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Reliable and accurate in situ sensors capable of detecting and quantifying troublesome marine biofilms on metallic surfaces are increasingly necessary. A 0.2 mm diameter gold electrochemical sensor was fully characterized using cyclic voltammetry in abiotic and biotic artificial seawater media within a continuous culture flow cell to detect the growth and development of an aerobic Pseudoalteromonas sp. biofilm. Deconvolution of the abiotic and biotic responses enable the constituent extracellular electron transfer and biofilm responses to be resolved. Differentiation of enhanced oxygen reduction kinetics within the aerobic bacterial biofilm is linked to enzyme and redox mediator activities.

Bacterial capsular polysaccharides (CPSs) participate in environmental adaptation in diverse bacteria species. However, the role and regulation of CPS production in marine bacteria have remained largely unexplored. We previously reported that both wrinkled and translucent Pseudoalteromonas lipolytica variants with altered polysaccharide production were generated in pellicle biofilm-associated cells. In this study, we observed that translucent variants were generated at a rate of ∼20% in colony biofilms of P. lipolytica cultured on HSLB agar plates for 12 days. The DNA sequencing results revealed that nearly 90% of these variants had an IS5-like element inserted within the coding or promoter regions of nine genes in the cps operon. In contrast, IS5 insertion into the cps operon was not detected in planktonic cells. Furthermore, we demonstrated that the IS5 insertion event inactivated CPS production, which leads to a translucent colony morphology. The CPS-deficient variants showed an increased ability to form attached biofilms but exhibited reduced resistance to sublethal concentrations of antibiotics. Moreover, deleting the DNA repair gene recA significantly decreased the frequency of occurrence of CPS-deficient variants during biofilm formation. Thus, IS insertion into the cps operon is an important mechanism for the production of genetic variants during biofilm formation of marine bacteria., (© FEMS 2019.)

Biofouling is a phenomenon that describes the fouling organisms attached to man-made surfaces immersed in water over a period of time. It has emerged as a chronic problem to the oceanic industries, especially the shipping and aquaculture fields. The metal-containing coatings that have been used for many years to prevent and destroy biofouling are damaging to the ocean and many organisms. Therefore, this calls for the critical need of natural product-based antifoulants as a substitute for its toxic counterparts. In this study, the antibacterial and antibiofilm activities of the bioactive compounds of Pseudoalteromonas sp. IBRL PD4.8 have been investigated against selected fouling bacteria. The crude extract has shown strong antibacterial activity against five fouling bacteria, with inhibition zones ranging from 9.8 to 13.7 mm and minimal inhibitory concentrations of 0.13 to 8.0 mg/ml. Meanwhile, the antibiofilm study has indicated that the extract has attenuated the initial and pre-formed biofilms of Vibrio alginolyticus FB3 by 45.37 ± 4.88% and 29.85 ± 2.56%, respectively. Moreover, micrographs from light and scanning electron microscope have revealed extensive structural damages on the treated biofilms. The active fraction was fractionated with chromatographic methods and liquid chromatography-mass spectroscopy analyses has further disclosed the presence of a polyunsaturated fatty acid 4,7,10,13-hexadecatetraenoic acid (C 16 H 24 O 2 ). Therefore, this compound was suggested as a potential bioactive compound contributing to the antibacterial property. In conclusion, Pseudoalteromonas sp. IBRL PD4.8 is a promising source as a natural antifouling agent that can suppress the growth of five fouling bacteria and biofilms of V. alginolyticus FB3., Biofouling is a phenomenon that describes the fouling organisms attached to man-made surfaces immersed in water over a period of time. It has emerged as a chronic problem to the oceanic industries, especially the shipping and aquaculture fields. The metal-containing coatings that have been used for many years to prevent and destroy biofouling are damaging to the ocean and many organisms. Therefore, this calls for the critical need of natural product-based antifoulants as a substitute for its toxic counterparts. In this study, the antibacterial and antibiofilm activities of the bioactive compounds of Pseudoalteromonas sp. IBRL PD4.8 have been investigated against selected fouling bacteria. The crude extract has shown strong antibacterial activity against five fouling bacteria, with inhibition zones ranging from 9.8 to 13.7 mm and minimal inhibitory concentrations of 0.13 to 8.0 mg/ml. Meanwhile, the antibiofilm study has indicated that the extract has attenuated the initial and pre-formed biofilms of Vibrio alginolyticus FB3 by 45.37 ± 4.88% and 29.85 ± 2.56%, respectively. Moreover, micrographs from light and scanning electron microscope have revealed extensive structural damages on the treated biofilms. The active fraction was fractionated with chromatographic methods and liquid chromatography-mass spectroscopy analyses has further disclosed the presence of a polyunsaturated fatty acid 4,7,10,13-hexadecatetraenoic acid (C 16 H 24 O 2 ). Therefore, this compound was suggested as a potential bioactive compound contributing to the antibacterial property. In conclusion, Pseudoalteromonas sp. IBRL PD4.8 is a promising source as a natural antifouling agent that can suppress the growth of five fouling bacteria and biofilms of V. alginolyticus FB3.

