofilm as inoculum ensured almost optimal performance of the biofilm anode as soon as it was set up; the procedure also proved able to form biofilms in fully aerated media, which provided up to 0.7 A/m². The current density was finally raised to 8.2 A per square meter projected surface area using a stainless steel grid. The inoculating procedure used here combined with the control of the potential revealed, for the first time, stainless steel as a very competitive material for forming bioanodes with natural microbial consortia.     

Influence of materials and precoating on initial anaerobic biofilm development

Summary This study investigated the possibility of reducing anaerobic reactor start-up times through the use of various support surfaces or through the use of biological precoating (denitrifying biofilm) and chemical precoating (polymer precoating) as means of enhancing anaerobic biofilm development. Results from the support media variation experiment indicated significant differences among the materials. Support media precoating with denitrifying bacterial biofilms and the various polymers tested did not appear to enhance the rate of initial anaerobic biofilm accumulation.     

Influence of surface chemistry on the hygienic status of industrial stainless steel

Coupons of fourteen different stainless steels were investigated in terms of surface chemistry and ease of cleaning. Steel surfaces were exposed to Bacillus cereus spores in static saline solution for 2 h. Surfaces were rinsed and then covered with whole milk and allowed to dry. Surfaces were then cleaned in an experimental flow system that mimics an industrial application. After cleaning, remaining spores were released by sonication, spores cultured and colony forming units determined. Surfaces with higher levels of Fe in the outer surface of the passive film cleaned more easily. There was a relation between the polar component and ease of cleaning. The higher the polar component the more easily the surface cleaned. The cleaning mechanism involves dissolution of Fe enriched hydroxide films on the surface.     

Effectiveness of phages in the decontamination of Listeria monocytogenes adhered to clean stainless steel, stainless steel coated with fish protein, and as a biofilm

Listeria monocytogenes is a food-borne pathogen which causes listeriosis and is difficult to eradicate from seafood processing environments; therefore, more effective control methods need to be developed. This study investigated the effectiveness of three bacteriophages (LiMN4L, LiMN4p and LiMN17), individually or as a three-phage cocktail at ≈9 log₁₀ PFU/ml, in the lysis of three seafood-borne L. monocytogenes strains (19CO9, 19DO3 and 19EO3) adhered to a fish broth layer on stainless steel coupon (FBSSC) and clean stainless steel coupon (SSC), in 7-day biofilm, and dislodged biofilm cells at 15 ± 1 °C. Single phage treatments (LiMN4L, LiMN4p or LiMN17) decreased bacterial cells adhered to FBSSC and SSC by ≈3–4.5 log units. Phage cocktail reduced the cells on both surfaces (≈3.8–4.5 and 4.6–5.4 log₁₀ CFU/cm², respectively), to less than detectable levels after ≈75 min (detection limit = 0.9 log₁₀ CFU/cm²). The phage cocktail at ≈5.8, 6.5 and 7.5 log₁₀ PFU/cm² eliminated Listeria contamination (≈1.5–1.7 log₁₀ CFU/cm²) on SSC in ≈15 min. One-hour phage treatments (LiMN4p, LiMN4L and cocktail) in three consecutive applications resulted in a decrease of 7-day L. monocytogenes biofilms (≈4 log₁₀ CFU/cm²) by ≈2–3 log units. Single phage treatments reduced dislodged biofilm cells of each L. monocytogenes strain by ≈5 log₁₀ CFU/ml in 1 h. The three phages were effective in controlling L. monocytogenes on stainless steel either clean or soiled with fish proteins which is likely to occur in seafood processing environments. Phages were more effective on biofilm cells dislodged from the surface compared with undisturbed biofilm cells. Therefore, for short-term phage treatments of biofilm it should be considered that some disruption of the biofilm cells from the surface prior to phage application will be required.     

Influence of surface topography on biofilm development: Experiment and modeling

The effect of surface topography on the long-term development (≈10 weeks) of biofilms has been investigated using a monitoring technique based on images produced by a flat-bed scanner and initially developed for flat surfaces. The biofilm response to rotation speed changes in lab-scale rotating biological contactors (RBCs) has been studied. Two RBCs, each containing five discs (two with flat surfaces and three with rough surfaces) were run initially at two different rotation speeds: 4 rpm for reactor I and 40 rpm for reactor II. After 47 days, the rotation speed was increased in reactor I to 40 rpm and decreased in reactor II to 4 rpm. Prior to the rotation speed change, the biofilm on the flat discs underwent large detachments in both reactors, but the biofilm on rough discs was less extensively damaged. The increase in rotation speed induced large detachments of the biofilm in reactor I on all discs, but the biofilm on the rough discs recovered more effectively with faster regrowth. In reactor II, the decrease in rotation speed favored the development of the biofilm. Wall stress distributions obtained from CFD simulations on flat and rough discs at different rotation speeds were well correlated with experimental observations.     

