Aquatic Biofilms Research Articles

Resource constraints highlight complex microbial interactions during lake biofilm development

Abstract This study evaluated how the availability of nutrients and organic carbon interact to influence the associations between autotrophic and heterotrophic micro‐organisms during lake biofilm development. Considering that decomposers are often better competitors for nutrients than producers in aquatic environments, we hypothesized that heterotrophs would outcompete autotrophs for available nutrients unless heterotrophs were limited by organic carbon provided by autotrophs. To test our hypothesis, we evaluated autotrophic (algae) and heterotrophic (fungi, bacteria) biomass in response to a factorial enrichment of nutrients (nitrogen and phosphorus in combination) and glucose using nutrient‐diffusing substrates with either inorganic or organic discs in a subalpine lake. In the field, nutrient‐diffusing substrates were exposed to either natural sunlight or placed under a darkened experimental canopy to evaluate the response of heterotrophs to nutrients and carbon subsidies in the presence or absence of algae. We expected that heterotrophs would be limited by organic carbon on inorganic substrates in the absence of autotrophic production (i.e., dark treatments), and that organic substrates would provide a carbon subsidy for heterotrophic metabolism. Fungi were stimulated by nutrient enrichment on inorganic substrates in the presence of algae (light treatment), but not in the dark (without algae). The response of fungi to algal presence on inorganic substrates was similar in magnitude to the response of fungi to nutrients and glucose substrates incubated in the dark. In contrast to our expectations, elevated algal biomass did not stimulate heterotrophic bacteria in the presence of elevated nutrient levels on inorganic substrates, possibly owing to antagonistic interactions between bacteria and fungi. The positive effect of nutrients on algal biomass was significantly reduced in favour of heterotrophs when nutrients were combined with glucose, suggesting that heterotrophs were able to outcompete algae for available nutrients in the absence of carbon limitation. Synthesis. These results expand our understanding of how the availability of limiting resources governs the outcomes of complex interactions among micro‐organisms in aquatic biofilms, and suggests that background levels of organic carbon regulate producer and decomposer responses to nutrient availability during biofilm development.

A freshwater analog for the production of Epiphyton-like microfossils.

Calcified microbial microfossils-often interpreted as cyanobacteria-were important components of Precambrian and Paleozoic limestones, but their paucity in modern marine environments complicates our ability to make conclusive interpretations about their taxonomic affinity and geologic significance. Freshwater spring-associated limestones (e.g., travertine and tufa) serve as terrestrial analogs to investigate mineralization in and around aquatic biofilms on observable timescales. We document the diagenesis of calcite fabrics associated with the freshwater algae Oocardium stratum, an epiphytic colonial green algae (desmid) known for producing stalks of extracellular polymeric substances (EPS) and passively producing a bifurcating tubular calcite monocrystal. Bifurcating EPS stalks produced by Oocardium colonies can become calcified and preserved in ancient carbonate deposits. Calcified micritic EPS stalks have a filamentous morphology, show evidence of branching, and maintain uniformity in diameter thickness throughout the mm-scale colony, much like the enigmatic calcimicrobe Epiphyton. We provide a mechanism by which calcification associated with a colonial semispherical micro-organism produces microfossils that deceptively resemble filamentous forms. These findings have implications for the use of morphological traits when assigning taxonomic affinities to extinct microfossil groups and highlight the utility of calcifying freshwater modern environments to investigate microbial taphonomy.

Visualization of the Sorption of Nickel within Exopolymer Microdomains of Bacterial Microcolonies Using Confocal and Scanning Electron Microscopy.

The sorption and distribution of nickel, a common metal contaminant in aquatic systems, were assessed in bacterial microcolonies using a combination of fluorescent staining with Newport Green and confocal laser scanning microscopy (CLSM) with confirmation by scanning electron microscopy (SEM) and X-ray microprobe analyses. CLSM with Newport Green, selected fluor-conjugated lectins, and DNA staining allowed for the discrimination of the microdomains present in the microcolony exopolymeric matrix and detection of bound nickel. This approach avoided the artefacts associated with drying and fixation required by analytical electron microscopy. The results obtained indicated that specific microcolonies within river biofilms sorbed nickel within limited microdomains present in the complex tripartite exopolymeric matrix surrounding bacterial cells. Sorption occurred such that nickel was concentrated within the exopolymeric matrix, but not directly associated with cells. These microdomains appeared to have neutral pH and be dominated by negatively charged residues favoring the sorption of nickel and other cations. These results also suggest an important role for specific community members in the sorption and concentration of metals in aquatic biofilm communities.

