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     

Biofilms and corrosion interactions on stainless steel in seawater

Biofouling and biocorrosion lead to an important modification of the metal/ solution interface inducing changes in the type and concentration of ions, pH values, oxygen levels, flow velocity, etc. Metal dissolution in seawater is mainly conditioned by two different processes: (a) biofouling settlement and (b) corrosion products formation.Corrosion-resistant alloys such as stainless steel present an ideal substratum for microbial colonization, rather similar to inert non-metallic surfaces, due to the lack of corrosion products. Stainless steels are sensitive to pitting and other types of localized corrosion in chloride-containing media such as seawater. Biofilms and bacterial metabolism may accelerate the initiation of crevice attack by depletion of oxygen in the crevice solution due to microbial respiration. Bacterial colonization occurs within a period of 24–72 h on stainless steel samples exposed to natural seawater and, depending on environmental conditions, a copious and patchy biofilm is generally formed.Different interpretations of biofilms' effects on corrosion are critically discussed. A practical case, involving polluted harbour seawater, is reported to illustrate biofilm and corrosion interactions on stainless steel samples.     

Monitoring Microbial Attachment in Seawater

The attachment activity of microorganisms in seawater was studied by electrochemical and microscopic techniques. It was frequently observed that in natural seawater, the open circuit potential (E_ocp) of stainless steel was ennobled due to biofilm formation on the metal surface. Microscope observation revealed that the ennoblement of the E_ocp value of stainless steel changed linearly with the number of bacteria attached to the metal surface. Considering the fact that E_ocp of copper is almost a constant for a longer time in seawater, a compound electrode was made up of a stainless steel electrode and copper electrode for assessing the attachment of microbes in different seawater media according to the change in the potential difference between the two metals. The results demonstrated a good performance of the compound electrode for this purpose.     

Metal corrosion by aerobic bacteria isolated from stimulated corrosion systems: Effects of additional nitrate sources

Many microorganisms are reported to influence the corrosive behaviour of mild steel and stainless steel in different habitats. In this study, 40 bacterial strains were isolated from corroded mild steel and stainless steel coupons in the nitrate supplemented environments. The corrosion abilities of the isolates against the mild steel and stainless steel coupons were tested with or without additional nitrate sources. The presence of bacterial isolates alone stimulated the corrosion of mild steel coupons. Most of the bio-corrosion processes of mild steel coupons were mitigated by adding nitrate supplement with bacterial isolates. The effects of bacterial isolates and additional nitrogen sources on corrosion of stainless steels were varied. Not all bacterial isolates stimulated the corrosion on stainless steel during the study period. Unlike the effects on mild steel coupons, additional NaNO₃ might stimulate, retard the corrosion rate by the bacterial isolates or have limited effects. Similar results were obtained when NH₄NO₃ was used. Phylogenetic analysis demonstrated that all isolates were closely related. The majority of the bacterial isolates from corroded metal coupons were identified as Bacillus species. Others were identified as Pseudomonas sp., Marinobacter sp., and Halomonas species. The results prove that the isolated aerobic microorganisms do play a role in the corrosion process of stainless and mild steel. Adding additional nitrate sources might be a tool to mitigate corrosion of mild steel which was stimulated by the presence of bacteria. However, to prevent the corrosion of stainless steels, it might need a trial and errors approach in each case.     

Marine prosthecate bacteria involved in the ennoblement of stainless steel

Ennoblement, a phenomenon in which open-circuit potential is elevated to a noble value, triggers metal corrosion in the environment and is considered to be biologically catalysed. This study investigated the involvement of marine microorganisms in the ennoblement of stainless steel coupons in sea water pumped from Kamaishi Bay. Scanning electron microscopy (SEM) showed significant attachment of prosthecate bacteria on the surfaces of stainless steel coupons in the course of ennoblement. In denaturing gradient gel electrophoresis (DGGE) analyses of polymerase chain reaction-amplified bacterial 16S rDNA fragments, several major bands were detected from the surface of the ennobled coupons, including those affiliated with the alpha and gamma subclasses of the Proteobacteria. After these observations, bacterial strains were isolated from the surface of the ennobled coupon. The 16S rDNA analysis revealed that a bacterial isolate (designated PWB3) corresponded to a major DGGE band representing an alpha-Proteobacterial population; a database analysis showed that its closest relative was Rhodobium spp., albeit with low homology ( approximately 89%). SEM indicated that this bacterium was a prosthecate bacterium that was morphologically similar to those observed on the ennobled coupons. In pure culture of strain PWB3, stainless steel coupons were ennobled when the culture was supplemented with MnCl2. Manganese was recovered from the surface of the ennobled coupons after treatment with a reducing agent. These results suggest that the attachment of manganese-oxidizing prosthecate bacteria triggered the ennoblement of stainless steel in Kamaishi Bay sea water.     

