Marine Biofilm Formation Research Articles

Effects of dissolved oxygen on marine biofilm formation and its on microstructure and chloride ion permeability of concrete

Effects of dissolved oxygen on marine biofilm formation and its on microstructure and chloride ion permeability of concrete

Influence of Elution Characteristics of Steelmaking Slags on Major Bacterial Communities in Biofilms

Steelmaking slags are prospective base materials for seaweed beds, resulting from a continuous process of biofouling, starting from biofilm formation and leading to growing algae. While focusing on biofilm formation, we investigated specific features of steelmaking slags when utilized as a base for seaweed beds by comparing the bacterial communities in marine biofilms between steelmaking slags and artificially produced ones. Genomic DNA was extracted from the biofilms collected on days 3 and 7, and partial 16S rRNA libraries were generated and sequenced by second-generation next-generation sequencing. The read sequences were analyzed using QIIME 2™, then heatmaps and non-metric multidimensional scaling based on the Bray–Curtis dissimilarity index in the R program. Rhodobacteraceae and Flavobacteriaceae were the most dominant family members in all samples on both days 3 and 7. However, Mariprofundus, comprising iron-oxidative bacteria, was predominantly detected in the samples of steelmaking slags on day 7. This suggested that the growth of Mariprofundus was dependent on Fe(II) ion concentration and that steelmaking slags eluted Fe(II) ions more easily than artificial slags. In contrast, Sulfurovaceae, sulfur-oxidizing bacteria, were dominantly present in all samples on day 3, but decreased by day 7, regardless of the sulfur content. It was supposed that engine oil-derived sulfur compounds strongly influenced Sulfurovaceae growth, whereas slag-derived sulfur compounds did not. Heatmap analysis indicated that the submersion period significantly influenced the bacterial communities, regardless of the differences in the main slag content ratios. Summarizing these results, the elution characteristics of steelmaking slags have the potential to influence the formation of marine biofilms, and this formation is significantly influenced by environmental conditions.

Diatom adhesive trail proteins acquired by horizontal gene transfer from bacteria serve as primers for marine biofilm formation.

Biofilm-forming benthic diatoms are key primary producers in coastal habitats, where they frequently dominate sunlit intertidal substrata. The development of gliding motility in raphid diatoms was a key molecular adaptation that contributed to their evolutionary success. However, the structure-function correlation between diatom adhesives utilized for gliding and their relationship to the extracellular matrix that constitutes the diatom biofilm is unknown. Here, we have used proteomics, immunolocalization, comparative genomics, phylogenetics and structural homology analysis to investigate the evolutionary history and function of diatom adhesive proteins. Our study identified eight proteins from the adhesive trails of Craspedostauros australis, of which four form a new protein family called Trailins that contain an enigmatic Choice-of-Anchor A (CAA) domain, which was acquired through horizontal gene transfer from bacteria. Notably, the CAA-domain shares a striking structural similarity with one of the most widespread domains found in ice-binding proteins (IPR021884). Our work offers new insights into the molecular basis for diatom biofilm formation, shedding light on the function and evolution of diatom adhesive proteins. This discovery suggests that there is a transition in the composition of biomolecules required for initial surface colonization and those utilized for 3D biofilm matrix formation.

Comparative Study of the Marinobacter hydrocarbonoclasticus Biofilm Formation on Antioxidants Containing Siloxane Composite Coatings.

