Biofilm diversity, structure and matrix seasonality in a full-scale cooling tower.

Biofilms commonly colonise cooling water systems, causing equipment damage and interference with the operational requirements of the systems. In this study, next-generation sequencing (NGS), catalysed reporter deposition fluorescence in situ hybridisation (CARD-FISH), lectin staining and microscopy were used to evaluate temporal dynamics in the diversity and structure of biofilms collected seasonally over one year from an open full-scale cooling tower. Water samples were analysed to evaluate the contribution of the suspended microorganisms to the biofilm composition and structure. Alphaproteobacteria dominated the biofilm communities along with Beta- and Gammaproteobacteria. The phototrophic components were mainly cyanobacteria, diatoms and green algae. Bacterial biodiversity decreased from winter to autumn, concurrently with an increase in cyanobacterial and microalgal richness. Differences in structure, spatial organisation and glycoconjugates were observed among assemblages during the year. Overall, microbial variation appeared to be mostly affected by irradiance and water temperature rather than the source of the communities. Variations in biofilms over seasons should be evaluated to develop specific control strategies.

Osteopontin adsorption to Gram-positive cells reduces adhesion forces and attachment to surfaces under flow.

The bovine milk protein osteopontin (OPN) may be an efficient means to prevent bacterial adhesion to dental tissues and control biofilm formation. This study sought to determine to what extent OPN impacts adhesion forces and surface attachment of different bacterial strains involved in dental caries or medical device-related infections. It further investigated if OPN's effect on adhesion is caused by blocking the accessibility of glycoconjugates on bacterial surfaces. Bacterial adhesion was determined in a shear-controlled flow cell system in the presence of different concentrations of OPN, and interaction forces of single bacteria were quantified using single-cell force spectroscopy before and after OPN exposure. Moreover, the study investigated OPN's effect on the accessibility of cell surface glycoconjugates through fluorescence lectin-binding analysis. OPN strongly affected bacterial adhesion in a dose-dependent manner for all investigated species (Actinomyces naeslundii, Actinomyces viscosus, Lactobacillus paracasei subsp. paracasei, Staphylococcus epidermidis, Streptococcus mitis, and Streptococcus oralis). Likewise, adhesion forces decreased after OPN treatment. No effect of OPN on the lectin-accessibility to glycoconjugates was found. OPN reduces the adhesion and adhesion force/energy of a variety of bacteria and has a potential therapeutic use for biofilm control. OPN acts upon bacterial adhesion without blocking cell surface glycoconjugates.

The acid soluble extracellular polymeric substance of aerobic granular sludge dominated by Defluviicoccus sp.

A new acid soluble extracellular polymeric substance (acid soluble EPS) was extracted from an acetate fed aerobic granular sludge reactor operated at 35 °C. Acid soluble EPS dominated granules exhibited a remarkable and distinctive tangled tubular morphology. These granules are dominated by Defluviicoccus Cluster II organisms. Acetic acid instead of the usually required alkaline extraction medium was needed to dissolve the granules and solubilise the polymeric matrix. The extracted acid soluble EPS was analysed and identified using various instrumental analysis including 1H and 13C Nuclear Magnetic Resonance, Fourier Transform Infrared Spectroscopy and Raman spectroscopy. In addition, the glycoconjugates were characterized by fluorescence lectin-binding analysis. The acid soluble EPS is α-(1 â†’ 4) linked polysaccharide, containing both glucose and galactose as monomers. There are OCH3 groups connected to the glucose monomer. Transmission and scanning electron microscopy (TEM, SEM) as well as confocal laser scanning microscopy (CLSM) showed that the acid soluble EPS was present as a tightly bound capsular EPS around bacterial cells ordered into a sarcinae-like growth pattern. The special granule morphology is decided by the acid soluble EPS produced by Defluviicoccus Cluster II organisms. This work shows that no single one method can be used to extract all possible extracellular polymeric substances. Results obtained here can support the elucidation of biofilm formation and structure in future research.