Comparative studies of bacterial biofilms on steel surfaces using atomic force microscopy and environmental scanning electron microscopy

Environmental scanning electron microscopy (ESEM) and atomic force microscopy (AFM) were compared as tools for the observation of bacterial biofilms developed on carbon steel and AISI 316 stainless steel surfaces under stagnant conditions. Biofilms were generated in batch cultures of two different isolates of marine sulphate reducing bacteria (SRB) and in cultures consisting of mixed populations of acidophilic bacteria, known as "acid streamers";. Imaging of single SRB cells on mica was also carried out to reveal the surface topography of individual bacterial cells at nanometre resolution. Following the removal of biofilms, the stainless steel surfaces were profiled using AFM to determine the degree of steel deterioration. ESEM and AFM studies of bacterial biofilms in-situ, gave both qualitative and quantitative information on biofilm structure at high resolution. The use of AFM image analysis software allowed estimation of the width and height of bacterial cells, the thickness and width of exopolymeric (EPS) capsule and bacterial flagella, as well as characterisation of the surface roughness of the steel, including measurements of depth and diameter of individual pits. Exposure of stainless steel specimens to acid streamers resulted in a significant increase in the surface roughness of the steel, compared to specimens placed in sterile medium.     

Influence of copper-alloying of austenitic stainless steel on multi-species biofilm development

AIMS: To investigate the bactericidal influence of copper-alloying of stainless steel on microbial colonization. METHODS AND RESULTS: Inhibition of bacterial adherence was investigated by monitoring (192 h) the development of a multi-species biofilm on Cu-alloyed (3.72 wt%) stainless steel in a natural surface water. During the first 120 h of exposure, lower numbers of viable bacteria in the water in contact with copper-containing steel relative to ordinary stainless steel were observed. Moreover, during the first 48 h of exposure, lower colony counts were found in the biofilm adhering to the Cu-alloyed steel. No lower colony or viable counts were found throughout the remainder of the experimental period. CONCLUSION: The presence of Cu in the steel matrix impedes the adhesion of micro-organisms during an initial period (48 h), while this bactericidal effect disappears after longer incubation periods. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of Cu-alloyed stainless steels for bactericidal purposes should be restricted to regularly-cleaned surfaces.     

The growth of Bacillus stearothermophilus on stainless steel

AIMS: To determine the potential for Bacillus stearothermophilus cells to form biofilms of significance in dairy manufacture. METHODS AND RESULTS: The ability of isolates of B. stearothermophilus from dairy manufacturing plants to attach to stainless steel surfaces was demonstrated by exposing stainless steel samples to suspensions of spores or vegetative cells and determining the numbers attaching using impedance microbiology. Spores attached more readily than vegetative cells. The attachment of cells to stainless steel was increased 10-100-fold by the presence of milk fouling the stainless steel. The growth of B. stearothermophilus as a biofilm on stainless steel surfaces was determined using a continuously flowing experimental reactor. Vegetative cells were released in greater numbers than spores from biofilms of most strains studied. Biofilms of one strain (B11) were studied in detail. Biofilms of > 106 cells cm-2 formed in the reactor and released approximately 106 cells ml-1 into milk passing over the biofilm. A doubling time of 25 min was calculated for this organism grown as a biofilm. CONCLUSION: The formation of biofilms of thermophilic Bacillus species within the plant appears to be a likely cause of contamination of manufactured dairy products. Methods to control the formation of biofilms in dairy manufacturing plants are required to reduce the contamination of dairy products with thermophilic bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilms of B. stearothermophilus growing in dairy manufacturing plants can explain the contamination of dairy products with these bacteria.     