No systematic study of antioxidant containing coatings and their anti-biofilm action has been reported so far. The utilization of antioxidants in protective coatings to inhibit marine biofilm formation is a current challenge. The aim of this preliminary study was to prepare, characterize and compare the efficiency of low adhesive siloxane composite coatings equally loaded with different antioxidants against mono-species biofilms formation. Most often participating in the marine biofilms formation, Marinobacter hydrocarbonoclasticus was the test bacterium. Both the biofilm covered surface area (BCSA) and corrected total cell fluorescence (CTCF) (by fluorescent microscopy) were selected as the parameters for quantification of the biofilm after 1 h and 4 h incubation. Differing extents of altered surface characteristics (physical-chemical; physical-mechanical) and the specific affection of M. hydrocarbonoclasticus biofilm formation in both reduction and stimulation, were found in the studied antioxidant containing coatings, depending on the chemical nature of the used antioxidant. It was concluded that not all antioxidants reduce mono-species biofilm formation; antioxidant chemical reactivity stipulates the formation of an altered vulcanization network of the siloxane composites and thus microbial adhesion which influences the surface characteristics of the vulcanized coatings; and low surface energy combined with a low indentation elastic modulus are probably pre-requisites of low microbial adhesion.

Anti-bacterial performance evaluation of hydrophobic poly (dimethylsiloxane)-ZnO coating using Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) has been implicated in the attachment and formation of marine biofilms which initiate biofouling and biocorrosion. To this end, several hydrophobic and superhydrophobic coatings have been suggested for anti-bacterial application, but some possess toxic substances which affect useful aquatic lives. Thus, the aim of this study was to appraise the anti-bacterial and anti-corrosion performance of hydrophobic perfluorodecyltrichlorosilane (FDTS)-modified poly (dimethylsiloxane) (PDMS)-ZnO coating using P. aeruginosa. The surface analytical, physico-chemical and electrochemical techniques were used to investigate the properties of the coatings. Results show that FDTS-modified PDMS-ZnO coating displayed higher resistance to adhesion of P. aeruginosa biofilm and better corrosion resistance performance than the unmodified coating. The outstanding performance of FDTS-modified coating was attributed to the low surface activity of FDTS which increased the kinetic barrier between the coating surface and biofilm. It is therefore anticipated that these results would provide insight in the design of future anti-bacterial coatings for marine application.

Early stage of marine biofilm formation on duplex stainless steel.

The aim of this work was to investigate the bacteria-surface interactions occurring during the first hour of adhesion of marine Pseudoalteromonas NCIMB 2021 at the surface of 2304 lean duplex stainless steel in artificial seawater. A complete characterization of the biofilm and the passive film was performed coupling epifluorescence microscopy, scanning electron microscopy (SEM), x-ray photoelectron spectroscopy (XPS), and time of flight secondary ion mass spectrometry (ToF-SIMS). The coupling of XPS and ToF-SIMS analyses revealed that (1) protein and polysaccharide contents in the biofilm are similar in the presence or absence of nutrients, (2) the biofilm is mainly composed of proteins and the protein content is similar to the one of Tightly Bound EPS, (3) increased bacterial activity due to nutrients leads to chromium enrichment in the passive film in close vicinity to the bacteria.

Marine biofilms: diversity of communities and of chemical cues.

Surfaces immersed in seawater are rapidly colonized by various microorganisms, resulting in the formation of heterogenic marine biofilms. These communities are known to influence the settlement of algae spores and invertebrate larvae, triggering a succession of fouling events, with significant environmental and economic impacts. This review covers recent research regarding the differences in composition of biofilms isolated from different artificial surface types and the influence of environmental factors on their formation. One particular phenomenon - bacterial quorum sensing (QS) - allows bacteria to coordinate swarming, biofilm formation among other phenomena. Some other marine biofilm chemical cues are believed to modulate the settlement and the succession of macrofouling organisms, and they are also reviewed here. Finally, since the formation of a marine biofilm is considered to be an initial, QS-dependent step in the development of marine fouling events, QS inhibition is discussed on its potential as a tool for antibiofouling control in marine settings.

