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.     

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.     

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     

Electrochemical concepts and techniques in the study of stainless steel ennoblement

Electrochemical theory and technique used to investigate microbially influenced corrosion is discussed with a focus on methods used to demonstrate the manganic-oxide mechanism of stainless steel Ennoblement. The concept of mixed potential and its relationship to the current-voltage behavior of stainless steel is developed. This concept is used to interpret microbially induced changes in corrosion potential, polarization behavior, surface-oxide abundance, and the redox environment at submerged metal surfaces. Microelectrode, capacitance, and coulometric methods are described that can be used to discriminate electrochemical effects caused by changes in solution properties from those caused by mineral deposition at the metal surface. The variety of electrochemical, wet-chemical, microbiological, and surface analytical techniques used to demonstrate the effect of biomineralized manganese dioxide on the electrochemical behavior of stainless steel are summarized.     

Defouling and cleaning using nanobubbles on stainless steel

The present work demonstrates that nanobubbles can be used as cleaning agents on stainless steel (SS) surfaces. Cleaning efficiency has been quantified. Using an Atomic Force Microscope (AFM), it was demonstrated that nanobubbles can be produced by electrochemical treatment on a SS surface either with or without adsorbed bovine serum albumin (BSA). After allowing adsorption on SS overnight, radio-labeled BSA was removed by electrochemically generated nanobubbles, and then the remaining BSA on the surface was quantified by radioactivity measurement. The results indicate that nanobubbles can remove >10% of the protein in each 3-min electrochemical treatment while in a control group, washing with water and electrolyte resulted in no more than 3% of the protein being removed each time. Cleaning of conducting surfaces by nanobubbles is promising in any system where fouling occurs in biomedia.     

Adhesion and removal kinetics of Bacillus cereus biofilms on Ni-PTFE modified stainless steel

Biofilm control remains a challenge to food safety. A well-studied non-fouling coating involves codeposition of polytetrafluoroethylene (PTFE) during electroless plating. This coating has been reported to reduce foulant build-up during pasteurization, but opportunities remain in demonstrating its efficacy in inhibiting biofilm formation. Herein, the initial adhesion, biofilm formation, and removal kinetics of Bacillus cereus on Ni-PTFE-modified stainless steel (SS) are characterized. Coatings lowered the surface energy of SS and reduced biofilm formation by > 2 log CFU cm^?2. Characterization of the kinetics of biofilm removal during cleaning demonstrated improved cleanability on the Ni-PTFE coated steel. There was no evidence of biofilm after cleaning by either solution on the Ni-PTFE coated steel, whereas more than 3 log and 1 log CFU cm^?2 of bacteria remained on the native steel after cleaning with water and an alkaline cleaner, respectively. This work demonstrates the potential application of Ni-PTFE non-fouling coatings on SS to improve food safety by reducing biofilm formation and improving the cleaning efficiency of food processing equipment.     

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.     

Continuous flow system for biofilm formation using controlled concentrations of Pseudomonas putida from chicken carcass and coupled to electrochemical impedance detection

Abstract

Most studies dealing with monitoring the dynamics of biofilm formation use microbial suspensions at high concentrations. These conditions do not always represent food or water distribution systems. A continuous flow system capable of controlling the concentration of the microbial suspension stream from 10⁴ to 10⁶ CFU ml^?1 is reported. Pseudomonas putida biofilms formed using 100-fold, 1,000-fold or 10,000-fold diluted bacterial suspensions were monitored in-line by electrochemical impedance spectroscopy (EIS) and total plate counts. Randles equivalent circuit model and a modified Randles model with biofilm elements were used to fit the EIS data. In Randles equivalent circuit, the charge transfer resistance decreased as the biofilm formed. The log colony counts of the biofilm correlated to the charge transfer resistance. In the biofilm model, the biofilm resistance and the double layer capacitance decreased as the biofilm formed. The log colony counts of the biofilm correlated to the biofilm resistance.     

Analysis of a microbial electrochemical cell using the proton condition in biofilm (PCBIOFILM) model

Common to all microbial electrochemical cells (MXCs) are the anode-respiring bacteria (ARB), which transfer electrons to an anode and release protons that must transport out of the biofilm. Here, we develop a novel modeling platform, Proton Condition in BIOFILM (PCBIOFILM), with a structure geared towards mechanistically explaining: (1) how the ARB half reaction produces enough acid to inhibit the ARB by low pH; (2) how the diffusion of alkalinity carriers (phosphates and carbonates) control the pH gradients in the biofilm anode; (3) how increasing alkalinity attenuates pH gradients and increases current; and (4) why carbonates enable higher current density than phosphates. Analysis of literature data using PCBIOFILM supports the hypothesis that alkalinity limits the maximum current density for MXCs. An alkalinity criterion for eliminating low-pH limitation - 12 mg CaCO₃/mg BOD - implies that a practical MXC can achieve a maximum current density with an effluent quality comparable to anaerobic digestion.     

