Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highestresolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.Scanning transmission X-ray microscopy (STXM) (1, 2), which uses near-edge X-ray absorption spectroscopy (NEXAFS) as its contrast mechanism, is a powerful new tool that can be applied to fully hydrated biological materials. This is possible due to the ability of soft X rays to penetrate water, the presence of suitable analytical core edges in the soft X-ray region, and reduced radiation damage (compared to that caused by electron beam techniques). Soft X rays also provide spatial resolution of better than 50 nm, which is suitable for imaging bacteria and bacterial biofilms. The spectral resolution is on the order of 100 meV, which in combination with their intrinsic spectral properties is sufficient to provide good differentiation of classes of biomolecules (26, 43; X. Zhang, T. Araki, A. P. Hitchcock, J. R. Lawrence, and G. G. Leppard, unpublished data). Through the application of tunable soft X rays and appropriate analysis of X-ray absorption spectra in the form of NEXAFS image sequences (15), quantitative chemical mapping at a spatial scale below 50 nm may be achieved. With use of the appropriate spectral range, NEXAFS microscopy provides detailed, quantitative speciation and elemental analysis with parts-per-thousand local and parts-per-million global sensitivities with transmission detection. Soft X-ray microscopy provides a combination of suitable spatial resolution and chemical information at a microscale. In addition, soft X rays interact with nearly all elements and also allow mapping of chemical species based on bonding structure (2). Further, the method uses the intrinsic X-ray absorption properties of the sample, thus eliminating the need for addition of reflective, absorptive, or fluorescent probes and markers that may introduce artifacts or complicate interpretation. It is...

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A scanning transmission X-ray microscope illuminated with synchrotron light was used to investigate the speciation and spatial distributions of metals in a microbial biofilm cultivated from river water. Metal 2p absorption edge signals were used to provide metal speciation (through shapes of the absorption spectra) and quantitative spatial distributions of the metal species. This paper presents sample data and describes methods for extracting quantitative maps of metal species from image sequences recorded in the region of the metal 2p edges. Comparisons were made with biochemical characterization of the same region using images recorded at the C 1s and O 1s edges. The method is applied to detailed quantitative analysis of ferrous and ferric iron in a river biofilm, in concert with mapping Ni-(II) and Mn(II) species in the same region. The distributions of the metal species are discussed in the context of the biofilm structure. These results demonstrate that soft X-ray STXM measurements at the metal 2p absorption edges can be used to speciate metals and to provide quantitative spatial distribution maps for metal species in environmental samples with 50 nm spatial resolution.

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Although pharmaceutical and therapeutic products are widely found in the natural environment, there is limited understanding of their ecological effects. Here we used rotating annular bioreactors to assess the impact of 10 microg.L(-1) of the selected pharmaceuticals ibuprofen, carbamazepine, furosemide, and caffeine on riverine biofilms. After 8 weeks of development, community structure was assessed using in situ microscopic analyses, fluor-conjugated lectin binding, standard plate counts, fluorescent in situ hybridization, carbon utilization spectra, and stable carbon isotope analyses. The biofilm communities varied markedly in architecture although only caffeine treated biofilms were significantly thicker. Cyanobacteria were suppressed by all 4 compounds, whereas the nitrogen containing caffeine, furosemide, and carbamazepine increased algal biomass. Ibuprofen and carbamazepine reduced bacterial biomass, while caffeine and furosemide increased it. Exopolymer content and composition of the biofilms was also influenced. Significant positive and negative effects were observed in carbon utilization spectra. In situ hybridization analyses indicated all treatments significantly decreased the gamma-proteobacterial populations and increased beta-proteobacteria. Ibuprofen in particular increased the alpha-proteobacteria, beta-proteobacteria, cytophaga-flavobacteria, and SRB385 probe positive populations. Caffeine and carbamazepine additions resulted in significant increases in the high GC354c and low GC69a probe positive cells. Live-dead analyses of the biofilms indicated that all treatments influenced the ratio of live-to-dead cells with controls having a ratio of 2.4, carbamazepine and ibuprofen being 3.2 and 3.5, respectively, and furosemide and caffeine being 1.9 and 1.7, respectively. Stable isotope analyses of the biofilms indicated delta 13C values shifted to more negative values relative to control biofilms. This shift may be consistent with proportional loss of cyanobacteria and relative increase in algal biomass rather than incorporation of pharmaceutical carbon into microbial biofilm. Thus, at 10 microg.L(-1) levels pharmaceuticals exhibit both nutrient-like and toxic effects on riverine microbial 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.

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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.