Both sulfate-reducing bacteria and Enterobacteriaceae take part in marine biocorrosion of carbon steel

AIMS: In order to evaluate the part played in biocorrosion by microbial groups other than sulfate-reducing bacteria (SRB), we characterized the phylogenetic diversity of a corrosive marine biofilm attached to a harbour pile structure as well as to carbon steel surfaces (coupons) immersed in seawater for increasing time periods (1 and 8 months). We thus experimentally checked corroding abilities of defined species mixtures. METHODS AND RESULTS: Microbial community analysis was performed using both traditional cultivation techniques and polymerase chain reaction cloning-sequencing of 16S rRNA genes. Community structure of biofilms developing with time on immersed coupons tended to reach after 8 months, a steady state similar to the one observed on a harbour pile structure. Phylogenetic affiliations of isolates and cloned 16S rRNA genes (rrs) indicated that native biofilms (developing after 1-month immersion) were mainly colonized by gamma-proteobacteria. Among these, Vibrio species were detected in majority with molecular methods while cultivation techniques revealed dominance of Enterobacteriaceae such as Citrobacter, Klebsiella and Proteus species. Conversely, in mature biofilms (8-month immersion and pile structure), SRB, and to a lesser extent, spirochaetes were dominant. CONCLUSIONS: Corroding activity detection assays confirmed that Enterobacteriaceae (members of the gamma-proteobacteria) were involved in biocorrosion of metallic material in marine conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: In marine biofilms, metal corrosion may be initiated by Enterobacteriaceae.     

Molecular methods resolve the bacterial composition of natural marine biofilms on galvanically coupled stainless steel cathodes

Navy vessels consist of various metal alloys and biofilm accumulation at the metal surface is thought to play a role in influencing metal deterioration. To develop better strategies to monitor and control metallic biofilms, it is necessary to resolve the bacterial composition within the biofilm. This study aimed to determine if differences in electrochemical current could influence the composition of dominant bacteria in a metallic biofilm, and if so, determine the level of resolution using metagenomic amplicon sequencing. Current was generated by creating galvanic couples between cathodes made from stainless steel and anodes made from carbon steel, aluminum, or copper nickel and exposing them in the Delaware Bay. Stainless steel cathodes (SSCs) coupled to aluminum or carbon steel generated a higher mean current (0.39 mA) than that coupled to copper nickel (0.17 mA). Following 3 months of exposure, the bacterial composition of biofilms collected from the SSCs was determined and compared. Dominant bacterial taxa from the two higher current SSCs were different from that of the low-current SSC as determined by DGGE and verified by Illumina DNA-seq analysis. These results demonstrate that electrochemical current could influence the composition of dominant bacteria in metallic biofilms and that amplicon sequencing is sufficient to complement current methods used to study metallic biofilms in marine environments.     

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.     

Phylogenetic characterization of a corrosive consortium isolated from a sour gas pipeline

Biocorrosion is a common problem in oil and gas industry facilities. Characterization of the microbial populations responsible for biocorrosion and the interactions between different microorganisms with metallic surfaces is required in order to implement efficient monitoring and control strategies. Denaturing gradient gel electrophoresis (DGGE) analysis was used to separate PCR products and sequence analysis revealed the bacterial composition of a consortium obtained from a sour gas pipeline in the Gulf of Mexico. Only one species of sulfate-reducing bacteria (SRB) was detected in this consortium. The rest of the population consisted of enteric bacteria with different characteristics and metabolic capabilities potentially related to biocorrosion. Therefore, several types of bacteria may be involved in biocorrosion arising from natural biofilms that develop in industrial facilities. The low abundance of the detected SRB was evidenced by environmental scanning electron microscopy (ESEM). In addition, the localized corrosion of pipeline steel in the presence of the consortium was clearly observed by ESEM after removing the adhered bacteria.     

Effect of Hydrogenase and Mixed Sulfate-Reducing Bacterial Populations on the Corrosion of Steel

The importance of hydrogenase activity to corrosion of steel was assessed by using mixed populations of sulfate-reducing bacteria isolated from corroded and noncorroded oil pipelines. Biofilms which developed on the steel studs contained detectable numbers of sulfate-reducing bacteria (10⁴ increasing to 10⁷/0.5 cm²). However, the biofilm with active hydrogenase activity (i.e., corrosion pipeline organisms), as measured by a semiquantitative commercial kit, was associated with a significantly higher corrosion rate (7.79 mm/year) relative to noncorrosive biofilm (0.48 mm/year) with 10⁵ sulfate-reducing bacteria per 0.5 cm² but no measurable hydrogenase activity. The importance of hydrogenase and the microbial sulfate-reducing bacterial population making up the biofilm are discussed relative to biocorrosion.     

