Marine Microbial Biofilms Research Articles

Quorum Sensing and Antimicrobial Production Orchestrate Biofilm Dynamics in Multispecies Bacterial Communities.

Microbial interactions are often mediated by diffusible small molecules, including secondary metabolites, that play roles in cell-to-cell signaling and inhibition of competitors. Biofilms are often "hot spots" for high concentrations of bacteria and their secondary metabolites, which make them ideal systems for the study of small-molecule contributions to microbial interactions. Here, we use a five-member synthetic community consisting of Roseobacteraceae representatives to investigate the role of secondary metabolites on microbial biofilm dynamics. One synthetic community member, Rhodobacterales strain Y4I, possesses two acylated homoserine lactone (AHL)-based cell-to-cell signaling systems (pgaRI and phaRI) as well as a nonribosomal peptide synthase gene (igi) cluster that encodes the antimicrobial indigoidine. Through serial substitution of Y4I with mutants deficient in single signaling molecule pathways, the contribution of these small-molecule systems could be assessed. As secondary metabolite production is dependent upon central metabolites, the influence of growth substrate (i.e., complex medium versus defined medium with a single carbon substrate) on these dynamics was also considered. Depending on the Y4I mutant genotype included, community dynamics ranged from competitive to cooperative. The observed interactions were mostly competitive in nature. However, the community harboring a Y4I variant that was both impaired in quorum sensing (QS) pathways and unable to produce indigoidine (pgaR variant) shifted toward more cooperative interactions over time. These cooperative interactions were enhanced in the defined growth medium. The results presented provide a framework for deciphering complex, small-molecule-mediated interactions that have broad application to microbial biology. IMPORTANCE Microbial biofilms play critical roles in marine ecosystems and are hot spots for microbial interactions that play a role in the development and function of these communities. Roseobacteraceae are an abundant and active family of marine heterotrophic bacteria forming close associations with phytoplankton and carrying out key transformations in biogeochemical cycles. Group members are aggressive primary colonizers of surfaces, where they set the stage for the development of multispecies biofilm communities. Few studies have examined the impact of secondary metabolites, such as cell-to-cell signaling and antimicrobial production, on marine microbial biofilm community structure. Here, we assessed the impact of secondary metabolites on microbial interactions using a synthetic, five-member Roseobacteraceae community by measuring species composition and biomass production during biofilm growth. We present evidence that secondary metabolites influence social behaviors within these multispecies microbial biofilms, thereby improving understanding of bacterial secondary metabolite production influence on social behaviors within marine microbial biofilm communities.

Phylogenetic characterization of marine microbial biofilms associated with mammal bones in temperate and polar areas

Mammal bones sustain rich chemoautotrophic microbial communities that are consumed by a range of marine invertebrates, with bacteria playing a fundamental role making the organic matter retained in the bones available to other organisms. Our major aim here is to characterize the phylogenetic diversity of bacteria associated with Mediterranean shallow-water bones (whale, pig, and cow) examined at two different times after colonization, and compare it to bacterial communities developing on whale bones in the Southern Ocean. We sequenced 16S amplicons for approximately 50 clones from each of the 5 bone samples studied here, resulting in 215 OTUs. Our preliminary microbial community analysis resulted into two groups. Cluster 1 with cow and whale bones from the Mediterranean (3 months) and whale bones from the Southern Ocean (1 year), characterized by abundant Epsilon-proteobacteria, usually the first colonizers in anaerobic environments. Cluster 2 with Mediterranean cow (9 months) and pig bones (12 months), characterized by abundant Thiotrichales. Among the Thiotrichales, two different species of Beggiatoa spp. appeared in two different bones in the Mediterranean samples, indicating that Beggiatoa spp. can occur in the same area. Our work confirms that microbial mats associated with mammal bones in the shallow waters of the Mediterranean and the Southern Ocean are highly diverse with a predominance of Gamma- and Epsilon-proteobacteria.

