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.     

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.     

Influence of surface characteristics and microstructure on adhesion of bacterial cells onto a type 304 stainless steel

A study was carried out to understand the influence of the surface characteristics/microstructure of a type 304 stainless steel on bacterial adhesion by exposing solution-annealed, sensitized and air-oxidized stainless steel specimens in a culture of Pseudomonas sp. in dilute nutrient broth. Epifluorescence microscopy of the exposed surfaces revealed that the pattern of adhesion as well as number density of bacterial cells was different depending on the metallurgical condition of the substratum. Among the specimens with different microstructures, the sensitized specimens had the highest bacterial density, followed by the solution annealed and the oxidized specimens. The same trend was shown by the total viable counts on the various surfaces, estimated by a plate count technique. The study assumes significance in the context of the widely reported observation of preferential attack of the welded region during microbiologically influenced corrosion of fabricated components.     

Manganese biofouling and the corrosion behavior of stainless steel

Manganese- and iron-oxidizing bacteria (MFOB) are widely implicated in microbially influenced corrosion, often in association with sulfate-reducing bacteria (SRB). Traditionally MFOB have been assigned a passive role in the corrosion process, promoting differential aeration cells, and providing oxygen depleted conditions conducive to the growth and corrosive attack of SRB. Recent work, summarized in this article, demonstrates that manganese biofouling alters the electrochemical behavior of stainless steel (SS), and suggests that MFOB are more active in localized corrosion than traditionally held. The paper discusses the chemistry and potentially corrosive impact of manganese and iron oxides on SS, explores the possible relationship between MFOB and SRB, and proposes a model to describe the synergistic influence these organisms may exert in the corrosion process.     

Inhibiting mild steel corrosion from sulfate-reducing and iron-oxidizing bacteria using gramicidin-S-producing biofilms

A gramicidin-S-producing Bacillus brevis 18-3 biofilm was shown to reduce corrosion rates of mild steel by inhibiting both the sulfate-reducing bacterium Desulfosporosinus orientis and the iron-oxidizing bacterium Leptothrix discophora SP-6. When L. discophora SP-6 was introduced along with D. orientis to a non-antimicrobial-producing biofilm control, Paenibacillus polymyxa ATCC 10401, a corrosive synergy was created and mild steel coupons underwent more severe corrosion than when only D. orientis was present, showing a 2.3-fold increase via electrochemical impedance spectroscopy (EIS) and a 1.8-fold difference via mass-loss measurements. However, when a gramicidin-S-producing, protective B. brevis 18-3 biofilm was established on mild steel, the metal coupons were protected against the simultaneous attack of D. orientis and L. discophora SP-6. EIS data showed that the protective B. brevis 18-3 biofilm decreased the corrosion rate about 20-fold compared with the non-gramicidin-producing P. polymyxa ATCC 10401 biofilm control. The mass loss for the protected mild steel coupons was also significantly lower than that for the unprotected ones (4-fold decrease). Scanning electron microscope images corroborated the corrosion inhibition by the gramicidin-S-producing B. brevis biofilm on mild steel by showing that the metal surface remained untarnished, i.e., the polishing grooves were still visible after exposure to the simultaneous attack of the sulfate-reducing bacterium and the iron-oxidizing bacterium.     

Extracellular polysaccharides from Desulfovibrio desulfuricans and Pseudomonas fluorescens in the presence of mild and stainless steel