Biocorrosion: towards understanding interactions between biofilms and metals

The term microbially influenced corrosion, or biocorrosion, refers to the accelerated deterioration of metals owing to the presence of biofilms on their surfaces. The detailed mechanisms of biocorrosion are still poorly understood. Recent investigations into biocorrosion have focused on the influence of biomineralization processes taking place on metallic surfaces and the impact of extracellular enzymes, active within the biofilm matrix, on electrochemical reactions at the biofilm-metal interface.     

Role of beta 1-4 linked polymers in the biofilm structure of marine Pseudomonas sp. CE-2 on 304 stainless steel coupons

Pseudomonas sp CE-2 cells attach and form biofilms on 304-stainless steel (SS) coupons. A series of experiments were carried out in order to understand the role of exopolysaccharides (EPS) in the formation and maintenance of CE-2 biofilms on SS coupons. The biofilm density and EPS concentration increased over the period of incubation and the highest values for both were recorded after 72 h. Calcofluor and the lectin concanavalin A (Con A) showed a positive interaction with 72-h old biofilms, indicating the presence of beta 1-4 linked polymers, and alpha-d-glucose and alpha-d-mannose in the biofilm matrix of CE-2. When the CE-2 cells were grown in the presence of calcofluor (200 microg ml(-1)), biofilm formation was significantly reduced (approximately 85%). Conversely, the lectins Con A or WGA did not influence the CE-2 biofilms on the SS coupons. Furthermore, treatment with cellulase, an enzyme specific for the degradation of beta 1-4 linked polymers, removed substantial amounts of CE-2 biofilm from SS coupons. These results strongly suggest the involvement of beta 1-4 linked polymers in the formation and maintenance of Pseudomonas sp. CE-2 biofilms on SS coupons.     

The potential use of atomic force microscopy for studying corrosion of metals in the presence of bacterial biofilms — an overview

This communication outlines the principles of application of scanning probe microscopy (SPM) as a tool for studying physico-chemical and biological phenomena and discusses the potential use of atomic force microscopy (AFM) , a form of SPM, for investigation of bacterial biofilms formed on metal surfaces and for studying corrosion of these surfaces in the presence of such biofilms. AFM images showing biofilms developed in pure cultures of either Pseudomonas species on copper, or by a marine isolate of sulphate-reducing bacterium on 304 stainless steel are presented to demonstrate usefulness of the SPM technique for both quantitative and qualitative determination of biocorrosion.     

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.     

Mutual influences of Pseudomonas aeruginosa and Desulfovibrio desulfuricans on their adhesion to stainless steel

The mutual influences of Pseudomonas aeruginosa PAO1 and Desulfovibrio desulfuricans subsp. desulfuricans (ATCC 29577) on their adhesion to stainless steel were investigated in batch and column experiments. It was found that P. aeruginosa promoted the adhesion of D. desulfuricans under conditions of turbulence, but not under quiescent conditions. The enhancement involved the alignment of most D. desulfuricans along P. aeruginosa cells and was attributed to the additional interaction surface area provided by adhered P. aeruginosa to aligning D. desulfuricans cells. A slightly positive effect of preadhered D. desulfuricans on the adhesion of P. aeruginosa was found. Under condition of laminar flow, substantially better adhesion of D. desulfuricans to confluent P. aeruginosa biofilms than to steel was observed. The mutual influences are discussed in terms of more favorable adhesion energies and the influence of changed hydraulic conditions due to the roughness of P. aeruginosa biofilms.     

Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates

AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l(-1). Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into approximately 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l(-1) alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport clearly was retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.     

Physiology of biofilms of thermophilic bacilli—potential consequences for cleaning

Thermophilic Bacillus species readily attached and grew on stainless steel surfaces, forming mature biofilms of >10^6.0 cells/cm² in 6 h on a surface inoculated with the bacteria. Clean stainless steel exposed only to pasteurized skim milk at 55 °C developed a mature biofilm of >10^6.0 cells/cm² within 18 h. When bacilli were inoculated onto the steel coupons, 18-h biofilms were 30 m thick. Biofilm growth followed a repeatable pattern, with a reduction in the numbers of bacteria on the surface occurring after 30 h, followed by a recovery. This reduction in numbers was associated with the production of a substance that inhibited the growth of the bacteria. Variations in the environment, including pH and molarity, affected the viability of the cells. Chemicals that attack the polysaccharide matrix of the biofilm were particularly effective in killing and removing cells from the biofilm, demonstrating the importance of polysaccharides in the persistence of these biofilms. Treatment of either the biofilm or a clean stainless steel surface with lysozyme killed biofilm cells and prevented the attachment of any bacteria exposed to the surface. This suggests that lysozyme may have potential as an alternative control method for biofilms of these bacteria.     