Monitoring of Marine Biofilm Formation Dynamics at Submerged Solid Surfaces With Multitechnique Sensors

Biofouling on artificial and biotic solid substrata was studied in several locations in the Baltic Sea brackish water (Gulf of Gdansk) during a three-year period with contact angle wettability, confocal microscopy and photoacoustic spectroscopy techniques. As a reference, the trophic state of water body was determined from chemical analyses according to the following parameters: pH, dissolved O2, phosphate, nitrite, nitrate, ammonium etc. concentrations and further correlated to the determined biofilm characterizing parameters by means of Spearman’s rank correlation procedure. Biofilm adhesive surface properties (surface free energy, work of adhesion etc.) were obtained with the contact angle hysteresis (CAH) approach using an automatic captive bubble solid surface wettability sensor assigned for in-situ, on-line and quasi-continuous measurements of permanently submerged samples (Pogorzelski et al., 2013, Pogorzelski and Szczepańska, 2014). Structural and morphological biofilm features (biovolume, substratum coverage, area to volume ratio, spatial spreading, mean thickness and roughness) were determined from confocal reflection microscopy (COCRM) data. Photosynthetic properties (photosynthetic energy storage (ES), photoacoustic amplitude and phase spectra) of biofilm communities exhibited a seasonal variability as indicated by a novel closed-cell type photoacoustic spectroscopy (PAS) system. That allowed mathematical modeling of a marine biofilm under steady state, in particular the specific growth rates μi, and the conditioning or induction times λi to be derived from simultaneous multitechnique signals. A set of the established biofilm structural and physical parameters could be modern water body trophic state indexes.

Biofilm Inhibition by Novel Natural Product- and Biocide-Containing Coatings Using High-Throughput Screening.

The use of natural products (NPs) as possible alternative biocidal compounds for use in antifouling coatings has been the focus of research over the past decades. Despite the importance of this field, the efficacy of a given NP against biofilm (mainly bacteria and diatoms) formation is tested with the NP being in solution, while almost no studies test the effect of an NP once incorporated into a coating system. The development of a novel bioassay to assess the activity of NP-containing and biocide-containing coatings against marine biofilm formation has been achieved using a high-throughput microplate reader and highly sensitive confocal laser scanning microscopy (CLSM), as well as nucleic acid staining. Juglone, an isolated NP that has previously shown efficacy against bacterial attachment, was incorporated into a simple coating matrix. Biofilm formation over 48 h was assessed and compared against coatings containing the NP and the commonly used booster biocide, cuprous oxide. Leaching of the NP from the coating was quantified at two time points, 24 h and 48 h, showing evidence of both juglone and cuprous oxide being released. Results from the microplate reader showed that the NP coatings exhibited antifouling efficacy, significantly inhibiting biofilm formation when compared to the control coatings, while NP coatings and the cuprous oxide coatings performed equally well. CLSM results and COMSTAT analysis on biofilm 3D morphology showed comparable results when the NP coatings were tested against the controls, with higher biofilm biovolume and maximum thickness being found on the controls. This new method proved to be repeatable and insightful and we believe it is applicable in antifouling and other numerous applications where interactions between biofilm formation and surfaces is of interest.

Reduction of bacterial biofilm formation using marine natural antimicrobial peptides.

There is an important need for the development of new "environmentally-friendly" antifouling molecules to replace toxic chemicals actually used to fight against marine biofouling. Marine biomass is a promising source of non-toxic antifouling products such as natural antimicrobial peptides produced by marine organisms. The aim of this study was to demonstrate the efficiency of antimicrobial peptides extracted from snow crab (SCAMPs) to reduce the formation of marine biofilms on immerged mild steel surfaces. Five antimicrobial peptides were found in the snow crab hydrolysate fraction used in this study. SCAMPs were demonstrated to interact with natural organic matter (NOM) during the formation of the conditioning film and to limit the marine biofilm development in terms of viability and bacterial structure. Natural SCAMPs could be considered as a potential alternative and non-toxic product to reduce biofouling, and as a consequence microbial induced corrosion on immerged surfaces.

Anti-Biofilm Effect of Biodegradable Coatings Based on Hemibastadin Derivative in Marine Environment.