Influence of hydrodynamics and nutrients on biofilm structure

Hydrodynamic conditions control two interlinked parameters; mass transfer and drag, and will, therefore, significantly influence many of the processes involved in biofilm development. The goal of this research was to determine the effect of flow velocity and nutrients on biofilm structure. Biofilms were grown in square glass capillary flow cells under laminar and turbulent flows. Biofilms were observed microscopically under flow conditions using image analysis. Mixed species bacterial biofilms were grown with glucose (40 mg/l) as the limiting nutrient. Biofilms grown under laminar conditions were patchy and consisted of roughly circular cell clusters separated by interstitial voids. Biofilms in the turbulent flow cell were also patchy but these biofilms consisted of patches of ripples and elongated 'streamers' which oscillated in the flow. To assess the influence of changing nutrient conditions on biofilm structure the glucose concentration was increased from 40 to 400 mg/l on an established 21 day old biofilm growing in turbulent flow. The cell clusters grew rapidly and the thickness of the biofilm increased from 30 μ to 130 μ within 17 h. The ripples disappeared after 10 hours. After 5 d the glucose concentration was reduced back to 40 mg/l. There was a loss of biomass and patches of ripples were re-established within a further 2 d.     

The influence of environmental factors on the rate and extent of stainless steel ennoblement mediated by manganese‐oxidizing biofilms

The increase in the open circuit potential of passive metals in natural waters due to biofilm formation at the metal surface, termed ennoblement, has been reported for nearly 30 years. Although its occurrence is undoubtedly associated with microbial colonization, the underlying mechanism of ennoblement remains controversial. Recent work produced in the authors’ laboratory has provided convincing experimental evidence that ennoblement can be caused by deposition of biomineralized manganese produced by manganese‐oxidizing biofilms. The purpose of this study was to determine the effects of environmental factors on the rate and extent of ennoblement of 316L stainless steel exposed to natural waters. This was accomplished by exposing corrosion coupons to four freshwater systems over a 4‐yearperiod. The rate and extent of ennoblement observed in these locations was correlated with dissolved manganese concentrations, the mass of accumulated manganese oxides, organic carbon concentration, dissolved oxygen concentration, flow, conditions, temperature, and pH in these environments.     

Ennoblement of Stainless Steel by the Manganese-Depositing Bacterium Leptothrix discophora

The noble shift in open-circuit potential exhibited by microbially colonized stainless steel (ennoblement) was investigated by examining the relationship among surface colonization, manganese deposition, and open-circuit potential for stainless steel coupons exposed to batch cultures of the manganese-depositing bacterium Leptothrix discophora. Open-circuit potential shifted from -100 to +330 mV(infSCE) as a biofilm containing 75 nmol of MnO(infx) cm(sup-2) formed on the coupon surface but changed little further with continued MnO(infx) deposition up to 270 nmol cm(sup-2). Increased open-circuit potential corresponded to decreasing Mn(II) concentration in solution and to increased MnO(infx) accumulation and attached cell density on the coupon surfaces. MnO(infx) deposition was attributable to biological activity, and Mn(II) was observed to enhance cell attachment. The experimental results support a mechanism of ennoblement in which open-circuit potential is fixed near +350 mV(infSCE) by the cathodic activity of biomineralized MnO(infx).     

Studies on microbiologically influenced corrosion of SS304 by a novel manganese oxidizer,Bacillus flexus

A manganese oxidizing bacterium was isolated from the surface of steel scraps and biochemical tests and 16S rRNA sequencing analysis confirmed the isolate as Bacillus flexus. Potentiodynamic polarization curves showed ennoblement of open circuit potential, increased passive current, a lowering of breakdown potential, active re-passivation potential and enhanced cathodic current in the presence of B. flexus. Adhesion studies with B. flexus on SS304 specimens with different surface treatments demonstrated decreased adhesion on passivated and FeCl₃ treated specimens due to the removal of MnS inclusions. The present study provides evidence that surface treatment of stainless steels can reduce adhesion of this manganese oxidizing bacterium and decrease the probability of microbiologically influenced corrosion.     