Factors influencing attachment of thermophilic bacilli to stainless steel

AIMS: This project aimed to investigate the mechanism of attachment of the vegetative cells and spores of thermophilic bacilli to stainless steel with a view to devising strategies to limit biofilm development and survival. METHODS AND RESULTS: Spores and vegetative cells of bacterial isolates were exposed to protein denaturing agents (sodium dodecyl sulphate (SDS) and trypsin) and polysaccharide removing agents (sodium metaperiodate, trichloroacetic acid (TCA) and lysozyme). Treatment with sodium metaperiodate, TCA and lysozyme increased the number of vegetative cells attaching in many of the strains studied, while SDS and trypsin decreased attachment. Spores attached to stainless steel in greater numbers than vegetative cells, and the various treatments had less effect on this attachment than for vegetative cells. Viability of the cells or spores was not an important factor in attachment, as cells and spores rendered non-viable also attached to stainless steel in similar numbers. Coating the stainless steel with skim milk proteins decreased the attachment of both vegetative cells and spores. There was no correlation between the degree of attachment and the amount of extracellular polysaccharide (EPS) produced by each strain, surface hydrophobicity or zeta potential of vegetative cells or spores, though spores were found to be more hydrophobic than vegetative cells. CONCLUSIONS: The results suggest that biofilm formation by these thermophilic bacilli is probably a multifactorial process, and that cell-surface proteins play a very important role in the initial process of attachment during the formation of biofilms by these bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This information will provide direction for developing improved cleaning systems to control biofilms of thermophilic bacilli in dairy manufacturing plants.     

Role of 𝛃 1-4 linked polymers in the biofilm structure of marine Pseudomonas sp. CE-2 on 304 stainless steel coupons

Pseudomonas sp CE-2 cells attach and form biofilms on 304-stainless steel (SS) coupons. A series of experiments were carried out in order to understand the role of exopolysaccharides (EPS) in the formation and maintenance of CE-2 biofilms on SS coupons. The biofilm density and EPS concentration increased over the period of incubation and the highest values for both were recorded after 72 h. Calcofluor and the lectin concanavalin A (Con A) showed a positive interaction with 72-h old biofilms, indicating the presence of β 1-4 linked polymers, and α-d-glucose and α-d-mannose in the biofilm matrix of CE-2. When the CE-2 cells were grown in the presence of calcofluor (200 μg ml^?1), biofilm formation was significantly reduced (～85%). Conversely, the lectins Con A or WGA did not influence the CE-2 biofilms on the SS coupons. Furthermore, treatment with cellulase, an enzyme specific for the degradation of β 1-4 linked polymers, removed substantial amounts of CE-2 biofilm from SS coupons. These results strongly suggest the involvement of β 1-4 linked polymers in the formation and maintenance of Pseudomonas sp. CE-2 biofilms on SS coupons.     

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.     

Bacterial Community Structure of Biofilms on Artificial Surfaces in an Estuary

This study examined bacterial community structure of biofilms on stainless steel and polycarbonate in seawater from the Delaware Bay. Free-living bacteria in the surrounding seawater were compared to the attached bacteria during the first few weeks of biofilm growth. Surfaces exposed to seawater were analyzed by using 16S rDNA libraries, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE). Community structure of the free-living bacterial community was different from that of the attached bacteria according to FISH and DGGE. In particular, alpha-proteobacteria dominated the attached communities. Libraries of 16S rRNA genes revealed that representatives of the Rhodobacterales clade were the most abundant members of biofilm communities. Changes in community structure during biofilm growth were also examined by DGGE analysis. We hypothesized that bacterial communities on dissimilar surfaces would initially differ and become more similar over time. In contrast, the compositions of stainless steel and polycarbonate biofilms were initially the same, but differed after about 1 week of biofilm growth. These data suggest that the relationship between surface properties and biofilm community structure changes as biofilms grow on surfaces such as stainless steel and polycarbonate in estuarine water.     

Microbiologically influenced corrosion: looking to the future.

