Effects of zinc on microalgal biofilms in intertidal and subtidal habitats

Microalgal biofilms are sensitive to environmental conditions. Impacts of contaminants on assemblages of marine biofilm are often investigated in laboratories or in mesocosms. Such experiments are rarely representative of the effects of contaminants on biofilms under natural conditions. Studies in field situations, with enough power to detect impacts, are necessary to develop a better understanding of the effects of contaminants on ecological processes. Metals are a common contaminant of marine systems and can cause disturbances to assemblages. Using a new technique to experimentally deliver contaminants to microalgal assemblages, hypotheses were tested regarding the effects of zinc on microalgal biofilms growing on settlement panels in subtidal and intertidal habitats. PAM fluorometry was used to assess the amount and physiological state of biofilms on panels. Control panels deployed for 1 month in each habitat had significantly greater amounts of biofilm than those exposed to zinc. After deployment for 3 months, the results varied with location. The observed effects on the biofilm did not, however, cause significant changes in the macro-invertebrate assemblages that developed on the panels.     

Nutrient dynamics and successional changes in a lentic freshwater biofilm

SUMMARY 1. Colonisation, species composition, succession of microalgae and nutrient dynamics in biofilms grown under light and dark conditions were examined during the initial phases of biofilm development in a lentic freshwater environment.
2. Biofilms were developed on inert (perspex) panels under natural illuminated and experimental dark conditions and the panels were retrieved for analysis after different incubation periods. Analysed parameters included biofilm thickness, algal density, biomass, chlorophyll a , species composition, total bacterial density and nutrients such as nitrite, nitrate, phosphate and silicate.
3. Biofilm thickness, algal density, biomass, chlorophyll a and species richness were significantly higher in light-grown biofilms, compared with dark-grown biofilms. The light-grown biofilms showed a three-phased succession pattern, with an initial domination of Chlorophyceae followed by diatoms (Bacillariophyceae) and finally by cyanobacteria. Dark-grown biofilms were mostly dominated by diatoms.
4. Nutrients were invariably more concentrated in biofilms than in ambient water. Nutrient concentrations were generally higher in dark-grown biofilms except in the case of phosphate, which was more concentrated in light-grown biofilms. Significant correlations between nutrients and biofilm parameters were observed only in light-grown biofilms.
5. The N : P ratio in the biofilm matrix decreased sharply in the initial 4 days of biofilm growth; ensuing N-limitation status seemed to influence biofilm community structure. The N : P ratios showed significant positive correlations with the chlorophycean fraction in both light and dark-grown biofilms, and low N : P ratio in the older biofilms favoured cyanobacteria. Our data indicate that nutrient chemistry of biofilm matrix shapes community structure in microalgal biofilms.     

Impacts of hydrodynamic conditions and microscale surface roughness on the critical shear stress to develop and thickness of early-stage Pseudomonas putida biofilms

Biofilms can increase pathogenic contamination of drinking water, cause biofilm-related diseases, alter the sediment erosion rate, and degrade contaminants in wastewater. Compared with mature biofilms, biofilms in the early-stage have been shown to be more susceptible to antimicrobials and easier to remove. Mechanistic understanding of physical factors controlling early-stage biofilm growth is critical to predict and control biofilm development, yet such understanding is currently incomplete. Here, we reveal the impacts of hydrodynamic conditions and microscale surface roughness on the development of early-stage Pseudomonas putida biofilm through a combination of microfluidic experiments, numerical simulations, and fluid mechanics theories. We demonstrate that early-stage biofilm growth is suppressed under high flow conditions and that the local velocity for early-stage P. putida biofilms (growth time < 14 h) to develop is about 50 μm/s, which is similar to P. putida's swimming speed. We further illustrate that microscale surface roughness promotes the growth of early-stage biofilms by increasing the area of the low-flow region. Furthermore, we show that the critical average shear stress, above which early-stage biofilms cease to form, is 0.9 Pa for rough surfaces, three times as large as the value for flat or smooth surfaces (0.3 Pa). The important control of flow conditions and microscale surface roughness on early-stage biofilm development, characterized in this study, will facilitate future predictions and managements of early-stage P. putida biofilm development on the surfaces of drinking water pipelines, bioreactors, and sediments in aquatic environments.     