Study of Nanoparticles Impact on the Growth and Exopolysaccharides Production of Epiphytic Bacteria from Seaweeds

Upon entry into the environment, nanoparticles undergo agglomeration and biological or chemical transformation to other chemicals and results in toxic effects on marine biota. The Gulf of Mannar is the richest biological region and is found to be the habitat of seaweed resources. Recent investigations have established that significant accumulation of nanoparticles (NPs) occurs in aquatic biofilms. The most active fractions of bacteria are known as biofilms. In this study nine bacteria were screened from the surface of 10 different seaweeds and interacted with three nanoparticles (Silver, Zinc oxide, Titanium dioxide nanoparticles) at four different concentrations (0.1, 1, 10, 100 mg/L) each respectively. It has been found from the experiments that a reduction in the growth, biofilm formation and exopolysaccharides production was noted at higher concentration of nanoparticles (100 mg/L). Whereas at lower concentration (1, 10 mg/L) growth curve was similar to control but biofilm formation varied. Thus marine bacteria are tolerant to certain concentration of nanoparticles. Though engineered nanoparticles are best suited for various environmental works, it has its own limitations as it is found to be dangerous finally.

Characterization of Aquatic Biofilms with Flow Cytometry.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Linking biofilm spatial structure to real-time microscopic oxygen decay imaging

Two non-destructive techniques, confocal laser scanning microscopy (CLSM) and planar optode (VisiSens imaging), were combined to relate the fine-scale spatial structure of biofilm components to real-time images of oxygen decay in aquatic biofilms. Both techniques were applied to biofilms grown for seven days at contrasting light and temperature (10/20°C) conditions. The geo-statistical analyses of CLSM images indicated that biofilm structures consisted of small (~100 μm) and middle sized (~101 μm) irregular aggregates. Cyanobacteria and EPS (extracellular polymeric substances) showed larger aggregate sizes in dark grown biofilms while, for algae, aggregates were larger in light-20°C conditions. Light-20°C biofilms were most dense while 10°C biofilms showed a sparser structure and lower respiration rates. There was a positive relationship between the number of pixels occupied and the oxygen decay rate. The combination of optodes and CLMS, taking advantage of geo-statistics, is a promising way to relate biofilm architecture and metabolism at the micrometric scale.

On the use of high-throughput sequencing for the study of cyanobacterial diversity in Antarctic aquatic mats.

The study of Antarctic cyanobacterial diversity has been mostly limited to morphological identification and traditional molecular techniques. High-throughput sequencing (HTS) allows a much better understanding of microbial distribution in the environment, but its application is hampered by several methodological and analytical challenges. In this work, we explored the use of HTS as a tool for the study of cyanobacterial diversity in Antarctic aquatic mats. Our results highlight the importance of using artificial communities to validate the parameters of the bioinformatics procedure used to analyze natural communities, since pipeline-dependent biases had a strong effect on the observed community structures. Analysis of microbial mats from five Antarctic lakes and an aquatic biofilm from the Sub-Antarctic showed that HTS is a valuable tool for the assessment of cyanobacterial diversity. The majority of the operational taxonomic units retrieved were related to filamentous taxa such as Leptolyngbya and Phormidium, which are common genera in Antarctic lacustrine microbial mats. However, other phylotypes related to different taxa such as Geitlerinema, Pseudanabaena, Synechococcus, Chamaesiphon, Calothrix, and Coleodesmium were also found. Results revealed a much higher diversity than what had been reported using traditional methods and also highlighted remarkable differences between the cyanobacterial communities of the studied lakes. The aquatic biofilm from the Sub-Antarctic had a distinct cyanobacterial community from the Antarctic lakes, which in turn displayed a salinity-dependent community structure at the phylotype level.

Who eats who in biofilms? Exploring the drivers of microalgal and micro-meiofaunal abundance