In situ detection of bacteria involved in cathodic depolarization and stainless steel surface corrosion using microautoradiography

Aims: To examine the activity of bacteria involved in cathodic depolarization and surface corrosion on stainless steel in an in situ model system. Methods and Results: The microautoradiographic technique (MAR) was used to evaluate the activity of bacterial populations on stainless steel surfaces with a single cell resolution. Anaerobic uptake and fixation of ¹⁴C‐labelled bicarbonate occurred within corrosion sites in the absence of atmospheric hydrogen or other external electron donors, whereas it was taken up and fixed by bacteria at all other stainless steel surfaces in the presence of atmospheric hydrogen. This indicates that the bacteria utilized electrons originating from the corrosion sites due to the ongoing corrosion (cathodic depolarization). Conclusion: Under in situ conditions, bacteria were fixating ¹⁴C‐labelled bicarbonate at corrosion sites in the absence of atmospheric hydrogen. This indicates that electrons transferred to the bacteria provided energy for bicarbonate fixation due to cathodic depolarization. Significance and Impact of the Study: Application of the MAR method showed ongoing biocorrosion in the applied in situ model system and allowed in situ examination of bacterial activity on a single cell level directly on a metal surface providing information about potential corrosion mechanisms. Furthermore, application of fluorescence in situ hybridization in combination with MAR allows for identification of the active bacteria.     

The corrosion behaviour of mild steel and type 304 stainless steel in media from an anaerobic biodigestor

Mild steel and AISI 304 stainless steel samples were exposed to the aqueous solutions from an anaerobic biodigestor of wastewater, showing quite different behaviours. Carbon steel presented generalized corrosion whereas stainless steel, as-received or sensitized, tended to show some localized corrosion. Both materials presented bacteria attachment, particularly sulphate reducing bacteria (SRB), forming biofilms which were abundant and mixed with corrosion products on the mild steel surface and thin and patchy on stainless steel surfaces. Different types of anaerobic and aerobic bacteria were detected in the medium. Experiments were carried out both at 1 and at 7 atmospheres pressure, in presence of a gaseous phase containing N₂, CO₂ and CH₄. Potentiodynamic experiments were conducted in order to have a better insight on the electrochemical behaviour of the material in this medium.     

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.     

Cathodic behaviour of stainless steel in coastal Indian seawater: calcareous deposits overwhelm biofilms

Type-316 stainless steel (SS) was investigated as the cathode in galvanic couples in full-strength seawater from the Gulf of Mannar on the southeast coast of India. Tests were devised to examine the impact of SS cathodes on anode materials with or without the accrual of marine biofilms. Biofilmed SS cathodes significantly enhanced the rate of corrosion of nickel, causing noble shifts in the couple potentials. With mild steel and zinc as the anodes, calcareous deposits developed quite rapidly on the SS cathodes and led to a significant reduction of bacterial numbers. The calcareous deposits also caused substantial reduction of galvanic corrosion rates for mild steel, whereas there was no difference for zinc. The deposits were identified by XRD as essentially carbonates, oxides and hydroxides of calcium and magnesium. Potentiodynamic polarization performed on the actual couples after disconnection and equilibration provided reasonable interpretations of the galvanic corrosion trends. Data from this work suggest that a potential of about ?0.70 V vs. saturated calomel electrode (SCE) should provide optimum protection of SS in warmer, full-strength seawater that supports the precipitation of calcareous deposits. The criterion commonly recommended for temperate conditions of lower water temperature and estuarine waters of lower alkalinity is ?1.0 V (SCE).     

Homogenous photocatalytic decontamination of prion infected stainless steel and titanium surfaces

Prions are notorious for their extraordinary resistance to traditional methods of decontamination, rendering their transmission a public health risk. Iatrogenic Creutzfeldt–Jakob disease (iCJD) via contaminated surgical instruments and medical devices has been verified both experimentally and clinically. Standard methods for prion inactivation by sodium hydroxide or sodium hypochlorite have failed, in some cases, to fully remove prion infectivity, while they are often impractical for routine applications. Prion accumulation in peripheral tissues and indications of human-to-human bloodborne prion transmission, highlight the need for novel, efficient, yet user-friendly methods of prion inactivation. Here we show both in vitro and in vivo that homogenous photocatalytic oxidation, mediated by the photo-Fenton reagent, has the potential to inactivate the pathological prion isoform adsorbed on metal substrates. Photocatalytic oxidation with 224 μg mL⁻¹ Fe³⁺, 500 μg mL⁻¹ h⁻¹ H₂O₂, UV-A for 480 min lead to 100% survival in golden Syrian hamsters after intracranial implantation of stainless steel wires infected with the 263K prion strain. Interestingly, photocatalytic treatment of 263K infected titanium wires, under the same experimental conditions, prolonged the survival interval significantly, but failed to eliminate infectivity, a result that we correlate with the increased adsorption of PrP^Sc on titanium, in comparison to stainless steel. Our findings strongly indicate that our, user- and environmentally friendly protocol can be safely applied to the decontamination of prion infected stainless steel surfaces.     