In situ evidence for metabolic and chemical microdomains in the structured polymer matrix of bacterial microcolonies.

CLSM and fluorescent probes were applied to assess the structure, composition, metabolic activity and gradients within naturally occurring ß-proteobacteria microcolonies. Extracellular polymeric substances (EPS) as defined by lectin-binding analyses had three regions: (i) cell associated, (ii) intercellular and (iii) an outer layer covering the entire colony. We assessed structural, microenvironmental and metabolic implications of this complex EPS structure. Permeability studies indicated that the outer two layers were permeable to 20 nm beads, intercellular EPS to <40 nm beads and the outer layer was permeable to <100 nm beads. Phosphatase activity occurred at the cell surface and associated polymer. Glucose oxidase activity was only detected inside the cells and the cell-associated polymer. Rhodamine 123 suggested that activity was highest near the cell surface. The potential sensitive dye JC-1 concentrated within the outer EPS layer and the gradient was responsive to inhibition by KCN, dispersion using KCl and enhanced by addition of nutrients (nutrient broth). pH gradients occurred from the cell interior (pH 7) to the microcolony interior (pH 4+) with a gradient of increasing pH (pH 7+) to the colony exterior. The EPS provides a physical and chemical structuring mechanism forming microdomains that segregate extracellular activities at the microscale, possibly resulting in a microcolony with unitary structure and function.

Protistan predation interferes with bacterial long-term adaptation to substrate restriction by selecting for defence morphotypes.

Bacteria that are introduced into aquatic habitats face a low substrate environment interspersed with rare productive 'hotspots', as well as high protistan grazing. Whereas the former condition should select for growth performance, the latter should favour traits that reduce predation mortality, such as the formation of large cell aggregates. However, protected morphotypes often convey a growth disadvantage, and bacteria thus face a trade-off between investing in growth or defence traits. We set up an evolutionary experiment with the freshwater isolate Sphingobium sp. strain Z007 that conditionally increases aggregate formation in supernatants from a predator-prey coculture. We hypothesized that low substrate levels would favour growth performance and reduce the aggregated subpopulation, but that the concomitant presence of a flagellate predator might conserve the defence trait. After 26 (1-week) growth cycles either with (P+) or without (P-) predators, bacteria had evolved into strikingly different phenotypes. Strains from P- had low numbers of aggregates and increased growth yield, both at the original rich growth conditions and on various single carbon sources. By contrast, isolates from the P+ treatment formed elevated proportions of defence morphotypes, but exhibited lower growth yield and metabolic versatility. Moreover, the evolved strains from both treatments had lost phenotypic plasticity of aggregate formation. In summary, the (transient) residence of bacteria at oligotrophic conditions may promote a facultative oligotrophic life style, which is advantageous for survival in aquatic habitats. However, the investment in defence against predation mortality may constrain microbial adaptation to the abiotic environment.

Dominance of 'Gallionella capsiferriformans' and heavy metal association with Gallionella-like stalks in metal-rich pH 6 mine water discharge.

Heavy metal-contaminated, pH 6 mine water discharge created new streams and iron-rich terraces at a creek bank in a former uranium-mining area near Ronneburg, Germany. The transition from microoxic groundwater with ~5 mm Fe(II) to oxic surface water may provide a suitable habitat for microaerobic iron-oxidizing bacteria (FeOB). In this study, we investigated the potential contribution of these FeOB to iron oxidation and metal retention in this high-metal environment. We (i) identified and quantified FeOB in water and sediment at the outflow, terraces, and creek, (ii) studied the composition of biogenic iron oxides (Gallionella-like twisted stalks) with scanning and transmission electron microscopy (SEM, TEM) as well as confocal laser scanning microscopy (CLSM), and (iii) examined the metal distribution in sediments. Using quantitative PCR, a very high abundance of FeOB was demonstrated at all sites over a 6-month study period. Gallionella spp. clearly dominated the communities, accounting for up to 88% of Bacteria, with a minor contribution of other FeOB such as Sideroxydans spp. and 'Ferrovum myxofaciens'. Classical 16S rRNA gene cloning showed that 96% of the Gallionella-related sequences had ≥ 97% identity to the putatively metal-tolerant 'Gallionella capsiferriformans ES-2', in addition to known stalk formers such as Gallionella ferruginea and Gallionellaceae strain R-1. Twisted stalks from glass slides incubated in water and sediment were composed of the Fe(III) oxyhydroxide ferrihydrite, as well as polysaccharides. SEM and scanning TEM-energy-dispersive X-ray spectroscopy revealed that stalk material contained Cu and Sn, demonstrating the association of heavy metals with biogenic iron oxides and the potential for metal retention by these stalks. Sequential extraction of sediments suggested that Cu (52-61% of total sediment Cu) and other heavy metals were primarily bound to the iron oxide fractions. These results show the importance of 'G. capsiferriformans' and biogenic iron oxides in slightly acidic but highly metal-contaminated freshwater environments.