Summary This communication reports the presence of polysaccharides in biofilms formed by pure and mixed cultures of Desulfovibrio desulfuricans and Pseudomonas fluorescens on mild and stainless steel surfaces. The results of colorimetric assays, indicating significant differences between the amounts of neutral sugars present in these biofilms, were supported by gas chromatographic (GC)-mass spectrophotometric and GC-flame ionisation detection analyses. Neutral sugars in biofilms grown on mild steel surfaces were identified and quantified, revealing glucose as a major carbohydrate followed by mannose and galactose in all types of biofilm. Extracellular polymeric substances (EPS) precipitated from bacterial cultures grown with and without steel surfaces were also analysed for their carbohydrate content. The influence of the surfaces present in the cultures on the amount and type of sugars released into the bulk phase was established. There was significantly more carbohydrate in EPS harvested from pure and mixed cultures of D. desulfuricans incubated mild and stainless steel coupons than in EPS obtained from coupon-free cultures. No significant difference in sugar quantities was observed in EPS precipitated from cultures of P. fluorescens grown under different conditions (absence or presence of steel surfaces). The main carbohydrates identified in all types of EPS samples were mannose, glucose and galactose in order of prevalence. Offprint requests to: I. B. Beech     

The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor

The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor was investigated over a 10-month period in a hotel. Planktonic and sessile numbers of sulphate reducing bacteria (SRB) and heterotrophic bacteria were monitored. The corrosion rate was determined by the weight loss method. The corrosion products were analyzed by energy dispersive X-ray spectroscopy and X-ray diffraction. A mineralized, heterogeneous biofilm was observed on the coupons. Although a biocide and a corrosion inhibitor were regularly added to the cooling water, the results showed that microorganisms, such as SRB in the mixed species biofilm, caused corrosion of galvanized steel. It was observed that Zn layers on the test coupons were completely depleted after 3?months. The Fe concentrations in the biofilm showed significant correlations with the weight loss and carbohydrate concentration (respectively, p?<?0.01 and p?<?0.01).     

The effect of inorganic phosphate and hydrogenase on the corrosion of mild steel

The data reported in this investigation revealed that phosphate and hydrogenase can affect the corrosion of mild steel. Phosphate reacted on mild-steel with concomitant evolution of hydrogen gas (H₂) and the formation of vivianite. The enzyme hydrogenase was shown to accelerate this reaction by oxidizing the H₂ produced, its known substrate. The components of biological growth media, one of which has been identified as phosphate, can interact with mild steel and cause corrosion. The implications of these findings to the corrosion industry are discussed. Correspondence to: E. Laishley     

Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing biofilms in Three-Mile-Island process water

Biofilms were used to produce gramicidin S (a cyclic decapeptide) to inhibit corrosion-causing, sulfate-reducing bacteria (SRB). In laboratory studies these biofilms protected mild steel 1010 continuously from corrosion in the aggressive, cooling service water of the AmerGen Three-Mile-Island (TMI) nuclear plant, which was augmented with reference SRB. The growth of both reference SRB (Gram-positive Desulfosporosinus orientis and Gram-negative Desulfovibrio vulgaris) was shown to be inhibited by supernatants of the gramicidin-S-producing bacteria as well as by purified gramicidin S. Electrochemical impedance spectroscopy and mass loss measurements showed that the protective biofilms decreased the corrosion rate of mild steel by 2- to 10-fold when challenged with the natural SRB of the TMI process water supplemented with D. orientis or D. vulgaris. The relative corrosion inhibition efficiency was 50–90% in continuous reactors, compared to a biofilm control which did not produce the antimicrobial gramicidin S. Scanning electron microscope and reactor images also revealed that SRB attack was thwarted by protective biofilms that secrete gramicidin S. A consortium of beneficial bacteria (GGPST consortium, producing gramicidin S and other antimicrobials) also protected the mild steel.     

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.     

Cross-sectional transmission electron microscopy of TiN and Ti-TiN coatings on AISI 304 stainless steel

The formation of a TiN or Ti-TiN PVD coating on austenitic stainless steel AISI 304 substrates has been studied by means of cross-sectional transmission electron microscopy (XTEM). Three types of specimens were studied. Three different depositions have been used, which are classified as follows: Type 1 is the coating system AISI 304-Ti-TiN, type 2 is the coating system AISI 304-TiN (new way of deposition, with the substrate masked from the plasma during the initial stage of deposition) and finally type 3 is the system AISI 304-TiN (conventional way of deposition). The XTEM study was carried out using a Philips CM 12 microscope, operating at 120 kV, and equipped with an EDAX energy dispersive X ray analyser with a Be window.     