Marine Biofilms as Mediators of Colonization by Marine Macroorganisms: Implications for Antifouling and Aquaculture

In the marine environment, biofilms on submerged surfaces can promote or discourage the settlement of invertebrate larvae and macroalgal spores. The settlement-mediating effects of biofilms are believed to involve a variety of biofilm attributes including surface chemistry, micro-topography, and a wide range of microbial products from small-molecule metabolites to high-molecular weight extracellular polymers. The settled organisms in turn can modify microbial species composition of biofilms and thus change the biofilm properties and dynamics. A better understanding of biofilm dynamics and chemical signals released and/or stored by biofilms will facilitate the development of antifouling and mariculture technologies. This review provides a brief account of 1) existing knowledge of marine biofilms that are relevant to settlement mediation, 2) biotechnological application of biofilms with respect to developing non-toxic antifouling technologies and improving the operation of aquaculture facilities, and 3) challenges and future directions for advancing our understanding of settlement-mediating functions of biofilms and for applying this knowledge to real-life situations.     

Development of associations between microalgae and denitrifying bacteria in streams of contrasting anthropogenic influence

We compared the development of microalgal and bacterial-denitrifier communities within biofilms over 28 days in a restored-prairie stream (RP) and a stream receiving treated wastewater effluent (DER). Inorganic nutrient concentrations were an order of magnitude greater in DER, and stream waters differed in the quality of dissolved organics (characterized via pyrolysis-GC/MS). Biofilm biomass and the densities of algae and bacteria increased over time in both systems; however, algal and denitrifier community composition and the patterns of development differed between systems. Specifically, algal and denitrifier taxonomic composition stabilized more quickly in DER than RP, whereas the rates of algal and denitrifier succession were more closely coupled in RP than DER. We hypothesize that, under unenriched conditions, successional changes in algal assemblages influence bacterial denitrifiers due to their dependence on algal exudates, while under enriched conditions, this relationship is decoupled. Between-system differences in organic signatures supported this, as RP biofilms contained more labile, aliphatic compounds than DER. In addition, potential denitrification rates (DNP) were negatively correlated with the percentage of aromatic compounds within the biofilm organic signatures, suggesting a significant relationship between algal exudate composition and denitrification. These results are significant because anthropogenic factors that affect biofilm community composition may alter their capacity to perform critical ecosystem services.     

Transported biofilms and their influence on subsequent macrofouling colonization

Biofilm organisms such as diatoms are potential regulators of global macrofouling dispersal because they ubiquitously colonize submerged surfaces, resist antifouling efforts and frequently alter larval recruitment. Although ships continually deliver biofilms to foreign ports, it is unclear how transport shapes biofilm microbial structure and subsequent macrofouling colonization. This study demonstrates that different ship hull coatings and transport methods change diatom assemblage composition in transported coastal marine biofilms. Assemblages carried on the hull experienced significant cell losses and changes in composition through hydrodynamic stress, whereas those that underwent sheltered transport, even through freshwater, were largely unaltered. Coatings and their associated biofilms shaped distinct macrofouling communities and affected recruitment for one third of all species, while biofilms from different transport treatments had little effect on macrofouling colonization. These results demonstrate that transport conditions can shape diatom assemblages in biofilms carried by ships, but the properties of the underlying coatings are mainly responsible for subsequent macrofouling. The methods by which organisms colonize and are transferred by ships have implications for their distribution, establishment and invasion success.     

Copper affects biofilm inductiveness to larval settlement of the serpulid polychaete Hydroides elegans (Haswell)

Copper (Cu) contamination is a potential threat to the marine environment due to the use of Cu-based antifouling paints. Cu stress on larval settlement of the polychaete Hydroides elegans was investigated, and this was linked to Cu stress on biofilms and on the biofilm development process. The inductiveness of young biofilms was more easily altered by Cu stress than that of old biofilms, indicating the relative vulnerability of young biofilms. This might result from changes in bacterial survival, the bacterial community composition and the chemical profiles of young biofilms. Cu also affected biofilm development and the chemical high performance liquid chromatograph fingerprint profile. The results indicate that Cu affected larval settlement mainly through its effect on the process of biofilm development in the marine environment, and the chemical profile was crucial to biofilm inductiveness. It is strongly recommended that the effects of environmentally toxic substances on biofilms are evaluated in ecotoxicity bioassays using larval settlement of invertebrates as the end point.     