Characterisation of algal organic matter produced by bloom-forming marine and freshwater algae.

Algal blooms can seriously affect the operation of water treatment processes including low pressure (micro- and ultra-filtration) and high pressure (nanofiltration and reverse osmosis) membranes mainly due to accumulation of algal-derived organic matter (AOM). In this study, the different components of AOM extracted from three common species of bloom-forming algae (Alexandrium tamarense, Chaetoceros affinis and Microcystis sp.) were characterised employing various analytical techniques, such as liquid chromatography - organic carbon detection, fluorescence spectroscopy, fourier transform infrared spectroscopy, alcian blue staining and lectin staining coupled with laser scanning microscopy to indentify its composition and force measurement using atomic force microscopy to measure its stickiness. Batch culture monitoring of the three algal species illustrated varying characteristics in terms of growth pattern, cell concentration and AOM release. The AOM produced by the three algal species comprised mainly biopolymers (e.g., polysaccharides and proteins) but some refractory compounds (e.g., humic-like substances) and other low molecular weight acid and neutral compounds were also found. Biopolymers containing fucose and sulphated functional groups were found in all AOM samples while the presence of other functional groups varied between different species. A large majority (>80%) of the acidic polysaccharide components (in terms of transparent exopolymer particles) were found in the colloidal size range (<0.4 µm). The relative stickiness of AOM substantially varied between algal species and that the cohesion between AOM-coated surfaces was much stronger than the adhesion of AOM on AOM-free surfaces. Overall, the composition as well as the physico-chemical characteristics (e.g., stickiness) of AOM will likely dictate the severity of fouling in membrane systems during algal blooms.

Use of lectins to in situ visualize glycoconjugates of extracellular polymeric substances in acidophilic archaeal biofilms.

Biofilm formation and the production of extracellular polymeric substances (EPS) by meso- and thermoacidophilic metal-oxidizing archaea on relevant substrates have been studied to a limited extent. In order to investigate glycoconjugates, a major part of the EPS, during biofilm formation/bioleaching by archaea on pyrite, a screening with 75 commercially available lectins by fluorescence lectin-binding analysis (FLBA) has been performed. Three representative archaeal species, Ferroplasma acidiphilum DSM 28986, Sulfolobus metallicus DSM 6482(T) and a novel isolate Acidianus sp. DSM 29099 were used. In addition, Acidianus sp. DSM 29099 biofilms on elemental sulfur were studied. The results of FLBA indicate (i) 22 lectins bound to archaeal biofilms on pyrite and 21 lectins were binding to Acidianus sp. DSM 29099 biofilms on elemental sulfur; (ii) major binding patterns, e.g. tightly bound EPS and loosely bound EPS, were detected on both substrates; (iii) the three archaeal species produced various EPS glycoconjugates on pyrite surfaces. Additionally, the substratum induced different EPS glycoconjugates and biofilm structures of cells of Acidianus sp. DSM 29099. Our data provide new insights into interactions between acidophilic archaea on relevant surfaces and also indicate that FLBA is a valuable tool for in situ investigations on archaeal biofilms.

