Ambient laser ablation and solvent capture by aspiration (LASCA) mass spectrometric imaging was combined with metabolomics high-performance liquid chromatography (HPLC) mass spectrometry analysis and light profilometry to investigate the correlation between chemical composition of marine bacterial biofilms on surfaces of 1018 carbon steel and corrosion damage of steel underneath the biofilms. Pure cultures of Marinobacter sp. or a wild population of bacteria present in coastal seawater served as sources of biofilms. Profilometry data of biofilm-free surfaces demonstrated heterogeneous distributions of corrosion damage. LASCA data were correlated with areas on the coupons varying in the level of corrosion attack, to reveal differences in chemical composition within biofilm regions associated with corroding and corrosion-free zones. Putative identification of selected compounds was carried out based on HPLC results and subsequent database searches. This is the first report of successful ambient chemical and metabolomic imaging of marine biofilms on corroding metallic materials. The metabolic analysis of such biofilms is challenging due to the presence in the biofilm of large amounts of corrosion products. However, by using the LASCA imaging interface, images of more than 1000 ions (potential metabolites) are generated, revealing striking heterogeneities within the biofilm. In the two model systems studied here, it is found that some of the patterns observed in selected ion images closely correlate with the occurrence and extent of corrosion in the carbon steel substrate as revealed by profilometry, while others do not. This approach toward the study of microbially influenced corrosion (MIC) holds great promise for approaching a fundamental understanding of the mechanisms involved in MIC.
Severe corrosion found on steel mooring components (CSMC) at several sites worldwide has caused concern in recent years as to whether the components can safely meet their design life. A pilot study was initiated to understand the underlying corrosion causes with the aim of developing successful CSMC mitigation methods. In 2014, a field test was conducted offshore at two different locations in West Africa in order to confirm the contribution of microbiologically influenced corrosion (MIC) to CSMC. The study provided evidence that MIC is a root cause of the observed severe corrosion in the form of mega-pits at one of the two test sites. The tests consisted of deploying carbon steel coupons on a fiber rope, herein referred to as a Љmicrobial baiting kitЉ, at facilities near the mooring systems to capture the biofilm forming microorganisms. The kit was submerged approximately three meters below the water surface for an extended period of time allowing for free swimming microorganisms to colonize the coupons. The kit was the first of its kind to be used in the industry for investigating MIC of mooring systems. Upon recovery of the coupons, pitting damage was revealed underneath the fouling deposits. Following DNA extraction, subsequent analysis of sequences representing fragments of the bacterial 16S rRNA gene demonstrated that, regardless of the test location, the outer part of the biofilm formed on coupon surfaces had significantly different microbial community structure when compared to the surrounding seawater. In both test sites, biofilm DNA analysis confirmed that obtained bacterial sequences represented the initial colonizers of submerged structures in marine environments. Sequences identified as belonging to sulfate-reducing bacteria (SRB), which are considered major contributors to MIC in suboxic/anoxic aquatic environments, were more abundant in biofilms but scarce in water samples. A higher number of SRB sequences were associated with coupons retrieved from the test location where pitting attacks were prominent. Sequences indicative of acetic acid-producers and non-SRB hydrogen sulfide-producing microorganisms, that are also likely MIC contributors, were identified; however, further work is required to prove the involvement of these prokaryotes in steel deterioration. The results and finding from this pilot work set the stage for a comprehensive Joint Industry Project (JIP) launched by DeepStar ® and is entitled DeepStar ® CTR12402 Integrity Management of Mooring Systems Against Corrosion JIP. The aim of the DeepStar ® JIP is to determine possible measures of MIC mitigation.
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.
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