Exposure to Crude Oil and Chemical Dispersant May Impact Marine Microbial Biofilm Composition and Steel Corrosion

The release of hydrocarbons and chemical dispersant in marine environments may disrupt benthic ecosystems, including artificial reefs, formed by historic steel shipwrecks, and their associated organisms. Experiments were performed to determine the impacts of crude oil, dispersed crude oil, and dispersant on the community structure and function of microorganisms in seawater (SW) and biofilms formed on carbon steel, a common ship hull construction material. Steel corrosion was also monitored to illustrate how oil spills may impact preservation of steel shipwrecks. Microcosms were filled with seawater (SW) and incubated at 4oC. Carbon steel disks (CSDs) were placed in each tank, and tanks were amended with crude oil and/or dispersant or no treatment. SW and CSD biofilms were sampled biweekly for genetic analysis using Illumina sequencing of 16S ribosomal RNA gene amplicons. Predicted and sequenced bacterial metagenomes were analyzed to examine impacts of oil and dispersant on metabolic function. Gammaproteobacteria, Alphaproteobacteria, and Flavobacteriia dominated SW and biofilms. Bacterial community structure differed significantly between treatments for SW and biofilms. OTUs affiliated with known (Pseudomonas) and potential (Marinomonas) hydrocarbon-degraders were roughly twice as abundant in biofilms treated with oil and dispersed oil, and steel corrosion of CSDs in these treatments was higher compared to control and dispersant treatments. OTUs affiliated with the Rhodobacteraceae family (biofilm formers and potential oil degraders) were less abundant in the dispersant treatment compared to other treatments in biofilm and SW samples, but OTUs affiliated with the Pseudoalteromonas genus (biofilm formers and proposed hydrocarbon degraders) were more abundant in dispersant-treated biofilms. Overall, functional gene analyses revealed a decrease in genes (predicted using PICRUSt and observed in sequenced metagenomes) associated with hydrocarbon degradation in dispersant-treated biofilms. This study indicates that exposure to oil and dispersant could disrupt the composition and metabolic function of biofilms colonizing metal hulls, as well as corrosion processes, potentially compromising shipwrecks as ecological and historical resources.

Recent Advances in the Study of Marine Microbial Biofilm: From the Involvement of Quorum Sensing in Its Production up to Biotechnological Application of the Polysaccharide Fractions

The present review will explore the most relevant findings on marine microbial biofilm, with particular attention towards its polysaccharide fraction, namely exopolysaccharide (EPS). EPSs of microbial origin are ubiquitous in nature, possess unique properties and can be isolated from the bacteria living in a variety of habitats, including fresh water or marine environments, extreme environments or different soil ecosystems. These biopolymers have many application in the field of biotechnology. Several studies showed that the biofilm formation is closely related to quorum sensing (QS) systems, which is a mechanism relying on the production of small molecules defined as “autoinducers” that bacteria release in the surrounding environment where they accumulate. In this review, the involvement of microbial chemical communication, by QS mechanism, in the formation of marine biofilm will also be discussed.

Marine phototrophic consortia transfer electrons to electrodes in response to reductive stress.

This work studies how extracellular electron transfer (EET) from cyanobacteria-dominated marine microbial biofilms to solid electrodes is affected by the availability of inorganic carbon (Ci). The EET was recorded chronoamperometrically in the form of electrical current by a potentiostat in two identical photo-electrochemical cells using carbon electrodes poised at a potential of +0.6V versus standard hydrogen electrode under 12/12h illumination/dark cycles. The Ci was supplied by the addition of NaHCO3 to the medium and/or by sparging CO2 gas. At high Ci conditions, EET from the microbial biofilm to the electrodes was observed only during the dark phase, indicating the occurrence of a form of night-time respiration that can use insoluble electrodes as the terminal electron acceptor. At low or no Ci conditions, however, EET also occurred during illumination suggesting that, in the absence of their natural electron acceptor, some cyanobacteria are able to utilise solid electrodes as an electron sink. This may be a natural survival mechanism for cyanobacteria to maintain redox balance in environments with limiting CO2 and/or high light intensity.

Erratum: Sponge larval settlement cues: the role of microbial biofilms in a warming ocean

Microbial biofilms play important roles in initiating settlement of marine invertebrate larvae. Given the importance of habitat selection by the motile larval phase, understanding settlement choices is critical if we are to successfully predict the population dynamics of sessile adults. Marine microbial biofilms show remarkable variability in community composition, often mediated by environmental conditions and biofilm age. To determine if biofilm communities were influenced by the time allowed to establish (age) and/or seawater temperature, we manipulated experimental surfaces to firstly determine biofilm community composition and secondly test larval settlement responses for the abundant coral reef sponge Rhopaloeides odorabile. Microbial profiling of biofilms revealed different communities according to both age and temperature. Biofilm community composition, as a result of both elevated seawater temperature and biofilm age, contributed to settlement for sponge larvae with markedly higher numbers of larvae settling to biofilms developed over longer periods (10 d) and at temperatures 2–6°C above ambient.