Neutrophilic iron-oxidizing "zetaproteobacteria" and mild steel corrosion in nearshore marine environments

Microbiologically influenced corrosion (MIC) of mild steel in seawater is an expensive and enduring problem. Little attention has been paid to the role of neutrophilic, lithotrophic, iron-oxidizing bacteria (FeOB) in MIC. The goal of this study was to determine if marine FeOB related to Mariprofundus are involved in this process. To examine this, field incubations and laboratory microcosm experiments were conducted. Mild steel samples incubated in nearshore environments were colonized by marine FeOB, as evidenced by the presence of helical iron-encrusted stalks diagnostic of the FeOB Mariprofundus ferrooxydans, a member of the candidate class "Zetaproteobacteria." Furthermore, Mariprofundus-like cells were enriched from MIC biofilms. The presence of Zetaproteobacteria was confirmed using a Zetaproteobacteria-specific small-subunit (SSU) rRNA gene primer set to amplify sequences related to M. ferrooxydans from both enrichments and in situ samples of MIC biofilms. Temporal in situ incubation studies showed a qualitative increase in stalk distribution on mild steel, suggesting progressive colonization by stalk-forming FeOB. We also isolated a novel FeOB, designated Mariprofundus sp. strain GSB2, from an iron oxide mat in a salt marsh. Strain GSB2 enhanced uniform corrosion from mild steel in laboratory microcosm experiments conducted over 4 days. Iron concentrations (including precipitates) in the medium were used as a measure of corrosion. The corrosion in biotic samples (7.4 ± 0.1 mM) was significantly higher than that in abiotic controls (5.0 ± 0.1 mM). These results have important implications for the role of FeOB in corrosion of steel in nearshore and estuarine environments. In addition, this work shows that the global distribution of Zetaproteobacteria is far greater than previously thought.     

Adhesion of Staphylococcus aureus and Staphylococcus epidermidis to the Episkin reconstructed epidermis model and to an inert 304 stainless steel substrate

AIMS: The aim of this study was to evaluate the respective influence of the physicochemical interactions and the roughness involved in the first part of the biological substrate biocontamination. METHODS AND RESULTS: Therefore we compared the bioadhesion results obtained on the biological model substrate (Episkin) and on a commonly employed inert substrate (AISI 304 stainless steel), frequently used either in dermatology or in development of medical devices. The two studied strains presented different characteristics, both physicochemical and microbiological. Staphylococcus epidermidis, a relatively hydrophobic bacteria capable of exchanging interactions which are principally of the van der Waals type, adhered more to 304 steel than to the surface of reconstituted skin. As for S. aureus, an essentially basic, hydrophilic bacteria, was more adherent to Episkin (a bipolar, hydrophilic substrate) than to stainless steel (a unipolar, basic, hydrophilic substrate). CONCLUSIONS: In the absence of electrostatic interactions, the adhesion of substrate-dependent bacteria to the surface of reconstituted skin was dependent upon the balance between gamma(LW), gamma(+) and gamma(-). SIGNIFICANCE AND IMPACT OF THE STUDY: Consequently, so as to restrict microbial adhesion and reduce adhesive binding between micro-organisms and the surface of the skin, it would be preferable to render this substrate hydrophobic and apolar through the use of appropriate surface treatment.     