A substantial amount of research has been dedicated to aquatic biofilms, but unlike the influence of macrograzers, that of microscopic herbivores (2 μm to 2 mm) has been largely disregarded. The selective pressure that they may exert remains unexplored, despite its potential impact on microbial community structure and ultimately, function. We performed in situ experiments to assess the abundance of microflora and micro-meiofauna in mature biofilms, in four rivers located in southwest France, stressing their quantitative importance (averaging 50% of the total number of organisms counted). We highlighted that the balance between microflora and micro-meiofauna depends on local environmental conditions, and specific taxon selection is likely to provide information on river pollution. Indeed, microalgae were directly favoured by nutrients and indirectly by biocides targeting micrograzers. In contrast, micro-meiofauna was predominant in sites where herbicides were found, in particular photosynthesis inhibitors (e.g. in the River Ponteils, 97% micro-meiofauna). Although the prey–predator relationships in the laboratory experiment appeared to depend on the initial pools of microorganisms during the first days, they reached stable states within a week. Then, all cultures stabilized around a ratio of 20 ± 6 : 1 prey/potential predator. Further research is required to better understand the abiotic (water quality, habitat) and biotic (traits of the organisms present) drivers governing the coexistence of microalgae and their predators across the microbial landscape, and its consequences on the structure and function of the aquatic ecosystem.

Molecular detection of pathogenic leptospiral protein encoding gene (lipL32) in environmental aquatic biofilms.

Leptospirosis is a zoonotic disease often encountered during post-monsoon season due to exposure with contaminated water. Leptospires have long been regarded as solitary organisms that persist in soil and aquatic environments. Here, the presence of leptospires in the aquatic biofilm exposed in the paddy field, sewers and stagnant rain water was demonstrated. Biofilm samples from paddy field water, submerged paddy leaves, sewers and stagnant rain waters from urban and rural areas were collected. Total genomic DNA was extracted and pathogenic leptospiral specific gene amplification was carried out to determine the spatial distribution of the bacteria. The degree of pathogenic Leptospira in biofilms from paddy field surface water, submerged leaf, were 33·3% and 27·2% respectively, whereas in rural and urban area, the sampling sites such as stagnant rain water, domestic sewer and collective sewers showed 11·1%, 13% and 16·6% with leptospires respectively. Higher proportion of pathogenic Leptospira in aquatic ecosystems, such as paddy field, could be one of the main factors for the occurrence of disease, more among the agricultural workers. This study would help to identify various survival strategies of leptospires in the environment and thus disease transmission. Little is known regarding the mechanisms by which pathogenic leptospires persist in aqueous environment, outside the mammalian host. In this view this is the first report of the distribution of Leptospira in environmental biofilm such as sewers and paddy leaf surfaces. This ability of pathogenic Leptospira to survive in aquatic ecosystems especially in biofilms could be one of the main factors which facilitate its survival in the environment, and thus disease transmission among the risk groups, such as sewage and agriculture worker. This study will encourage researchers in the field to consider biofilm as an important factor, when detecting leptospires in environment.

Memory Effect of Aquatic Biofilms in the Partitioning of Polycyclic Aromatic Hydrocarbons (PAHs) and Polychlorinated Biphenyls (PCBs) in Water Streams

Memory Effect of Aquatic Biofilms in the Partitioning of Polycyclic Aromatic Hydrocarbons (PAHs) and Polychlorinated Biphenyls (PCBs) in Water Streams

2,4-D and Glyphosate affect aquatic biofilm accrual, gross primary production, and community respiration

2,4-Dichlorophenoxyacetic acid (2,4-D) and glyphosate are widely used agricultural herbicides commonly found in surface waters near cultivated land. Field experiments were conducted to determine the effects of 2,4-D and glyphosate on biofilms in a pond next to agricultural land in Athabasca, Alberta. Contaminant-exposure substrates (CES) consisted of GF/C glass fiber or a cellulose filter paper substrates placed on specimen jars filled with agar that contained low levels of nitrogen and phosphorus, and different concentrations (15, 9.0, 1.5 mM) of either 2,4-D or glyphosate. Nutrients and herbicide diffused freely through the agar to the substrate surface. CES arrays were deployed 15 cm below the water surface for 22 days, after which biofilms were collected and biomass (chlorophyll a ), autotroph gross primary production (GPP), and heterotroph community respiration (CR) were measured. 2,4-D (15 mM) caused significant decreases in rates of biomass accrual (−22%), GPP (−34%), and CR(−63%). Glyphosate (15 mM) also caused significant decreases in rates of biomass accrual (−50%), GPP (−67%), and CR (−47%). For the contaminant concentrations used, mean flux rates are estimated to be between 50–700 ng cm −2 min −1 .

When nanoparticles meet biofilms-interactions guiding the environmental fate and accumulation of nanoparticles.