Dibromohemibastadin-1 (DBHB) is an already known potent inhibitor of blue mussel phenoloxidase (which is a key enzyme involved in bioadhesion). Within this study, the potentiality of DBHB against microfouling has been investigated. The activity of DBHB was evaluated on key strains of bacteria and microalgae involved in marine biofilm formation and bioassays assessing impact on growth, adhesion and biofilm formation were used. To assess the efficiency of DBHB when included in a matrix, DBHB varnish was prepared and the anti-microfouling activity of coatings was assessed. Both in vitro and in situ immersions were carried out. Confocal Laser Scanning Microscopy (CLSM) was principally used to determine the biovolume and average thickness of biofilms developed on the coatings. Results showed an evident efficiency of DBHB as compound and varnish to reduce the biofilm development. The mode of action seems to be based principally on a perturbation of biofilm formation rather than on a biocidal activity in the tested conditions.

Low-concentration diffusible molecules affect the formation of biofilms by mixed marine communities

AbstractBiofilm formation is a major concern in any venture in the marine environment and often precedes the establishment of fouling by macro-organisms. In this study, the effects of three known cell-to-cell signalling molecules, nitric oxide (NO), cis-2-decenoic acid (CDA) and patulin, on the formation of marine biofilms were investigated. Each of the molecules has been shown to affect biofilms and this is the first study to investigate their effect on mixed communities of marine biofilm-forming micro-organisms. Studies of the biomass of those biofilms grown in the presence of the molecules showed that all three reduced biofilm formation by marine communities, with both NO and patulin reducing biofilm formation by more than 90% at the highest concentrations studied. However, colony counts revealed that the effect of patulin is likely due to toxicity. Analyses of the biofilm communities were also carried out using DGGE to determine whether there was any variation in the effects of each molecule on differ...

Characterization of self-generated variants in Pseudoalteromonas lipolytica biofilm with increased antifouling activities

Pseudoalteromonas is widespread in various marine environments, and most strains can affect invertebrate larval settlement and metamorphosis by forming biofilms. However, the impact and the molecular basis of population diversification occurring in Pseudoalteromonas biofilms are poorly understood. Here, we show that morphological diversification is prevalent in Pseudoalteromonas species during biofilm formation. Two types of genetic variants, wrinkled (frequency of 12 ± 5 %) and translucent (frequency of 5 ± 3 %), were found in Pseudoalteromonas lipolytica biofilms. The inducing activities of biofilms formed by the two variants on larval settlement and metamorphosis of the mussel Mytilus coruscus were significantly decreased, suggesting strong antifouling activities. Using whole-genome re-sequencing combined with genetic manipulation, two genes were identified to be responsible for the morphology alternations. A nonsense mutation in AT00_08765 led to a wrinkled morphology due to the overproduction of cellulose, whereas a point mutation in AT00_17125 led to a translucent morphology via a reduction in capsular polysaccharide production. Taken together, the results suggest that the microbial behavior on larval settlement and metamorphosis in marine environment could be affected by the self-generated variants generated during the formation of marine biofilms, thereby rendering potential application in biocontrol of marine biofouling.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6865-x) contains supplementary material, which is available to authorized users.

In situ colonization of marine biofilms on UNS S32760 duplex stainless steel coupons in areas with different water qualities: Implications for corrosion potential behavior

In the presence of biofilms, stainless steels (SS) exhibits an increase in corrosion potential, called ennoblement. In the present study, the corrosion potential (E corr) behavior of the duplex SS UNS S32760 was recorded simultaneously with the in situ marine biofilm formation in two areas at Arraial do Cabo, Southeastern Brazil. The biofilm at Forno Harbor (an anthropogenically disturbed area) was characterized by higher relative abundances of Bacteria at day 2, followed by diatoms (especially Navicula sp.) on day 10 and dinoflagellates on day 18, whereas no clear trend was recorded at Cabo Frio Island (an undisturbed area). The ennoblement of E corr values was site-dependent. In a complementary laboratory assay, biofilms were removed and the E corr values registered in sterile conditions for the subsequent 10 days and corroborated in situ results. Understanding biofilms and SS interactions has important implications for materials science and engineering decisions as well as helping to fill in important gaps in this knowledge.