The effect of extracellular polymeric substances on the attachment of Pseudomonas NCIMB 2021 to AISI 304 and 316 stainless steel

Surfaces of AISI 304 and 316 stainless steels were pre-treated with three different types of extracellular polymeric substances, viz. (i) exopolymers released into the culture medium ("free"; or planktonic exopolymers), (ii) capsular exopolymers, and (iii) biofilm exopolymers, produced by continuous cultures of marine Pseudomonas NCIMB 2021. The initial attachment of Pseudomonas cells to exopolymer-conditioned steel surfaces varied with the exopolymer type and concentration. Results gained from wettability studies of exopolymer-treated steel using contact angle measurements, as well as from the surface roughness measurements conducted employing atomic force microscopy analysis, could not account for the observed, statistically significant differences (p < 0.1) in the level of bacterial surface colonisation. It is therefore proposed that neither surface hydrophobicity nor roughness play an important part in the early attachment of Pseudomonas NCIMB 2021 to the conditioned steel surfaces and that a difference in the chemistry of the exopolymers is most likely a key parameter influencing initial cell adhesion to pre-treated steel.     

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.     

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).     

Support media for microbial adhesion in an anaerobic fluidized-bed reactor

The most significant variable in anaerobic digestion in an anaerobic fluidized-bed reactor (AFBR) is the selection of the support medium for microbial adhesion. Using eight kinds of media, cristobalite, zeolite, vermiculite, granular active carbon, granulated clay, pottery stone, volcanic ash, and slag, we examined the physical properties of each medium, microbial adhesion, loading rates of organic matter and removal efficiencies in an AFBR. It appeared that good performance as a support medium was associated with rougher surfaces rather than with larger surface areas, because, although cristobalite had a much smaller surface area (50 m²/g) than that of the granular active carbon (1,125 m²/g), it had a very rough surface with many tubercular processes, by which a maximum loading rate of TOC of 8 g/l·d could be achieved in a synthetic wastewater. Moreover, it appeared to be important that the surface of the medium has a positive charge judging from the difference in performance between cristobalite and zeolite. That is, the two media were charged positively and negatively at pH 7, respectively. As a result, microorganisms, charged negatively in general, could adhere more easily to cristobalite than to zeolite, which was confirmed under a scanning electron microscope (SEM) and by amounts of microbial cells adhering (85 mg cells/g). The upflow linear velocity to allow twice the expanded volume (defined as the ratio of the expanded height to the static height) was decreased to half (0.13 cm/s) by microbial adhesion. In conclusion, a suitable medium for adherence of microorganisms in AFBR should have a rough and positively charged surface rather than a large surface area.     

The influence of macrofouling on the corrosion behaviour of API 5L X65 carbon steel

Seawater is a complex corrosive system, and biofouling is one of the factors that influences corrosion processes. The behaviour of corrosion associated with the development of macrofouling was investigated during the first 6 months of the successional process. Three treatments were compared: the 'Control' treatment (absence of macrofouling); 'Community' treatment, and 'Barnacle' treatment, where other macroorganisms were excluded. In the Community treatment, the dominant organisms were filamentous macroalgae (23.73%), barnacles (17.51%), hydroids (16.96%) and encrusting bryozoans (9.58%). In the Barnacle treatment, the cover varied between 39.38% and 62.50%. The corrosion potential ranged from -665.75 to -517.50 mV(Ag/AgC l((KCl))) and could not be associated with fouling development. The highest corrosion rate in the control suggests that macrofouling provides a protection against mass loss. The highest percentage of localised attacks was found in the Community treatment. This may indicate that not only barnacles, but also other organisms induce localised corrosion.     

Bacterial corrosion of mild steel under the condition of simultaneous formation of ferrous and sulphide ions

Summary Corrosion of mild steel in cultures of a Pseudomonas species under the condition of simultaneous formation of Fe(II) and S^2- was initially inhibited by inhibiting the anodic reaction, but after long incubation the corrosion process was allowed to continue. When only S^2- was produced, the initial corrosion rate increased for up to 60 h but later declined, probably due to a protective FeS film formed on the metal. Cathodic reactions were affected in a similar fashion as the anode.Extensive pitting corrosion was observed when the mild steel coupons were immersed in bacterial culture producing Fe(IIO) and S^2-, but not in the uninoculated control.