A number of bacteria adopt various lifestyles such as planktonic free-living or sessile biofilm stages. This enables their survival and development in a wide range of contrasting environments. With the aim of highlighting specific metabolic shifts between these phenotypes and to improve the overall understanding of marine bacterial adhesion, a dual metabolomics/proteomics approach was applied to planktonic and biofilm cultures of the marine bacterium Pseudoalteromonas lipolytica TC8. The liquid chromatography mass spectrometry (LC-MS) based metabolomics study indicated that membrane lipid composition was highly affected by the culture mode: phosphatidylethanolamine (PEs) derivatives were over-produced in sessile cultures while ornithine lipids (OLs) were more specifically synthesized in planktonic samples. In parallel, differences between proteomes revealed that peptidases, oxidases, transcription factors, membrane proteins and the enzymes involved in histidine biosynthesis were over-expressed in biofilms while proteins involved in heme production, nutrient assimilation, cell division and arginine/ornithine biosynthesis were specifically up-regulated in free-living cells.

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti- S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis .

Microbial biofilms are mainly studied due to detrimental effects on human health but they are also well established in industrial biotechnology for the production of chemicals. Moreover, biofilm can be considered as a source of novel drugs since the conditions prevailing within biofilm can allow the production of specific metabolites. Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 when grown in biofilm condition produces an anti-biofilm molecule able to inhibit the biofilm of the opportunistic pathogen Staphylococcus epidermidis. In this paper we set up a P. haloplanktis TAC125 biofilm cultivation methodology in automatic bioreactor. The biofilm cultivation was designated to obtain two goals: (1) the scale up of cell-free supernatant production in an amount necessary for the anti-biofilm molecule/s purification; (2) the recovery of P. haloplanktis TAC125 cells grown in biofilm for physiological studies. We set up a fluidized-bed reactor fermentation in which floating polystyrene supports were homogeneously mixed, exposing an optimal air-liquid interface to let bacterium biofilm formation. The proposed methodology allowed a large-scale production of anti-biofilm molecule and paved the way to study differences between P. haloplanktis TAC125 cells grown in biofilm and in planktonic conditions. In particular, the modifications occurring in the lipopolysaccharide of cells grown in biofilm were investigated.

This study investigated soluble (Sol-EPS), loosely bound (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS) harvested from biofilm and planktonic cultures of the marine bacterium Pseudoalteromonas ulvae TC14. The aim of the characterization (colorimetric methods, FTIR, GC-MS, NMR, HPGPC, and AFM analyses) was to identify new anti-biofilm compounds; activity was assessed using the BioFilm Ring Test®. A step-wise separation of EPS was designed, based on differences in water-solubility and acidity. An acidic fraction was isolated from TB-EPS, which strongly inhibited biofilm formation by marine bacterial strains in a concentration-dependent manner. The main constituents of this fraction were characterized as two glucan-like polysaccharides. An active poly(glutamyl-glutamate) fraction was also recovered from TB-EPS. The distribution of these key EPS components in Sol-EPS, LB-EPS, and TB-EPS was distinct and differed quantitatively in biofilm vs planktonic cultures. The anti-biofilm potential of the fractions emphasizes the putative antifouling role of EPS in the environment.