Chip-calorimetry provides real time insights into the inactivation of biofilms by predatory bacteria.

Control or removal of undesired biofilms has frequently been found to be quite difficult. In addition to biocidal or antibiotic chemicals or materials designed to prevent biofouling, biological control agents appear to be promising. Reports of bacterial predators eradicating biofilms or eliminating pathogens motivate a more systematic screening of biofilm-eliminating bacterial predators. Unfortunately, the analysis of the eradication process is demanding. In the present study, chip-calorimetry was applied to monitor the elimination of Pseudomonas sp. biofilms by Bdellovibrio bacteriovorus. The method uses metabolic heat as a real-time parameter for biofilm activity. The method is non-invasive, fast and convenient due to real-time data acquisition. In addition, heat-production data can reveal information about the energetics of the predator-prey interaction. The calorimetric results were validated by confocal laser scanning microscopy. The approach described may be useful for the screening of biofilm susceptibility to different predators.

Characterization of glycoconjugates of extracellular polymeric substances in tufa-associated biofilms by using fluorescence lectin-binding analysis.

Freshwater tufa deposits are the result of calcification associated with biofilms dominated by cyanobacteria. Recent investigations highlighted the fact that the formation of microbial calcium carbonates is mainly dependent on the saturation index, which is determined by pH, the ion activity of Ca(2+) and CO(3)(2-), and the occurrence of extracellular polymeric substances (EPS) produced by microorganisms. EPS, which contain carboxyl and/or hydroxyl groups, can strongly bind cations. This may result in inhibition of CaCO(3) precipitation. In contrast, the formation of templates for crystal nucleation was reported by many previous investigations. The purposes of this study were (i) to characterize the in situ distribution of EPS glycoconjugates in tufa-associated biofilms of two German hard-water creeks by employing fluorescence lectin-binding analysis (FLBA), (ii) to verify the specific lectin-binding pattern by competitive-inhibition assays, and (iii) to assess whether carbonates are associated with structural EPS domains. Three major in situ EPS domains (cyanobacterial, network-like, and cloud-like structures) were detected by FLBA in combination with laser scanning microscopy (LSM). Based on lectin specificity, the EPS glycoconjugates produced by cyanobacteria contained mainly fucose, amino sugars (N-acetyl-glucosamine and N-acetyl-galactosamine), and sialic acid. Tufa deposits were irregularly covered by network-like EPS structures, which may originate from cyanobacterial EPS secretions. Cloud-like EPS glycoconjugates were dominated by sialic acid, amino sugars, and galactose. In some cases calcium carbonate crystals were associated with cyanobacterial EPS glycoconjugates. The detection of amino sugars and calcium carbonate in close association with decaying sheath material indicated that microbially mediated processes might be important for calcium carbonate precipitation in freshwater tufa systems.

Effective diffusivities and mass fluxes in fungal biopellets.

Mass transport within biological aggregates is a key process that can determine overall turnover rates in submerged cultivations. A parameter commonly used for its description is the effective diffusion coefficient D(eff), which is highly dependent on biomass density and structure. Different approaches have been used to estimate or measure D(eff), yet the data still shows broad scattering. This study provides experimental data on effective diffusivities of oxygen within fungal pellets. A correlation is found with the hyphal gradient (dh/dr), which is a morphological parameter describing the structure of the pellet periphery. Furthermore, the dependency of D(eff) on fluid dynamic conditions at the pellet is investigated. The comparison of the results with data from literature clearly demonstrates the influence of the experimental methodology applied for determination of D(eff). Moreover, it is shown that while diffusion limitation of whole pellets is mainly a function of size, the influence of advection in the outer zone of pellets that is supplied with oxygen is actually rather high. Thus, it is concluded that the effective diffusion coefficient might not be sufficient for the description of mass transport within the pellet periphery for a broad range of realistic fluid dynamic conditions during cultivation. Nevertheless, although actual mass transport rates inside pellets are unknown, mass fluxes can be calculated on the basis of spatially resolved data of oxygen and biomass distribution within the pellet.