Antifouling Activity of Synthetic Alkylpyridinium Polymers Using the Barnacle Model

Polymeric alkylpyridinium salts (poly-APS) isolated from the Mediterranean marine sponge, Haliclona (Rhizoniera) sarai, effectively inhibit barnacle larva settlement and natural marine biofilm formation through a non-toxic and reversible mechanism. Potential use of poly-APS-like compounds as antifouling agents led to the chemical synthesis of monomeric and oligomeric 3-alkylpyridinium analogues. However, these are less efficient in settlement assays and have greater toxicity than the natural polymers. Recently, a new chemical synthesis method enabled the production of poly-APS analogues with antibacterial, antifungal and anti-acetylcholinesterase activities. The present study examines the antifouling properties and toxicity of six of these synthetic poly-APS using the barnacle (Amphibalanus amphitrite) as a model (cyprids and II stage nauplii larvae) in settlement, acute and sub-acute toxicity assays. Two compounds, APS8 and APS12-3, show antifouling effects very similar to natural poly-APS, with an anti-settlement effective concentration that inhibits 50% of the cyprid population settlement (EC50) after 24 h of 0.32 mg/L and 0.89 mg/L, respectively. The toxicity of APS8 is negligible, while APS12-3 is three-fold more toxic (24-h LC50: nauplii, 11.60 mg/L; cyprids, 61.13 mg/L) than natural poly-APS. This toxicity of APS12-3 towards nauplii is, however, 60-fold and 1200-fold lower than that of the common co-biocides, Zn- and Cu-pyrithione, respectively. Additionally, exposure to APS12-3 for 24 and 48 h inhibits the naupliar swimming ability with respective IC50 of 4.83 and 1.86 mg/L.

A survey of culturable aerobic and anaerobic marine bacteria in de novo biofilm formation on natural substrates in St. Andrews Bay, Scotland

This study reports a novel study of marine biofilm formation comprising aerobic and anaerobic bacteria. Samples of quartz and feldspar, minerals commonly found on the earth, were suspended 5m deep in the North Sea off the east coast of St. Andrews, Scotland for 5weeks. The assemblage of organisms attached to these stones was cultivated under aerobic and anaerobic conditions in the laboratory. Bacteria isolated on Marine Agar 2216 were all Gram-negative and identified to genus level by sequencing the gene encoding 16S rRNA. Colwellia, Maribacter, Pseudoaltermonas and Shewanella were observed in aerobically-grown cultures while Vibrio was found to be present in both aerobic and anaerobic cultures. The obligate anaerobic bacterium Psychrilyobacter atlanticus, a recently defined genus, was identified as a close relative of isolates grown anaerobically. The results provide valuable information as to the main players that attach and form de novo biofilms on common minerals in sea water.

Anti-microfouling activity of lipidic metabolites from the invasive brown alga Sargassum muticum (Yendo) Fensholt.

The purification of the chloroform extract from the brown invasive macroalga Sargassum muticum, through a series of chromatographic separations, yielded 12 fractions that were tested against strains of bacteria, microalgae, and fungi involved in marine biofilm formation. The chemical composition of four (a, c, g, and k) out of the six fractions that exhibited anti-microfouling activity was investigated. Fraction a contained saturated and unsaturated linear hydrocarbons (C12-C27). Arachidonic acid was identified as the major metabolite in fraction c whereas fraction g contained mainly palmitic, linolenic, and palmitoleic acids. Fraction k was submitted to further purification yielding the fraction kAcaF1e that was composed of galactoglycerolipids, active against the growth of two of the four bacterial strains (Shewanella putrefaciens and Polaribacter irgensii) and all tested fungi. These promising results, in particular the isolation and the activity of galactoglycerolipids, attest the potential of the huge biomass of S. muticum as a source of new environmentally friendly antifouling compounds.