Staphylococcus epidermidis is recognized as cause of biofilm-associated infections and interest in the development of new approaches for S. epidermidis biofilm treatment has increased. In a previous paper we reported that the supernatant of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 presents an anti-biofilm activity against S. epidermidis and preliminary physico-chemical characterization of the supernatant suggested that this activity is due to a polysaccharide. In this work we further investigated the chemical nature of the anti-biofilm P. haloplanktis TAC125 molecule. The production of the molecule was evaluated in different conditions, and reported data demonstrated that it is produced in all P. haloplanktis TAC125 biofilm growth stages, also in minimal medium and at different temperatures. By using a surface coating assay, the surfactant nature of the anti-biofilm compound was excluded. Moreover, a purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is not due to a polysaccharide molecule but that it is due to small hydrophobic molecules that likely work as signal. The enriched fraction was also used to evaluate the effect on S. epidermidis biofilm formation in dynamic condition by BioFlux system., (© The Author(s) 2015.)

This review is a retrospective of ecological effects of bioactivities produced by biofilms of surface-dwelling marine/intertidal microbes as well as of the industrial and environmental biotechnologies developed exploiting the knowledge of biofilm formation. Some examples of significant interest pertaining to the ecological aspects of biofilm-forming species belonging to the Roseobacter clade include autochthonous bacteria from turbot larvae-rearing units with potential application as a probiotic as well as production of tropodithietic acid and indigoidine. Species of the Pseudoalteromonas genus are important examples of successful surface colonizers through elaboration of the AlpP protein and antimicrobial agents possessing broad-spectrum antagonistic activity against medical and environmental isolates. Further examples of significance comprise antiprotozoan activity of Pseudoalteromonas tunicata elicited by violacein, inhibition of fungal colonization, antifouling activities, inhibition of algal spore germination, and 2-n-pentyl-4-quinolinol production. Nitrous oxide, an important greenhouse gas, emanates from surface-attached microbial activity of marine animals. Marine and intertidal biofilms have been applied in the biotechnological production of violacein, phenylnannolones, and exopolysaccharides from marine and tropical intertidal environments. More examples of importance encompass production of protease, cellulase, and xylanase, melanin, and riboflavin. Antifouling activity of Bacillus sp. and application of anammox bacterial biofilms in bioremediation are described. Marine biofilms have been used as anodes and cathodes in microbial fuel cells. Some of the reaction vessels for biofilm cultivation reviewed are roller bottle, rotating disc bioreactor, polymethylmethacrylate conico-cylindrical flask, fixed bed reactor, artificial microbial mats, packed-bed bioreactors, and the Tanaka photobioreactor.

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer-membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer-membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB-dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT-PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacterial biofilms occur on all submerged structures in marine environments. The authors previously reported that the marine bacterium Pseudoalteromonas sp. 3J6 secretes antibiofilm activity. Here, it was discovered that another Pseudoalteromonas sp. strain, D41, inhibited the development of strain 3J6 in mixed biofilms. Confocal laser scanning microscope observations revealed that the culture supernatant of strain D41 impaired biofilm formation of strain 3J6 and another marine bacterium. A microtiter plate assay of the antibiofilm activity was set up and validated with culture supernatants of Pseudoalteromonas sp. 3J6. This assay was used to determine the spectra of action of strains D41 and 3J6. Each culture supernatant impaired the biofilm development of 13 marine bacteria out of 18. However, differences in the spectra of action and the physical behaviours of the antibiofilm molecules suggest that the latter are not identical. They nevertheless share the originality of being devoid of antibacterial activity against planktonic bacteria.

The red-pigmented marine bacterium Pseudoalteromonas sp. sf57 forms a biofilm that deters larval settlement of the tube-building polychaete Hydroides elegans. To investigate the correlation between pigmentation and larval settlement deterrence, mutants of sf57 with deficient or altered pigmentation were generated by transposon mutagenesis. Five groups of pigmented mutants were obtained, viz. white, yellow, pink, dark red, and white-to-red. The white mutant WM1, which exhibited a substantial increase in bacterial density in the biofilm, became inductive to larval settlement. The other mutants that showed a lesser increase in bacterial density in their biofilms either retained their deterrence or induced higher larval settlement rates, but did not become inductive strains. Analysis of the disrupted genes in these mutants suggests that the type II secretion pathway, the LysR transcriptional regulator, NAD(P)-binding proteins, exonuclease, pyruvate metabolism, flagella assembly, and cell membrane processes may play a role in the regulation of pigmentation in sf57.