Evaluation of biocathodes in freshwater and brackish sediment microbial fuel cells

Biofilms on biocathodes can catalyze the cathodic oxygen reduction and accordingly guarantee high cathode redox potentials. The present research assessed the use of biocathodes in full-sediment microbial fuel cells. Carbon felt-based biocathodes were evaluated in freshwater systems, and an extension of their application to brackish systems and/or stainless steel webs as base material was considered. Efficient biocathodes could be developed within days through inoculation with active microorganisms. Carbon felt was found most suited for the biocathodes in freshwater with increased performance at salinities around 80–250 mM. Maximum long-term performance reached 12.3 μW cm⁻² cathode. The relative benefit of stainless steel seemed to increase with increasing salinity. A combination of stainless steel cathodes with biofilms could, however, also result in decreased electrical performance. In an efficiently catalyzing cathodic biofilm, an enrichment with an uncultured Proteobacterium—previously correlated with steel waste—was observed.     

Bactericidal Activity of Copper and Niobium–Alloyed Austenitic Stainless Steel

Biofouling and microbiologically influenced corrosion are processes of material deterioration that originate from the attachment of microorganisms as quickly as the material is immersed in a nonsterile environment. Stainless steels, despite their wide use in different industries and as appliances and implant materials, do not possess inherent antimicrobial properties. Changes in hygiene legislation and increased public awareness of product quality makes it necessary to devise control methods that inhibit biofilm formation or to act at an early stage of the biofouling process and provide the release of antimicrobial compounds on a sustainable basis and at effective level. These antibacterial stainless steels may find a wide range of applications in fields, such as kitchen appliances, medical equipment, home electronics, and tools and hardware. The purpose of this study was to obtain antibacterial stainless steel and thus mitigate the microbial colonization and bacterial infection. Copper is known as an antibacterial agent; in contrast, niobium has been demonstrated to improve the antimicrobial effect of copper by stimulating the formation of precipitated copper particles and its distribution in the matrix of the stainless steel. Thus, we obtained slides of 3.8% copper and 0.1% niobium alloyed stainless steel; subjected them to three different heat treatment protocols (550°C, 700°C, and 800°C for 100, 200, 300, and 400 hours); and determined their antimicrobial activities by using different initial bacterial cell densities and suspending solutions to apply the bacteria to the stainless steels. The bacterial strain used in these experiments was Escherichia coli CCM 4517. The best antimicrobial effects were observed in the slides of stainless steel treated at 700°C and 800°C using an initial cell density of approximately 10⁵ cells ml⁻¹ and phosphate-buffered saline as the solution in which the bacteria came into contact with copper and niobium–containing steel.     

The effect of gold and silver nanoparticles,chitosan and their combinations on bacterial biofilms of food-borne pathogens

Abstract

The antimicrobial activity of gold and silver nanoparticles (AuNPs, AgNPs), chitosan (CS) and their combinations was established by determining the minimum inhibitory concentration for planktonic (MICPC₈₀) and biofilm growth (MICBC₈₀), for biofilm formation (MICBF₈₀), metabolic activity (MICBM₈₀) and reduction (MICBR₈₀), and for the metabolic activity of preformed biofilm (MICMPB₈₀). Biofilms were quantified in microtitre plates by crystal violet staining and metabolic activity was evaluated by the MTT assay. Chitosan effectively suppressed biofilm formation (0.31–5?mg ml^?1) in all the tested strains, except Salmonella enterica Infantis (0.16–2.5?mg ml^?1) where CS and its combination with AgNPs induced biofilm formation. Nanoparticles inhibited biofilm growth only when the highest concentrations were used. Even though AuNPs, AgNPs and CS were not able to remove biofilm mass, they reduced its metabolic activity by at least 80%. The combinations of nanoparticles with CS did not show any significant positive synergistic effect on the tested target properties.     

Silver as a Residual Disinfectant To Prevent Biofilm Formation in Water Distribution Systems

Biofilms can have deleterious effects on drinking water quality and may harbor pathogens. Experiments were conducted using 100 μg/liter silver to prevent biofilm formation in modified Robbins devices with polyvinyl chloride and stainless steel surfaces. No significant difference was observed on either surface between the silver treatment and the control.     