Biofilm shows spatially stratified metabolic responses to contaminant exposure

Biofilms are core to a range of biological processes, including the bioremediation of environmental contaminants. Within a biofilm population, cells with diverse genotypes and phenotypes coexist, suggesting that distinct metabolic pathways may be expressed based on the local environmental conditions in a biofilm. However, metabolic responses to local environmental conditions in a metabolically active biofilm interacting with environmental contaminants have never been quantitatively elucidated. In this study, we monitored the spatiotemporal metabolic responses of metabolically active Shewanella oneidensis MR‐1 biofilms to U(VI) (uranyl, UO₂ ²⁺) and Cr(VI) (chromate, CrO₄ ^2?) using non‐invasive nuclear magnetic resonance imaging (MRI) and spectroscopy (MRS) approaches to obtain insights into adaptation in biofilms during biofilm‐contaminant interactions. While overall biomass distribution was not significantly altered upon exposure to U(VI) or Cr(VI), MRI and spatial mapping of the diffusion revealed localized changes in the water diffusion coefficients in the biofilms, suggesting significant contaminant‐induced changes in structural or hydrodynamic properties during bioremediation. Finally, we quantitatively demonstrated that the metabolic responses of biofilms to contaminant exposure are spatially stratified, implying that adaptation in biofilms is custom‐developed based on local microenvironments.     

A methodological review on the characterization of microalgal biofilm and its extracellular polymeric substances

Biofilm secreted by microalgae are extracellular polymeric substances (EPSs) composed mainly of polysaccharides, proteins, nucleic acids and lipids. These EPSs immobilize the cells and stabilize biofilm, mediating adhesion towards solid surfaces. The EPSs valorization through industrial exploitations and scientific works is becoming more popular, but the bottleneck of such studies is the lack of consensus among researchers on the selection of detection techniques to be used, especially for novice researchers. It is a daunting task for any inexperienced researcher when they fail to identify the right tools needed for microalgal biofilm studies. In this review, a well-refined analysis protocol about microalgal biofilm and EPSs were prepared including its extraction and characterization. Pros and cons of various detection techniques were addressed and cutting-edge methods to study biofilm EPSs were highlighted. Future perspectives were also presented at the end of this review to bridge research gaps in studying biofilm adhesion via EPSs production. Ultimately, this review aims to assist novice researchers in making the right choices in their research studies on microalgal biofilms in accordance to the available technologies and needs.     

Species and material considerations in the formation and development of microalgal biofilms

The development of microalgal biofilms has received very limited study despite its relevance in the design of photobioreactors where film growth may be advantageous for biomass separation or disadvantageous in fouling surfaces. Here, the effects of species selection, species control, and substrate properties on biofilms of Scenedesmus obliquus and Chlorella vulgaris were investigated. Experiments were conducted in batch culture and in continuous culture modes in a flow cell. Cell growth was monitored using confocal laser scanning microscopy and gravimetrically. Species selection and species control had significant effects on biofilm development. On non-sterile wastewater, C. vulgaris shifted from primarily planktonic (23.7% attachment) to primarily sessile (79.8% attachment) growth. The biofilms that developed in non-sterile conditions were thicker (52 ± 19 μm) than those grown in sterile conditions (7 ± 6 μm). By contrast, S. obliquus attained similar thicknesses (54 ± 31 and 53 ± 38 μm) in both sterile and non-sterile conditions. Neither species was able to dominate a non-sterile biofilm. The effect of substrate surface properties was minimal. Both species grew films of similar thickness (∼30 μm for S. obliquus, <10 μm for C. vulgaris) on materials ranging from hydrophilic (glass) to hydrophobic (polytetrafluoroethylene). Surface roughness created by micropatterning the surface with 10 μm grooves did not translate into long-term increases in biofilm thickness. The results indicate that species selection and control are more important than surface properties in the development of microalgal biofilms.     

An image-based 384-well high-throughput screening method for the discovery of biofilm inhibitors in Vibrio cholerae

Bacterial biofilms are assemblages of bacterial cells and extracellular matrix that result in the creation of surface-associated macrocolony formation. Most bacteria are capable of forming biofilms under suitable conditions. Biofilm formation by pathogenic bacteria on medical implant devices has been linked to implant rejection in up to 10% of cases, due to biofilm-related secondary infections. In addition, biofilm formation has been implicated in both bacterial persistence and antibiotic resistance. In this study, a method has been developed for the discovery of small molecule inhibitors of biofilm formation in Vibrio cholerae, through the use of high-throughput epifluorescence microscopy imaging. Adaptation of a strategy for the growth of bacterial biofilms in wellplates, and the subsequent quantification of biofilm coverage within these wells, provides the first example of an image-based 384-well format system for the evaluation of biofilm inhibition in V. cholerae. Application of this method to the high-throughput screening of small molecule libraries has lead to the discovery of 29 biofilm lead structures, many of which eliminate biofilm formation without altering bacterial cell viability.     