Influence of Cr3+ on microbial cluster formation in biofilm and on steel corrosion

Pit corrosion of mild steel in seawater increased with Cr³⁺ concentration. SEM observations showed that increasing Cr³⁺ concentration caused microbes in biofilm on the steel surface to aggregate forming clusters. AFM images suggested that pit corrosion occurred largely on the mild steel surface between clusters, and only little corrosion on the surface covered by microbes.     

Influence of physicochemical properties on the hygienic status of stainless steel with various finishes

The relative hygienic status of 16 stainless steel surfaces, characterised by topography and surface free energy was investigated. B. thuringiensis spores suspended in Bechamel sauce was chosen as the test fouling suspension. Surface topography was assessed using 10 standardised roughness parameters, along with scanning electron microscope observations. The number of residual adhering spores after a fouling and cleaning in place procedure was found to be influenced by the topography of the stainless steel surface, but not by the surface free energy. Among the various roughness parameters, R_A, R_RR R_PK and R_VK were shown to be related to the hygienic status. Microscopic observations demonstrated the influence of the shape and size of surface irregularities on the level of residual soil after cleaning. This confirms that the use of only one roughness parameter, usually R_A, is not sufficient in defining the hygienic status of stainless steel surfaces.     

Comparison of fluorinated polymers against stainless steel, glass and polypropylene in microbial biofilm adherence and removal

Biofilm formation is a long-standing problem in ultrapure water and bioprocess fluid transport lines. The standard materials used in these applications (316L stainless steel, polypropylene and glass) have long been known to be good surfaces for the attachment of bacteria and other biological materials. To compare the relative tenacity of biofilms grown on materials used in manufacturing processes, a model system for biofilm attachment was constructed that approximates the conditions in industrial process systems. New fluorinated polymers were compared to the above materials by evaluating the surface area coverage of bacterial populations on materials before and after mild chemical treatment. In addition, contact angle studies compared the relative hydrophobicity of surfaces to suspensions of bacteria in growth media, and scanning electron microscopy and atomic force microscopy studies were used to characterize surface smoothness and surface defects. Biofilm adherence to polymer-based substrata was determined to be a function of both surface finish and surface chemistry. Specifically, materials that are less chemically reactive, as indicated by higher contact angle, can have rougher surface finishes and still be amenable to biofilm removal. Received 20 March 1997/ Accepted in revised form 15 July 1997     

Impact of the surface roughness of AISI 316L stainless steel on biofilm adhesion in a seawater-cooled tubular heat exchanger-condenser

The present study evaluated biofilm growth in AISI 316L stainless steel tubes for seawater-cooled exchanger-condensers that had four different arithmetic mean surface roughness values ranging from 0.14 μm to 1.2 μm. The results of fluid frictional resistance and heat transfer resistance regarding biofilm formation in the roughest surface showed increases of 28.2% and 19.1% respectively, compared with the smoothest surface. The biofilm thickness taken at the end of the experiment showed variations of up to 74% between the smoothest and roughest surfaces. The thermal efficiency of the heat transfer process in the tube with the roughest surface was 17.4% greater than that in the tube with the smoothest surface. The results suggest that the finish of the inner surfaces of the tubes in heat exchanger-condensers is critical for improving energy efficiency and avoiding biofilm adhesion. This may be utilised to reduce biofilm adhesion and growth in the design of heat exchanger-condensers.     

"Escherichia coli-milk" biofilm removal from stainless steel surfaces: synergism between ultrasonic waves and enzymes

Three different methods to standardize biofilm removal for in situ sanitary control of closed surfaces in the food industry have been developed and compared, i.e. sonication, enzymatic treatment and a combined treatment which involved the application of ultrasound to enzyme preparations. The biofilm studied was an Escherichia coli model biofilm, made with milk on stainless steel sheets. Plate counting and epifluorescence microscopy were used to assess the efficiency of each treatment. The results are expressed in percentages, 100% denoting total removal, obtained with a flat ultrasonic transducer (T1) developed and presented in a previous study. The application of ultrasound by a patented curved transducer, T2 (10 s, 40 kHz), specifically devised for closed surfaces, was not sufficient to completely remove the biofilm (30 +/- 7%). This biofilm was dislodged by two proteolytic enzyme preparations tested by immersion, viz. a 15-min application of protease (84 +/- 1%) and a 30-min trypsin application (95 +/- 8%). Using a combined treatment, the results showed a synergism between ultrasonic waves and proteolytic or glycolytic enzyme preparations, with removal of a significant amount of biofilm, i.e. 61-96% depending on the conditions tested, i.e. two to three times greater compared to sonication alone (30%). This application was in agreement with an industrial control, i.e. a good reproducible recovery of the biofilm in 10 s compared with 30 or 15 min with the enzyme alone.