Miniaturized calorimetry - a new method for real-time biofilm activity analysis.

The partial dissipation of Gibbs energy as heat reflects the metabolic dynamic of biofilms in real time and may also allow quantitative conclusions about the chemical composition of the biofilm via Hess' law. Presently, the potential information content of heat is hardly exploited due to the low flexibility, the low throughput and the high price of conventional calorimeters. In order to overcome the limitations of conventional calorimetry a miniaturized calorimeter for biofilm investigations has been evaluated. Using four thermopiles a heat production with spatial and temporal resolutions of 2.5 cm(-1) and 2 s(-1) could be determined. The limit of detection of the heat flow measurement was 20 nW, which corresponds to the cell density of an early stage biofilm (approx. 3x10(5) cells cm(-2)). By separating biofilm cultivation from the actual heat measurement, a high flexibility and a much higher throughput was achieved if compared with conventional calorimeters. The approach suggested allows cultivation of biofilms in places of interest such as technological settings as well as in nature followed by highly efficient measurements in the laboratory. Functionality of the miniaturized calorimeter was supported by parallel measurements with confocal laser scanning microscopy and a fiber optic based oxygen sensor using the oxycaloric equivalent (-460 kJ mol-O2(-1)).

In situ evidence for microdomains in the polymer matrix of bacterial microcolonies.

Confocal laser scanning microscopy and fluorescent lectin-binding analyses (FLBA) were used to study the form, arrangement, and composition of exopolymeric substances (EPS) surrounding naturally occurring microcolonies in biofilms. FLBA, using multiple lectin staining and multichannel imaging, indicated that the EPS of many microcolonies exhibit distinct multiple binding regions. A common pattern in the microcolonies is a three zone arrangement with cell-associated, intercellular, and an outer layer of EPS covering the exterior of the colony. Differential binding of lectins suggests that there are differences in the glycoconjugate composition or their arrangement in the EPS of microcolonies. The combination of FLBA with fluorescent in situ hybridization (FISH) indicates that the colonies consist of the major groups, alpha- and beta-Proteobacteria. It is suggested that the EPS arrangement observed provides a physical structuring mechanism that can segregate extracellular activities at the microscale.

Characterization of adhesion threads of Deinococcus geothermalis as type IV pili.

Deinococcus geothermalis E50051 forms tenuous biofilms on paper machine surfaces. Field emission electron microscopy analysis revealed peritrichous appendages which mediated cell-to-surface and cell-to-cell interactions but were absent in planktonically grown cells. The major protein component of the extracellular extract of D. geothermalis had an N-terminal sequence similar to the fimbrial protein pilin annotated in the D. geothermalis DSM 11300 draft sequence. It also showed similarity to the type IV pilin sequence of D. radiodurans and several gram-negative pathogenic bacteria. Other proteins in the extract had N-terminal sequences identical to D. geothermalis proteins with conservative motifs for serine proteases, metallophosphoesterases, and proteins whose function is unknown. Periodic acid-Schiff staining for carbohydrates indicated that these extracellular proteins may be glycosylated. A further confirmation for the presence of glycoconjugates on the cell surface was obtained by confocal laser scanning imaging of living D. geothermalis cells stained with Amaranthus caudatus lectin, which specifically binds to galactose residues. The results indicate that the thread-like appendages of D. geothermalis E50051 are glycosylated type IV pili, bacterial attachment organelles which have thus far not been described for the genus Deinococcus.

An endolithic microbial community in dolomite rock in central Switzerland: characterization by reflection spectroscopy, pigment analyses, scanning electron microscopy, and laser scanning microscopy.