Complete Nucleotide Sequence and Analysis of Two Conjugative Broad Host Range Plasmids from a Marine Microbial Biofilm

The complete nucleotide sequence of plasmids pMCBF1 and pMCBF6 was determined and analyzed. pMCBF1 and pMCBF6 form a novel clade within the IncP-1 plasmid family designated IncP-1 ς. The plasmids were exogenously isolated earlier from a marine biofilm. pMCBF1 (62 689 base pairs; bp) and pMCBF6 (66 729 bp) have identical backbones, but differ in their mercury resistance transposons. pMCBF1 carries Tn5053 and pMCBF6 carries Tn5058. Both are flanked by 5 bp direct repeats, typical of replicative transposition. Both insertions are in the vicinity of a resolvase gene in the backbone, supporting the idea that both transposons are “res-site hunters” that preferably insert close to and use external resolvase functions. The similarity of the backbones indicates recent insertion of the two transposons and the ongoing dynamics of plasmid evolution in marine biofilms. Both plasmids also carry the insertion sequence ISPst1, albeit without flanking repeats. ISPs1is located in an unusual site within the control region of the plasmid. In contrast to most known IncP-1 plasmids the pMCBF1/pMCBF6 backbone has no insert between the replication initiation gene (trfA) and the vegetative replication origin (oriV). One pMCBF1/pMCBF6 block of about 2.5 kilo bases (kb) has no similarity with known sequences in the databases. Furthermore, insertion of three genes with similarity to the multidrug efflux pump operon mexEF and a gene from the NodT family of the tripartite multi-drug resistance-nodulation-division (RND) system in Pseudomonas aeruginosa was found. They do not seem to confer antibiotic resistance to the hosts of pMCBF1/pMCBF6, but the presence of RND on promiscuous plasmids may have serious implications for the spread of antibiotic multi-resistance.

Micro-fabricated polydimethyl siloxane (PDMS) surfaces regulate the development of marine microbial biofilm communities

This study explored an antifouling (AF) concept based on deployment of microfabricated polydimethyl siloxane (PDMS) surfaces with 1–10 μm periodicity corrugated topographies in temperate marine waters. The effect of the surfaces on the development of microbial biofilms over 28 days and during different seasons, including both summer and winter, was examined using confocal laser scanning microscopy (CLSM) as well as terminal restriction fragment (T-RF) analysis for phylogenetic fingerprinting. The microscale topography significantly impacted biofilm development by altering the attachment pattern and reducing microcolony formation on the 1, 2 and 4 μm PDMS surfaces. Also, field deployments over 28 days showed a significant reduction in biovolume on the 4 and 10 μm PDMS surfaces despite altered environmental conditions. The microfabricated PDMS surfaces further significantly impacted on the community composition of the biofilms, as revealed by changes in T-RF profiles, at different stages of development. Moreover, altered biofilm resistance was demonstrated by exposing pre-established biofilms on 10 μm micro-fabricated surfaces to enhanced flagellate predation by a heterotrophic protist, Rhynchomonas nasuta. Pronounced changes in the overall marine microbial biofilm development as well as community composition warrant exploring substratum modification for marine AF applications.

Sponge larval settlement cues: the role of microbial biofilms in a warming ocean

Microbial biofilms play important roles in initiating settlement of marine invertebrate larvae. Given the importance of habitat selection by the motile larval phase, understanding settlement choices is critical if we are to successfully predict the population dynamics of sessile adults. Marine microbial biofilms show remarkable variability in community composition, often mediated by environmental conditions and biofilm age. To determine if biofilm communities were influenced by the time allowed to establish (age) and/or seawater temperature, we manipulated experimental surfaces to firstly determine biofilm community composition and secondly test larval settlement responses for the abundant coral reef sponge Rhopaloeides odorabile. Microbial profiling of biofilms revealed different communities according to both age and temperature. Biofilm community composition, as a result of both elevated seawater temperature and biofilm age, contributed to settlement for sponge larvae with markedly higher numbers of larvae settling to biofilms developed over longer periods (10 d) and at temperatures 2–6°C above ambient.