Aim: The major objective of the present study was the partial characterization of the exopolysaccharides (EPS) produced by a marine biofilm-forming bacterium Pseudoalteromonas ruthenica under shake culture conditions., Methods and Results: EPS-producing bacterial cultures were isolated from the sea water collected from the vicinity of coastal electric power station. Zobell marine broth medium was used for growth of the cultures and the EPS produced was quantified using phenol sulfuric acid method. Chemical characterization of the EPS was carried out using Fourier transform infrared spectroscopy (FTIR), and capillary gas chromatography (GC). Further, viscosity and rheological properties of the purified EPS were studied. The FTIR spectrum revealed prominent peaks of various groups of OH and CH(3) bending. GC analysis showed the presence of eight individual sugars. Rheological studies of the aqueous EPS showed good shearing property., Conclusions: Pseudoalteromonas ruthenica isolated from marine environment produced copious amount of EPS under shake culture conditions. GC analysis of the EPS revealed the presence of eight individual sugars and the EPS had good shearing property., Significance and Impact of the Study: The EPS produced by P. ruthenica is pseudoplastic in nature and is stable at higher pH levels. These properties suggest that the EPS may have potential applications in the oil, textiles and food industries.

Biofilms of bacteria, frequently in association with algae, protozoa and fungi, are found on all submerged structures in the marine environment. Although it is likely that for the majority of organisms a biofilmed surface is not a pre-requisite for settlement, in practice, colonization by spores and larvae of fouling organisms almost always takes place via a biofilmed surface. Therefore, the properties of the latter may be expected to influence colonization, positively or negatively. Biofilms are responsible for a range of surface-associated and diffusible signals, which may moderate the settling behaviour of cells, spores and larvae. However, there is no consensus view regarding either cause and effect or the mechanism(s) by which biofilms moderate settlement. Studies with mixed biofilms, especially field experiments, are difficult to interpret because of the conflicting signals produced by different members of the biofilm community as well as their spatial organisation. Molecular techniques highlight the deficiencies of culture methods in identifying biofilm bacteria; hence, the strains with the most impact on settlement of spores and larvae may not yet have been isolated and cultured. Furthermore, secondary products isolated from cultured organisms may not reflect the situation that pertains in nature. The evidence that bacterial quorum sensing signal molecules stimulate settlement of spores of the green macroalga, Ulva, is discussed in some detail. New molecular and analytical tools should provide the opportunity to improve our fundamental understanding of the interactions between fouling organisms and biofilms, which in turn may inform novel strategies to control biofouling.

Pseudoalteromonas tunicata is a biofilm-forming marine bacterium that is often found in association with the surface of eukaryotic organisms. It produces a range of extracellular inhibitory compounds, including an antibacterial protein (AlpP) thought to be beneficial for P. tunicata during competition for space and nutrients on surfaces. As part of our studies on the interactions between P. tunicata and the epiphytic bacterial community on the marine plant Ulva lactuca, we investigated the hypothesis that P. tunicata is a superior competitor compared with other bacteria isolated from the plant. A number of U. lactuca bacterial isolates were (i) identified by 16S rRNA gene sequencing, (ii) characterized for the production of or sensitivity to extracellular antibacterial proteins, and (iii) labeled with a fluorescent color tag (either the red fluorescent protein DsRed or green fluorescent protein). We then grew single- and mixed-species bacterial biofilms containing P. tunicata in glass flow cell reactors. In pure culture, all the marine isolates formed biofilms containing microcolony structures within 72 h. However, in mixed-species biofilms, P. tunicata removed the competing strain unless its competitor was relatively insensitive to AlpP (Pseudoalteromonas gracilis) or produced strong inhibitory activity against P. tunicata (Roseobacter gallaeciensis). Moreover, biofilm studies conducted with an AlpP- mutant of P. tunicata indicated that the mutant was less competitive when it was introduced into preestablished biofilms, suggesting that AlpP has a role during competitive biofilm formation. When single-species biofilms were allowed to form microcolonies before the introduction of a competitor, these microcolonies coexisted with P. tunicata for extended periods of time before they were removed. Two marine bacteria (R. gallaeciensis and P. tunicata) were superior competitors in this study. Our data suggest that this dominance can be attributed to the ability of these organisms to rapidly form microcolonies and their ability to produce extracellular antibacterial compounds.