A community of endolithic microorganisms dominated by phototrophs was found as a distinct band a few millimeters below the surface of bare exposed dolomite rocks in the Piora Valley in the Alps. Using in situ reflectance spectroscopy, we detected chlorophyll a (Chl a), phycobilins, carotenoids, and an unknown type of bacteriochlorophyll-like pigment absorbing in vivo at about 720 nm. In cross sections, the data indicated a defined distribution of different groups of organisms perpendicular to the rock surface. High-performance liquid chromatography analyses of pigments extracted with organic solvents confirmed the presence of two types of bacteriochlorophylls besides chlorophylls and various carotenoids. Spherical organisms of varying sizes and small filaments were observed in situ with scanning electron microscopy and confocal laser scanning microscopy (one- and two-photon technique). The latter allowed visualization of the distribution of phototrophic microorganisms by the autofluorescence of their pigments within the rock. Coccoid cyanobacteria of various sizes predominated over filamentous ones. Application of fluorescence-labeled lectins demonstrated that most cyanobacteria were embedded in an exopolymeric matrix. Nucleic acid stains revealed a wide distribution of small heterotrophs. Some biological structures emitting a green autofluorescence remain to be identified.

Oxygen profiles and biomass distribution in biopellets of Aspergillus niger.

Morphology of fungal pellets has a significant influence on mass transfer and turnover processes in submerged cultures. There are many reports in literature that biomass is not distributed homogeneously over the pellet radius, yet quantitative data is rare. This study presents a method for the quantification of fungal pellet structure (Aspergillus niger). Confocal laser scanning microscopy (CLSM) is used in combination with image analysis freeware (Image J). Hyphal distribution is resolved spatially in radial direction. Quantitative morphological parameters are derived from digital images especially from the peripheral regions of the pellet that are not oxygen limited. This morphological information is combined with data of microelectrode measurements in the same pellets. Results show that the morphological parameters obtained can describe the impact of pellet structure on oxygen gradients much better than average biomass density. It is concluded that CLSM and image analysis are powerful tools not only to generate valuable data for quantitative description of pellet morphology. In addition, this data may be used in mathematical models to improve predictions of mass transfer and substrate conversion in mycelial aggregates.

Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms.

In this study an enrichment culture developed from activated sludge was used to investigate the architecture of fully hydrated multispecies biofilms. The assessment of biofilm structure and volume was carried out using confocal laser scanning microscopy (CLSM). Bacterial cell distribution was determined with the nucleic acid-specific stain SYTO 60, whereas glycoconjugates of extracellular polymeric substances (EPS) were stained with the Alexa-488-labeled lectin of Aleuria aurantia. Digital image analysis was employed for visualization and quantification of three-dimensional CLSM data sets. The specific volumes of the polymeric and cellular biofilm constituents were quantified. In addition, gravimetric measurements were done to determine dry mass and thickness of the biofilms. The data recorded by the CLSM technique and the gravimetric data were then compared. It was shown that the biofilm thicknesses determined with both methods agree well for slow-growing heterotrophic and chemoautotrophic biofilms. In addition, for slow-growing biofilms, the volumes and masses calculated from CLSM and the biomass calculated from gravimetric measurements were also comparable. For fast-growing heterotrophic biofilms cultivated with high glucose concentrations the data sets fit to a lesser degree, but still showed the same common trend. Compared with traditional gravimetric measurements, CLSM allowed differential recording of multiple biofilm parameters with subsequent three-dimensional visualization and quantification. The quantitative three-dimensional results recorded by CLSM are an important basis for understanding, controlling, exploiting, and modeling of biofilms.

Growth, structure and oxygen penetration in particle supported autotrophic biofilms.

Particle supported autotrophic biofilms were cultivated in external-loop airlift reactors at two different pumice concentrations. Oxygen microelectrodes were used to investigate substrate transport and conversion. A special flow cell was designed for the measurement of oxygen concentration profiles in the particle supported biofilms under defined hydrodynamic conditions. The oxygen concentration profiles inside the biofilms were found to be steeper at high flow velocities in the bulk phase of the flow cell compared to those at low flow velocities. Furthermore, the oxygen flux increased and the thickness of the concentration boundary layer decreased with increasing flow velocity. This dependence was found to be more pronounced in less dense biofilms out of airlift reactors with lower pumice concentrations. In addition confocal laser scanning microscopy (CLSM) was used to visualize the biofilm structure. The volume fractions of bacteria and extracellular polymeric substances (lectin-specific EPS-glycoconjugates) were measured in living fully hydrated biofilms. Both the microelectrode and CLSM measurement showed the influence of shear stress on particle supported biofilms. A higher particle concentration led to dense biofilms with a homogeneous surface, lower thickness of the concentration boundary layer and steeper oxygen concentration profiles. The combination of both techniques allows a detailed and quantitative characterisation of particle associated biofilm structure and function.