Acyl-homoserine lactones modulate the settlement rate of zoospores of the marine alga Ulva intestinalis via a novel chemokinetic mechanism

Bacteria utilize quorum sensing to regulate the expression of cell density-dependant phenotypes such as biofilm formation and virulence. Zoospores of the marine alga Ulva intestinalis exploit the acyl-homoserine lactone (AHL) quorum sensing system to identify bacterial biofilms for preferential settlement. Here, we demonstrate that AHLs act as strong chemoattractants for Ulva zoospores. Chemoattraction does not involve a chemotactic orientation towards the AHL source. Instead, it occurs through a chemokinesis in which zoospore swimming speed is rapidly decreased in the presence of AHLs. The chemoresponse to AHLs was dependant on the nature of the acyl side chain, with N-(3-oxododecanoyl)-homoserine lactone (30-C12-HSL) being the most effective signal molecule. Mean zoospore swimming speed decreased more rapidly over wild-type biofilms of the marine bacteria Vibrio anguillarum relative to biofilms of the vanM mutant, in which AHL synthesis is disrupted. These data implicate a role for AHL-mediated chemokinesis in the location and preferential settlement of Ulva zoospores on marine bacterial assemblages. Exposure to AHLs did not inhibit the negative phototaxis of Ulva zoospores, indicating that chemoattraction to bacterial biofilms does not preclude the response to a light stimulus in substrate location.     

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the calgary biofilm device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.     

Effects of different biotic substrata on mussel attachment

Surface colonization by invertebrates can be stimulated or inhibited by cues produced by biofilms, conspecifics or other macroorganisms. To study the effects of living substrata on the attachment of the brown mussel, Perna perna, two different approaches were employed: (1) mussels were distributed in sets of Petri dishes consisting of one sterile set (controls), three sets in which marine biofilms were allowed to develop in aquaria for 1, 7 or 15 days and another set that had been immersed in a natural marine environment for 1-day. There was no significant effect of biofilms on attachment, suggesting that neither age nor the source of the biofilm influenced attachment. (2) Mussels were suspended over PVC panels (controls) and over panels on which Balanus trigonus (Crustacea), Schizoporella errata (Bryozoa), Symplegma rubra or Didemnum speciosum (Ascidiacea) were present. Attachment was significantly higher on the controls and on B. trigonus than on colonial taxa such as S. rubra, S. errata and D. speciosum, probably due to antifouling defenses of these species. The results show that the composition of the biological substratum is an important factor affecting mussel behavior.     

Comparative study of biofilm formation on biocidal antifouling and fouling-release coatings using next-generation DNA sequencing

The bacterial and eukaryotic communities forming biofilms on six different antifouling coatings, three biocidal and three fouling-release, on boards statically submerged in a marine environment were studied using next-generation sequencing. Sequenced amplicons of bacterial 16S ribosomal DNA and eukaryotic ribosomal DNA internal transcribed spacer were assigned taxonomy by comparison to reference databases and relative abundances were calculated. Differences in species composition, bacterial and eukaryotic, and relative abundance were observed between the biofilms on the various coatings; the main difference was between coating type, biocidal compared to fouling-release. Species composition and relative abundance also changed through time. Thus, it was possible to group replicate samples by coating and time point, indicating that there are fundamental and reproducible differences in biofilms assemblages. The routine use of next-generation sequencing to assess biofilm formation will allow evaluation of the efficacy of various commercial coatings and the identification of targets for novel formulations.     