Effects of marine microbial biofilms in the steel carbon SAE 1008

Effects of marine microbial biofilms in the steel carbon SAE 1008

Effects of marine microbial biofilms on the biocide release rate from antifouling paints—A model-based analysis

The antifouling (AF) paint model of Kiil et al. [S. Kiil, C.E. Weinell, M.S. Pedersen, K. Dam-Johansen, Analysis of self-polishing antifouling paints using rotary experiments and mathematical modelling, Ind. Eng. Chem. Res. 40 (2001) 3906–3920] and the simplified biofilm growth model of Gujer and Wanner [W. Gujer, O. Wanner, Modeling mixed population biofilms, in: W.G. Characklis, K.C. Marshall (Eds.), Biofilms, Wiley–Interscience, New York, 1990] are used to provide a reaction engineering-based insight to the effects of marine microbial slimes on biocide leaching and, to a minor extent, polishing behaviour of AF paints. It is concluded that the perturbation of the local sea water conditions (e.g. pH), as a consequence of the metabolic activity of the biofilm should not affect the net biocide leaching and binder reaction rates significantly. This results from the thin and poorly active biofilms which presumably grow onto the highly effective modern AF paints. According to simulations, the experimental decrease in the biocide leaching rate caused by biofilm growth must be mainly attributed to adsorption of the biocide by the exopolymeric substances secreted by the microorganisms. The effects of biofilms on the leaching of any generic active compound (e.g. natural antifoulants) are discussed in relation to their potential release mechanisms. The largest influence of biofilms is predicted for those active compounds that are released by a diffusion-controlled mechanism (typically tin-free algaecides).

Site‐specific variation in Antarctic marine biofilms established on artificial surfaces

The community structure and composition of marine microbial biofilms established on glass surfaces was investigated across three differentially contaminated Antarctic sites within McMurdo Sound. Diverse microbial communities were revealed at all sites using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques. Sequencing of excised DGGE bands demonstrated close affiliation with known psychrophiles or undescribed bacteria also recovered from the Antarctic environment. The majority of bacterial sequences were affiliated to the Gammaproteobacteria, Cytophaga/Flavobacteria of Bacteroidetes (CFB), Verrucomicrobia and Planctomycetales. Principal components analysis of quantitative FISH data revealed distinct differences in community composition between sites. Each of the sites were dominated by different bacterial groups: Alphaproteobacteria, Gammaproteobacteria and CFB at the least impacted site, Cape Armitage; green sulfur and sulfate reducing bacteria near the semi-impacted Scott Base and Planctomycetales and sulfate reducing bacteria near the highly impacted McMurdo Station. The highest abundance of archaea was detected near Scott Base (2.5% of total bacteria). Multivariate analyses (non-metric multidimensional scaling and analysis of similarities) of DGGE patterns revealed greater variability in community composition between sites than within sites. This is the first investigation of Antarctic biofilm structure and FISH results suggest that anthropogenic impacts may influence the complex composition of microbial communities.

Presence and effects of marine microbial biofilms on biocide-based antifouling paints

Marine microorganisms are capable of successfully colonizing toxic surfaces through the formation of biofilm structures. In this article, most of the literature reporting the presence of marine biofilms on chemically-active antifouling paints is briefly reviewed. Of special concern is the influence of the dense extracellular polymeric substances (EPS) matrix on the release rate of the compounds involved in antifouling paint performance (i.e. active compounds and controlled-release binder molecules). A deeper understanding of these phenomena is of interest for both environmental legislators and paint formulators.

Application of EIS with Au–Hg microelectrode in determining electron transfer mechanisms

The objective of this study was to investigate the application of microelectrodes with electrochemical impedance spectroscopy (EIS) for distinguishing between one- and two-electron transfer electrochemical reactions occurring within marine microbial biofilms. In our study, both gold and gold–mercury amalgam microelectrodes with tip diameters of 100 μm were used. EIS measurements were performed at the half-wave potentials of the chemicals of interest in 0.5 M NaCl aqueous solutions containing 100 μM each of FeCl 3, [Fe(Cat) 2] −, [Fe(Cat) 3] 3−, [Fe(4NCat) 3] 3−, MnCl 2, FeCl 2 and mixed solutions with two of those chemicals in NaCl solution or seawater. The results showed that there was a linear relationship between the impedance Z re, Z im and angular frequency ω −1/2 obtained at phase angle of 45° for both one- and two-electron transfer reactions. One-electron transfer reactions could then be distinguished from two-electron transfer reactions by the slopes.

Viability of a marine microbial biofilm

The formation of a microbial biofilm on glass surfaces arranged in lamellar piles parallel with circulating sea water (3 cm·sec−1) was studied. The increase in dry weight, protein content, nucleotide content (ATP, ADP), and diatoms was followed over a period of 62 days. Dry weight and protein were estimates of the total biofilm development, whereas the nucleotide measurements revealed the viability of the biofilm and reflected the dynamics in the community structure.