Mutagenicity of fume particles from metal arc welding on stainless steel in the Salmonella/microsome test.

Mutagenic activity of fume particles produced by metal arc welding on stainless steel (ss) is demonstrated by using the Salmonella/microsome mutagenicity test described by Ames et al., with strain TA100 (base-pair substitution) and TA98 (frame-shift reversion). Results of a representative but limited selection of processes and materials show that mutagenic activity is a function of process and process parameters. Welding on stainless steel produces particles that are mutagenic, whereas welding on mild steel (ms) produces particles that are not. Manual metal arc (MMA) welding on stainless steel produces particles of higher mutagenic activity than does metal inert gas (MIG) welding, and fume particles produced by MIG welding under short-arc transfer. Further studies of welding fumes (both particles and gases) must be performed to determine process parameters of significance for the mutagenic activity.     

Bacterial attachment to stainless steel welds: Significance of substratum microstructure

AISI Type 304 L stainless steel (SS) is a widely used material in industry due to its strength and resistance to corrosion. However, corrosion on SS is reported largely at welds or adjacent areas. Bacteria were observed to colonize preferentially near welds as a result of surface roughness. In the present study, the influence of another important metal surface condition on bacterial adhesion has been evaluated, i.e. substratum microstructure. Type 304 L SS weld samples were prepared and machined to separate weld metal, the heat affected zone (HAZ) and base metal regions. The coupons were molded in resin so that only the surfaces polished to a 3 p.m finish were exposed to the experimental medium with Pseudomonas sp. isolated from a corrosive environment in Japan. The coupons were exposed for varying durations. The area of bacterial attachment showed significant differences with time of exposure and; the type of coupons. Generally, the weld metal samples showed more attachment whilst the base metal showed the least. The area of attachment was inversely proportional to the average grain size of the three samples. As the bacteria started colonizing, attachment mainly occurred on the grain boundaries of the base metal (after 8h, 84.62% and 15.38% of the total number of bacteria attached in the field of view (FOV) at the grain boundary and matrix, respectively) and on the austenite‐ferrite interface in the weld metal (after 8h, 88.33% and 11.77% of the total number of bacteria attached in the FOV at the boundary and matrix, respectively). The weld area had more grains and hence more grain boundary/ unit area than the base metal, resulting in more bacterial attachment. SEM observations showed this increased attachment of Pseudomonas sp. resulted in the initiation of microbiologically influenced corrosion (MIC) on the weld coupons by 16 d. Therefore, the results provide data to support the fact that substratum microstructure influences bacterial attachment, which in turn leads to corrosion.     

Adsorption of chemically synthesized mussel adhesive peptide sequences containing DOPA on stainless steel

The adsorption of proteins at solid–liquid interfaces is important in biosensor and biomaterial applications. Marine mussels affix themselves to surfaces using a highly cross‐linked, protein‐based adhesive containing a high proportion of L‐3,4‐dihydroxyphenylalanine (DOPA) residues. In this work, the effect of DOPA residues on protein adhesion on stainless steel surfaces was studied using a quartz crystal microbalance with dissipation system. The adsorption of two repetitive peptide motifs, KGYKYYGGSS and KGYKYY, from the mussel Mytilus edulis foot protein 5 on stainless steel was studied before and after chemo‐enzymatic modification of tyrosine residues to DOPA using mushroom tyrosinase. Conversion from tyrosine to DOPA, evaluated by HPLC, was in the range 70–99%. DOPA‐modified sequences showed fourfold greater adhesion than unmodified M. edulis foot protein 5 motifs. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Impact of biological factors on the ennoblement of stainless steel in Baltic seawater

Open circuit potentials of stainless steels increased when immersed in the Baltic Sea. The ennoblement potential was +200 mV_sce in 40 to 50 days when sea water temperature was below 52°C and +300–400 mV_sce within <40 days at around 102°C. Ennoblement occurred in a laboratory ecosystem at 232°C in 20 to 30 days, and at 262°C in <20 days, but no ennoblement occurred at A322°C within 40 days. By the time the ennoblement was complete, compact microcolonies covered 1–10% of the steel surface. Nutrient enrichment of Baltic Sea water by twofold above the natural levels increased microbial growth but attenuated open circuit potential increase of the stainless steels. Exposure of the ennobled stainless steels to similar levels of nutrients did not reverse the already developed open circuit potentials. Attenuation of the ennobling response of the stainless steels by increases of temperature and eutrophication suggests a role for microorganisms which is crucial for the electrochemical behaviour of steels in brackish Baltic Sea water. Journal of Industrial Microbiology & Biotechnology (2000) 24, 410–420. Received 02 November 1999/ Accepted in revised form 24 March 2000