Bacterial communities in the initial stage of marine biofilm formation on artificial surfaces

Succession of bacterial communities during the first 36 h of biofilm formation in coastal water was investigated at 3 approximately 15 h intervals. Three kinds of surfaces (i.e., acryl, glass, and steel substratum) were submerged in situ at Sacheon harbor, Korea. Biofilms were harvested by scraping the surfaces, and the compositions of bacterial communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP), and cloning and sequencing of 16S rRNA genes. While community structure based on T-RFLP analysis showed slight differences by substratum, dramatic changes were commonly observed for all substrata between 9 and 24 h. Identification of major populations by 16S rRNA gene sequences indicated that gamma-Proteobacteria (Pseudomonas, Acinetobacter, Alteromonas, and uncultured gamma-Proteobacteria) were predominant in the community during 0 approximately 9 h, while the ratio of alpha-Proteobacteria (Loktanella, Methylobacterium, Pelagibacter, and uncultured alpha-Proteobacteria) increased 2.6 approximately 4.8 folds during 24 approximately 36 h of the biofilm formation, emerging as the most predominant group. Previously, alpha-Proteobacteria were recognized as the pioneering organisms in marine biofilm formation. However, results of this study, which revealed the bacterial succession with finer temporal resolution, indicated some species of gamma-Proteobacteria were more important as the pioneering population. Measures to control pioneering activities of these species can be useful in prevention of marine biofilm formation.

A marine bacterial adhesion microplate test using the DAPI fluorescent dye: a new method to screen antifouling agents

To develop a method to screen antifouling agents against marine bacterial adhesion as a sensitive, rapid and quantitative microplate fluorescent test. Our experimental method is based on a natural biofilm formed by mono-incubation of the marine bacterium Pseudoalteromonas sp. D41 in sterile natural sea water in a 96-well polystyrene microplate. The 4'6-diamidino-2-phenylindole dye was used to quantify adhered bacteria in each well. The total measured fluorescence in the wells was correlated with the amount of bacteria showing a detection limit of one bacterium per 5 microm(2) and quantifying 2 x 10(7) to 2 x 10(8) bacteria adhered per cm(2). The antifouling properties of three commercial surface-active agents and chlorine were tested by this method in the prevention of adhesion and also in the detachment of already adhered bacteria. The marine bacterial adhesion inhibition rate depending on the agent concentration showed a sigmoid shaped dose-response curve. This test is well adapted for a rapid and quantitative first screening of antifouling agents directly in seawater in the early steps of marine biofilm formation. In contrast to the usual screenings of antifouling products which detect a bactericidal activity, this test is more appropriate to screen antifouling agents for bacterial adhesion removal or bacterial adhesion inhibition activities. This screening test focuses on the antifouling properties of the products, especially the initial steps of marine biofilm formation.

Electron microscopic examination of wastewater biofilm formation and structural components.

This research documents in situ wastewater biofilm formation, structure, and physiochemical properties as revealed by scanning and transmission electron microscopy. Cationized ferritin was used to label anionic sites of the biofilm glycocalyx for viewing in thin section. Wastewater biofilm formation paralleled the processes involved in marine biofilm formation. Scanning electron microscopy revealed a dramatic increase in cell colonization and growth over a 144-h period. Constituents included a variety of actively dividing morphological types. Many of the colonizing bacteria were flagellated. Filaments were seen after primary colonization of the surface. Transmission electron microscopy revealed a dominant gram-negative cell wall structure in the biofilm constituents. At least three types of glycocalyces were observed. The predominant glycocalyx possessed interstices and was densely labeled with cationized ferritin. Two of the glycocalyces appeared to mediate biofilm adhesion to the substratum. The results suggest that the predominant glycocalyx of this thin wastewater biofilm serves, in part, to: (i) enclose the bacteria in a matrix and anchor the biofilm to the substratum and (ii) provide an extensive surface area with polyanionic properties.