Effect of copper on multiple successional stages of a marine fouling assemblage

Copper based paints are used to prevent fouling on the hulls of ships. The widely documented effect of copper on hull assemblages may be primarily due to direct effects on the invertebrates themselves or indirect effects from copper absorbed into the microbial biofilm before settlement has commenced. Artificial units of habitat were exposed to varied regimes of copper to examine (1) the photosynthetic efficiency and pigments of early-colonising biofilms, and (2) subsequent macroinvertebrate assemblage change in response to the different regimes of copper. Macroinvertebrate assemblages were found to be less sensitive to the direct effects of copper than indirect effects as delivered through biofilms that have been historically exposed to copper, with some species more tolerant than others. This raises further concern for the efficacy of copper as a universal antifoulant on the hulls of ships, which may continue to assist the invasion of copper-tolerant invertebrate species.     

Response of biofilm-dwelling nematodes to habitat changes in the Garonne River,France: influence of hydrodynamics and microalgal availability

Lotic epilithic biofilms are submitted to seasonal disturbances (e.g. flood events, self-detachment), which influence the biomass, diversity and viability of their algal and bacterial communities. The objective of this study is to examine whether (1) biofilm-dwelling nematodes respond to such seasonal changes in terms of diversity and community structure, (2) nematode species and feeding-types distribution respond to the varied trophic situations within the biofilm, since variations in biofilm microalgal composition may represent a variation in available food. The biofilm-dwelling nematode community was monitored in a temperate river over an 18 month period with a high sampling frequency. These data were linked to environmental abiotic and biofilm biotic factors. Nematode density was positively correlated to biofilm and microalgal biomass, but was dampened by floods. A clear seasonal pattern of the community was detected (summer shift), so that two nematode groups stand out: (1) the epistrate-feeders Chromadorina bioculata (Schultze in Carus, 1857) and Chromadorina viridis (Linstow, 1876) were primarily related to diatom availability, and dominated the nematode assemblage most of the time, (2) seven species from various feeding types (deposit-feeders, suction-feeders and chewers) grew mainly under summer conditions concomitantly to a change of biofilm trophic status and microalgal composition. Overall, the results suggested that, in addition to abiotic disturbances, the availability of potential preys in the biofilm might represent an important driver of nematode community patterns.     

A new coupon design for simultaneous analysis of in situ microbial biofilm formation and community structure in drinking water distribution systems

This study presents a new coupon sampling device that can be inserted directly into the pipes within water distribution systems (WDS), maintaining representative near wall pipe flow conditions and enabling simultaneous microscopy and DNA-based analysis of biofilms formed in situ. To evaluate this sampling device, fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate changes in biofilms on replicate coupons within a non-sterile pilot-scale WDS. FISH analysis demonstrated increases in bacterial biofilm coverage of the coupon surface over time, while the DGGE analysis showed the development of increasingly complex biofilm communities, with time-specific clustering of these communities. This coupon design offers improvements over existing biofilm sampling devices in that it enables simultaneous quantitative and qualitative compositional characterization of biofilm assemblages formed within a WDS, while importantly maintaining fully representative near wall pipe flow conditions. Hence, it provides a practical approach that can be used to capture the interactions between biofilm formation and changing abiotic conditions, boundary shear stress, and turbulent driven exchange within WDS.     

Biofilms: implications in bioremediation

Biofilms are assemblages of single or multiple populations that are attached to abiotic or biotic surfaces through extracellular polymeric substances. Gene expression in biofilm cells differs from planktonic stage expression and these differentially expressed genes regulate biofilm formation and development. Biofilm systems are especially suitable for the treatment of recalcitrant compounds because of their high microbial biomass and ability to immobilize compounds. Bioremediation is also facilitated by enhanced gene transfer among biofilm organisms and by the increased bioavailability of pollutants for degradation as a result of bacterial chemotaxis. Strategies for improving bioremediation efficiency include genetic engineering to improve strains and chemotactic ability, the use of mixed population biofilms and optimization of physico-chemical conditions. Here, we review the formation and regulation of biofilms, the importance of gene transfer and discuss applications of biofilm-mediated bioremediation processes.     

Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms

Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga Ulva australis, were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates, Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii, were found to interact synergistically in biofilms formed in 96-well microtiter plates: biofilm biomass was observed to increase by >167% in biofilms formed by the four strains compared to biofilms composed of single strains. When exposed to the antibacterial agent hydrogen peroxide or tetracycline, the relative activity (exposed versus nonexposed biofilms) of the four-species biofilm was markedly higher than that in any of the single-species biofilms. Moreover, in biofilms established on glass surfaces in flow cells and subjected to invasion by the antibacterial protein-producing Pseudoalteromonas tunicata, the four-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.