Bacteria are essential components of all natural and many engineered systems. The most active fractions of bacteria are now recognized to occur as biofilms, where cells are attached and surrounded by a secreted matrix of “sticky” extracellular polymeric substances. Recent investigations have established that significant accumulation of nanoparticles (NPs) occurs in aquatic biofilms. These studies point to the emerging roles of biofilms for influencing partitioning and possibly transformations of NPs in both natural and engineered systems. While attached biofilms are efficient “sponges” for NPs, efforts to elucidate the fundamental mechanisms guiding interactions between NPs and biofilms have just begun. In this mini review, special attention is focused on NP–biofilm interactions within the aquatic environment. We highlight key physical, chemical, and biological processes that affect interactions and accumulation of NPs by bacterial biofilms. We posit that these biofilm processes present the likely possibility for unique biological and chemical transformations of NPs. Ultimately, the environmental fate of NPs is influenced by biofilms, and therefore requires a more in-depth understanding of their fundamental properties.

Dental unit water treatment with hydrogen peroxide and monovalent silver ions artificially contaminated with freshly isolated pathogens.

Dental unit water (DUW) could be contaminated by human pathogens coming from biological fluids penetrated during patient treatment and by opportunistic pathogens detached from aquatic biofilm. These microorganisms could be spread to following patients. We tested the disinfectant activity of hydrogen peroxide and monovalent silver ions (H(2)O(2)-Ag(+)) into DUW artificially contaminated with freshly isolated pathogens. The tested microorganisms were Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Pseudomonas aeruginosa, Legionella pneumophila, Mycobacterium chelonae, non-pathogenic Bacillus clausii spores. Bacterial suspensions were inoculated into the waterlines of pre-sterilized dental turbines. The test-turbines were connected to DUW and contaminated water was treated for 10 minutes with H(2)O(2)-Ag(+)-based disinfectant (H(2)O(2) 3% v/v, Ag(+) 0.001% w/v). The control-turbines were left untreated. Turbines were washed with sterile hard water used to assess the residual bacterial loads (expressed in colony forming units -cfu). Each strain was tested five times and the mean log loads were assessed. Following the European Standardization Committee, the disinfectant activity was evaluated as mean log load reduction, that is, the difference between the mean log load detected on the control-turbines and the mean log load detected on the test-turbines. Mean bacterial loads detected on the control-turbines ranged between 105-107 cfu. The mean log load reductions resulted 7.5 log cfu for S. aureus, E. faecalis, P. aeruginosa, 6.3 for C. albicans, 5.4 for L. pneumophila, 5.3 for M. chelonae, 2.9 for B. clausii spores. DUW disinfection with H(2)O(2)-Ag(+) could help minimize the risk that planktonic pathogens are spread to patients during dental treatment.

‘Synbiofilm’- A Friendly Microbial Association in Aquatic Ecosystem

‘Biofilm’ is a multi-community microbial association living on submerged substrates. Using the online search engine www.OJOSE.com to identify the 100 most recent journal articles, we found that the term ‘biofilm’ had been extensively used by journals from medical/pathological disciplines to mean virulent microbial associations in the human body. The question is, whether natural aquatic biofilms different from these virulent ones? Should we hold a separate view regarding natural aquatic biofilms? Ecological journals use ‘periphyton’ to address similar microbial associations living on submerged substrates. We have discussed the development of biofilm in water and emphasized that natural biofilm is a totally heterogeneous interdependent balanced ecological aggregation of different microbial organisms where ‘periphyton’ occupies an advanced successional form. Contrary to the pathogenic biofilms, natural biofilms from undisturbed aquatic ecosystems are exclusively ‘synbiotic’ in their mode of survival. We therefore propose the term ‘synbiofilm’ to maintain a classical distinction between natural aquatic biofilm and pathogenic biofilms.

Pharmaceuticals and personal care products alter growth and function in lentic biofilms

Environmental context Pharmaceuticals and personal care products are routinely found in waters discharged from treatment plants and in surrounding aquatic ecosystems. Despite the widespread occurrence of these biologically active agents, there is limited understanding of their potential effects on key ecosystem processes such as primary production, ecosystem respiration and algal growth. This paper examines the effects of five common pharmaceuticals on the rates of these fundamental processes. Abstract Pharmaceutical diffusing substrates were used to study in situ responses of aquatic biofilms in an urbanised lentic ecosystem to five pharmaceutical and personal care products (PPCPs; caffeine, cimetidine, ciprofloxacin, diphenhydramine and metformin). The pharmaceutical diffusing substrates consisted of porous biofilm substrates placed atop a mass of agar amended with 2.5mM of the PPCP compound of interest. Over 21 days, biofilms growing on the substrata were exposed to slow diffusion of the PPCP through the agar and porous substrate. Algal biomass was suppressed by exposure to diphenhydramine (–81%) and ciprofloxacin (–50%). Gross primary production was completely suppressed by diphenhydramine exposure but stimulated by caffeine (+39%) and cimetidine (+46%). For heterotroph biofilms, community respiration was suppressed by exposure to diphenhydramine (–24%). To characterise PPCP exposure, rates of diffusion from the pharmaceutical diffusing substrates were measured at 10, 20 and 30°C. Diffusion was Fickian for all compounds and all temperatures. Diffusion coefficients, D, were in the range 1.5×10–10 to 1.1×10–9m2s–1. From diffusion data, average release rates over 21 days were typically 30–50ngmin–1cm–2 at 20°C. The results show that PPCPs can dramatically affect rates of key ecological processes, and the relationship between release rate and ambient concentration of PPCPs is discussed.