Microscale and molecular assessment of impacts of nickel, nutrients, and oxygen level on structure and function of river biofilm communities.

Studies were carried out to assess the influence of nutrients, dissolved oxygen (DO) concentration, and nickel (Ni) on river biofilm development, structure, function, and community composition. Biofilms were cultivated in rotating annular reactors with river water at a DO concentration of 0.5 or 7.5 mg liter(-1), with or without a combination of carbon, nitrogen, and phosphorus (CNP) and with or without Ni at 0.5 mg liter(-1). The effects of Ni were apparent in the elimination of cyanobacterial populations and reduced photosynthetic biomass in the biofilm. Application of lectin-binding analyses indicated changes in exopolymer abundance and a shift in the glycoconjugate makeup of the biofilms, as well as in the response to all treatments. Application of the fluorescent live-dead staining (BacLight Live-Dead staining kit; Molecular Probes, Eugene, Oreg.) indicated an increase in the ratio of live to dead cells under low-oxygen conditions. Nickel treatments had 50 to 75% fewer 'live' cells than their corresponding controls. Nickel at 0.5 mg liter(-1) corresponding to the industrial release rate concentration for nickel resulted in reductions in carbon utilization spectra relative to control and CNP treatments without nickel. In these cases, the presence of nickel eliminated the positive influence of nutrients on the biofilm. Other culture-dependent analyses (plate counts and most probable number) revealed no significant treatment effect on the biofilm communities. In the presence of CNP and at both DO levels, Ni negatively affected denitrification but had no effect on hexadecane mineralization or sulfate reduction. Analysis of total community DNA indicated abundant eubacterial 16S ribosomal DNA (rDNA), whereas Archaea were not detected. Amplification of the alkB gene indicated a positive effect of CNP and a negative effect of Ni. The nirS gene was not detected in samples treated with Ni at 0.5 mg liter(-1), indicating a negative effect on specific populations of bacteria, such as denitrifiers, resulting in a reduction in diversity. Denaturing gradient gel electrophoresis revealed that CNP had a beneficial impact on biofilm bacterial diversity at high DO concentrations, but none at low DO concentrations, and that the negative effect of Ni on diversity was similar at both DO concentrations. Notably, Ni resulted in the appearance of unique bands in 16S rDNA from Ni, DO, and CNP treatments. Sequencing results confirmed that the bands belonged to bacteria originating from freshwater and marine environments or from agricultural soils and industrial effluents. The observations indicate that significant interactions occur between Ni, oxygen, and nutrients and that Ni at 0.5 mg liter(-1) may have significant impacts on river microbial community diversity and function.

Inhibition of lotic biofilms by Diclofenac.

Diclofenac, a common drug, was subjected to degradation studies using river biofilms grown in rotating annular reactors. Degradation of diclofenac was possible after acclimatisation as confirmed by liquid chromatography-mass spectrometry analyses. Adapted biofilms showed that degradation down to 10-25% of the initial concentration could be achieved within 4 days. In situ observation by confocal laser scanning microscopy, however, revealed slow biofilm development in the presence of diclofenac compared with control experiments grown in river water only. This was substantiated by low cell counts and isolation of fewer kinds of microorganisms from diclofenac-grown biofilms. Fluorescent in situ hybridisation analyses confirmed the presence of various bacterial groups, especially those belonging to the Cytophaga-Flavobacterium and gamma-Proteobacteria groups, in the biofilms. Quantification of image data indicated a negative effect of diclofenac on the growth of bacteria and algae. This is the first report on degradation of diclofenac by lotic biofilms.