Approaches to prevention, removal and killing of biofilms

Biofilms contribute to hygiene problems in the food industry and in the medical field. Biofilms are diverse and due to the development of special phenotypes, biofilm organisms are not as susceptible to biocides as planktonic microorganisms. Biofilms may be prevented by regular disinfection. Since the attachment of microbes to surfaces and the development of biofilm phenotypes is a very fast process, it is, however, almost impossible, to prevent biofilm formation completely. The removal and killing of established biofilms requires harsh treatments, mostly using oxidising biocides. Depending on the nature of the biofilms, different biocides may be useful and the best biocide for a specific biofilm still has to be determined under practical conditions. Another approach is the prevention of biofilm formation by selection of materials that do not support the attachment of microorganisms. Some materials like glass and stainless steel show less biofilm formation than others. The ranking of materials, however, depends on the conditions, under which they are tested. A novel approach is biofilm inhibition by supplementation of systems with nutrients, to inhibit attachment. First results on inhibition of biofouling in reversed osmosis systems are presented.     

Structure and on-site formation of biofilms in paper machine water flow

Paper machine biofilms formed in situ on stainless steel surfaces were studied. A robust flow cell was fitted to side stream (1.8 m s⁻¹) of the spray water circuit of a paper machine. This on-site tool allowed for assessing the efficacy of antifoulants and the adequacy of steel polishing under mill conditions. A rapid fluorescence-based assay was developed to quantify the biomass of shallow biofilms on machine steel. The fluorescence matched the ATP content measured for the same biofilms. Electrolytic polishing reduced the tendency of biofouling of 500 grit surface steel. Biofilm grew under machine conditions as clusters on the steels, showing uniformly coccoid, filaments or short rods; only one cell type in each cluster. The biofilm clusters excluded latex beads of 0.02 μm with hydrophilic or with hydrophobic surfaces from penetrating more than three to four layers of cells. Under the high hydraulic flow at the machine (1.8 m s⁻¹), the biofilm grew in 7 days 6–10 μm thick. The high flow rate guided the shape of the biofilm clusters emerging after the primary attachment of cells. Adhered individual bacteria were the platform on steel to which solids such as paper machine fines then accumulated. Journal of Industrial Microbiology & Biotechnology (2002) 28, 268–279 DOI: 10.1038/sj/jim/7000242 Received 04 October 2001/ Accepted in revised form 14 January 2002     

Cryosectioning of biofilms for microscopic examination

A method for rapid and minimally disruptive embedding and sectioning of bacterial biofilms has been developed and applied to binary population biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofilms were cryoembedded using a commercial tissue embedding medium. Frozen embedded biofilms could be removed easily from the substratum by gently flexing the steel coupon. Microscopic examination of the substratum surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five μm thick frozen sections were cut with a cryostat and examined by light or fluorescence microscopy. The cryoembedding technique preserved biofilm structural features including an irregular surface, water channels, local protrusions up to 500 μm thick, and a well‐defined substratum interface. The method requires minimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.     

Effects of diverse water pipe materials on bacterial communities and water quality in the annular reactor

To investigate the effects of pipe materials on biofilm accumulation and water quality, an annular reactor with the sample coupons of four pipe materials (steel, copper, stainless steel, and polyvinyl chloride) was operated under hydraulic conditions similar to a real plumbing system for 15 months. The bacterial concentrations were substantially increased in the steel and copper reactors with progression of corrosion, whereas those in stainless steel (STS) and polyvinyl chloride (PVC) reactors were affected mainly by water temperature. The heterotrophic plate count (HPC) of biofilms was about 100 times higher on steel pipe than other pipes throughout the experiment, with the STS pipe showing the lowest bacterial number at the end of the operation. Analysis of the 16S rDNA sequences of 176 cultivated isolates revealed that 66.5% was Proteobacteria and the others included unclassified bacteria, Actinobacteria, and Bacilli. Regardless of the pipe materials, Sphingomonas was the predominant species in all biofilms. PCR-DGGE analysis showed that steel pipe exhibited the highest bacterial diversity among the metallic pipes, and the DGGE profile of biofilm on PVC showed three additional bands not detected from the profiles of the metallic materials. Environmental scanning electron microscopy showed that corrosion level and biofilm accumulation were the least in the STS coupon. These results suggest that the STS pipe is the best material for plumbing systems in terms of the microbiological aspects of water quality.