Bacterial diversity and abundance in shell biofilms from the freshwater snail Pleurocera canaliculatum (cerithioidea: Pleuroceridae)

Mollusk shells provide a hard substrate for aquatic biofilm colonization. While most work has focused on bivalve shells and grazing, little work has focused on gastropod shells and the microbes growing on them. We sampled biofilms from 14 Pleuroceracanaliculatum and analyzed them using a metagenomic approach. Microbial diversity varied between individuals, and rarefaction suggested that 63 snails would need to be sampled to capture all of the estimated genus-level diversity. Cyanobacteria and species of Novosphingobium and Methylosoma were the most abundant taxa across all shells.

Extracellular excystation and development of Cryptosporidium: tracing the fate of oocysts within Pseudomonas aquatic biofilm systems.

BackgroundAquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions. With regard to Cryptosporidium, biofilms can serve as environmental reservoirs for oocysts, but may also support the growth of additional Cryptosporidium stages.ResultsHere we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture. We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle. Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium. These microscopy observations were further supported by flow cytometric analyses, where excysted oocyst populations were detected in 1, 3 and 6 day-old Cryptosporidium-exposed biofilms, but not in biofilm-free controls.ConclusionsThese observations not only highlight the risk that aquatic biofilms pose in regards to Cryptosporidium outbreaks from water distribution systems, but further indicate that even simple biofilms are able to stimulate oocyst excystation and support the extracellular multiplication and development of Cryptosporidium within aquatic environments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0281-8) contains supplementary material, which is available to authorized users.

Pseudomonas aeruginosa promotes Escherichia coli biofilm formation in nutrient-limited medium.

Biofilms have been implicated as an important reservoir for pathogens and commensal enteric bacteria such as Escherichia coli in natural and engineered water systems. However, the processes that regulate the survival of E. coli in aquatic biofilms have not been thoroughly studied. We examined the effects of hydrodynamic shear and nutrient concentrations on E. coli colonization of pre-established Pseudomonas aeruginosa biofilms, co-inoculation of E. coli and P. aeruginosa biofilms, and P. aeruginosa colonization of pre-established E. coli biofilms. In nutritionally-limited R2A medium, E. coli dominated biofilms when co-inoculated with P. aeruginosa, and successfully colonized and overgrew pre-established P. aeruginosa biofilms. In more enriched media, P. aeruginosa formed larger clusters, but E. coli still extensively overgrew and colonized the interior of P. aeruginosa clusters. In mono-culture, E. coli formed sparse and discontinuous biofilms. After P. aeruginosa was introduced to these biofilms, E. coli growth increased substantially, resulting in patterns of biofilm colonization similar to those observed under other sequences of organism introduction, i.e., E. coli overgrew P. aeruginosa and colonized the interior of P. aeruginosa clusters. These results demonstrate that E. coli not only persists in aquatic biofilms under depleted nutritional conditions, but interactions with P. aeruginosa can greatly increase E. coli growth in biofilms under these experimental conditions.

Bacterial Signaling Ecology and Potential Applications During Aquatic Biofilm Construction

In their natural environment, bacteria and other microorganisms typically grow as surface-adherent biofilm communities. Cell signal processes, including quorum signaling, are now recognized as being intimately involved in the development and function of biofilms. In contrast to their planktonic (unattached) counterparts, bacteria within biofilms are notoriously resistant to many traditional antimicrobial agents and so represent a major challenge in industry and medicine. Although biofilms impact many human activities, they actually represent an ancient mode of bacterial growth as shown in the fossil record. Consequently, many aquatic organisms have evolved strategies involving signal manipulation to control or co-exist with biofilms. Here, we review the chemical ecology of biofilms and propose mechanisms whereby signal manipulation can be used to promote or control biofilms.