This review discusses the state-of-the-art of research into biocorrosion and the biofouling of metals and alloys of industrial usage. The key concepts needed to understand the main effects of microorganisms on metal decay, and current trends in monitoring and control strategies to mitigate the deleterious effects of biocorrosion and biofouling are also described. Several relevant cases of biocorrosion studied by our research group are provided as examples: (i) biocorrosion of aluminum and its alloys by fungal contaminants of jet fuels; (ii) sulfate-reducing bacteria (SRB)-induced corrosion of steel; (iii) biocorrosion and biofouling interactions in the marine environment; (iv) monitoring strategies for assessing biocorrosion in industrial water systems; (v) microbial inhibition of corrosion; (vi) use and limitations of electrochemical techniques for evaluating biocorrosion effects. Future prospects in the field are described with respect to the potential of innovative techniques in microscopy (environmental scanning electron microscopy, confocal scanning laser microscopy, atomic force microscopy), new spectroscopic techniques for the study of corrosion products and biofilms (energy dispersion X-ray analysis, X-ray photoelectron spectroscopy, electron microprobe analysis) and electrochemistry (electrochemical impedance spectroscopy, electrochemical noise analysis).     

Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production

AIMS: The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel. METHODS AND RESULTS: Three curli-deficient strains (43895-, 43894- and E0018-) and three curli over-producing strains (43895+, 43894+ and E0018+) of E. coli O157:H7 were studied. Stainless steel coupons (SSC) were immersed in cell suspensions of each strain for 24 h at 4 degrees C. The number of cells attached to SSC was determined. To determine the ability of attached cells to form biofilm, SSC were immersed in 10% of tryptic soya broth up to 6 days at 22 degrees C. Curli-deficient and curli-producing strains did not differ in their ability to attach to SSC, but only curli-producing strains formed biofilms. CONCLUSIONS: Curli production by E. coli O157:H7 does not affect attachment of cells on stainless steel but curli-producing strains are better able to form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Curli production by E. coli O157:H7 enhances its ability to form biofilm on stainless steel, thereby potentially resulting in increased difficulty in removing or killing cells by routine cleaning and sanitizing procedures used in food-processing plants.     

Extracellular Electron Transfer Is a Bottleneck in the Microbiologically Influenced Corrosion of C1018 Carbon Steel by the Biofilm of Sulfate-Reducing Bacterium Desulfovibrio vulgaris

Carbon steels are widely used in the oil and gas industry from downhole tubing to transport trunk lines. Microbes form biofilms, some of which cause the so-called microbiologically influenced corrosion (MIC) of carbon steels. MIC by sulfate reducing bacteria (SRB) is often a leading cause in MIC failures. Electrogenic SRB sessile cells harvest extracellular electrons from elemental iron oxidation for energy production in their metabolism. A previous study suggested that electron mediators riboflavin and flavin adenine dinucleotide (FAD) both accelerated the MIC of 304 stainless steel by the Desulfovibrio vulgaris biofilm that is a corrosive SRB biofilm. Compared with stainless steels, carbon steels are usually far more prone to SRB attacks because SRB biofilms form much denser biofilms on carbon steel surfaces with a sessile cell density that is two orders of magnitude higher. In this work, C1018 carbon steel coupons were used in tests of MIC by D. vulgaris with and without an electron mediator. Experimental weight loss and pit depth data conclusively confirmed that both riboflavin and FAD were able to accelerate D. vulgaris attack against the carbon steel considerably. It has important implications in MIC failure analysis and MIC mitigation in the oil and gas industry.     

Inhibition of biofilms associated with dentures and toothbrushes by tetrasodium EDTA

AIMS: We examined the efficacy of tetrasodium EDTA in eradicating biofilms derived from salivary inocula or pure cultures of Candida albicans on discs of polymethyl methacrylate (PMMA) denture base or on toothbrushes that had been used normally for 4-8 weeks. Its efficiency in virus neutralization was also determined. METHODS AND RESULTS: Overnight (16 h) treatment with 4% (w/v) tetrasodium EDTA solution reduced salivary and C. albicans biofilm viable counts by > or =99%. Biofilm removal was confirmed using confocal laser scanning microscopy. Presence/absence of sucrose during biofilm formation had no effect on killing efficacy. Prolonged treatment of PMMA with tetrasodium EDTA did not influence subsequent formation of C. albicans biofilms or affect surface roughness of the PMMA, but it reduced subsequent biofilm formation from a salivary inoculum. Infectivities of herpes simplex virus and polio virus suspensions were reduced by >99.99% by treatment for 1 and 2 h, respectively. CONCLUSIONS: Tetrasodium EDTA solution efficiently disinfected toothbrushes and PMMA discs, with the detachment of biofilms, and rapidly neutralized both nonenveloped and enveloped viruses. SIGNIFICANCE AND IMPACT OF THE STUDY: Dentures and toothbrushes become contaminated by bacterial biofilms and by viruses. There is a need for disinfection methods that are rapidly effective, cost-effective, nontoxic and easily implemented. These studies indicate that tetrasodium EDTA solution has disinfection applications in the oral care field.