Investigation of lotic microbial aggregates by a combined technique of fluorescent in situ hybridization and lectin-binding-analysis

Böckelmann, Uta; Manz, Werner; Neu, Thomas R.; Szewzyk, Ulrich

doi:10.1016/s0167-7012(01)00354-2

Cited by 77 publications

(52 citation statements)

References 33 publications

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“…Using CLSM, it has been possible to delineate regions of space, cells, and exopolymeric materials, while optically sectioning and creating 3-D images of biofilms (3,28,30,42). However, as discussed by Neu et al (30), there are limitations-particularly to the chemical interpretation of fluorescent probe binding patterns in complex environments such as biofilms.…”

Section: Resultsmentioning

confidence: 99%

“…Although the N 1s region provides useful corroboration, only C 1s results are presented in this paper. For C 1s studies of organic material with a density of ϳ1 cm 3 , a sample thickness of a dry sample between 80 and 200 nm gives a peak optical density of 1 to 2, which is optimum with regard to statistical precision and avoiding absorption saturation (which in the STXM532 microscope starts at an optical density of around 3). This is typical of the preparation of solid polymer samples.…”

Section: Methodsmentioning

confidence: 99%

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Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

Lawrence¹,

Swerhone²,

Leppard³

et al. 2003

Appl Environ Microbiol

326

270

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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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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

Lawrence¹,

Swerhone²,

Leppard³

et al. 2003

Appl Environ Microbiol

326

270

View full text Add to dashboard Cite

show abstract

“…Concanavalin A and wheat germ agglutinin lectins labelled with FITC were used to stain the glycoconjugate fractions of the extracellular polymeric substances (EPSs) (7,28). Since these stains can also bind to protein and glycoconjugate groups associated with bacterial cell walls, counter-staining with PI was performed to distinguish the EPSs from the cell wall components.…”

Section: Staining Of Nucleic Acids and Lectinmentioning

confidence: 99%

Microbial Population Dynamics and Community Structure during the Formation of Nitrifying Granules to Treat Ammonia-Rich Inorganic Wastewater

et al. 2010

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Microbial population dynamics were investigated during the formation of nitrifying granules in an aerobic upflow fluidized bed (AUFB) reactor fed ammonia as a sole energy source. Analyses of clone libraries of 16S rRNA gene and the ammonia monooxygenase subunit A gene (amoA) revealed that although the clones obtained from the seed sludge were widely distributed among the ammonia-oxidizing bacteria (AOB) isolates, the community structure of AOB shifted towards the Nitrosomonas mobilis lineage as granulation proceeded. Quantitative fluorescence in situ hybridization showed that changes in the bacterial population occurred concomitantly with changes in nitrification performance and the size of granules. AOB associated with the N. mobilis lineage were predominant in the early stages as nitrifying granules formed (average diameter, 126 µm). In mature granules (average diameter, 270 µm), at least three types of AOB, N. mobilis, Nitrosomonas oligotropha, and Nitrosomonas europaea, formed different niches and coexisted. Nitrite-oxidizing bacteria (NOB) affiliated with Nitrospira spp. were detected in the start-up period, but were replaced by NOB affiliated with Nitrobacter spp. after granules formed.Key words: community structure, population dynamics, nitrifying bacteriaThe removal of biological nitrogen from various types of wastewater is becoming increasingly important owing to the implementation of strict regulations concerning nitrogen discharge. The process of nitrification involves the conversion of ammonia (NH3) or ammonium ions (NH4. Two types of nitrifying bacteria, namely, lithoautotrophic ammonia-oxidizing bacteria (AOB) and lithoautotrophic nitrite-oxidizing bacteria (NOB), play important roles in this process. AOB are classified into two phylogenetic lineages based on their 16S rRNA gene sequences; Nitrosomonas sp. and Nitrosospira sp. belonging to Betaproteobacteria, and Nitrosococcus oceani and Nitrosococcus halophilus belonging to Gammaproteobacteria (19). NOB are mainly classified into two phylogenetic lineages based on the cell morphology of isolated organisms and on 16S rRNA gene sequences. The most intensively studied NOB, Nitrobacter and Nitrospira, fall within the Alphaproeobacteria and separate phyla in Bacteria, respectively (2).It is generally accepted that nitrifying bacteria have a low growth rate and tend to be washed out from reactors. Thus, it is difficult to retain large numbers of them within a reactor. Therefore, there is a need for a simple yet effective method of immobilizing nitrifying bacteria within a reactor. Nitrifying granule production is one method of immobilization used to preserve a large population of nitrifying bacteria within a reactor (12,(40)(41)(42)(43). Tsuneda et al. (41) produced nitrifying granules in inorganic wastewater using an aerobic upflow fluidized bed (AUFB) reactor. These granules exhibited a good settling ability and a high rate of ammonia removal (1.5 kg-N m −3 d −1 ). Therefore, nitrifying granule production is a promising method for the effective remov...

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“…Thomas Neu presented his group's work using fluorescently labeled carbohydrate-specific lectins to analyze EPS distribution in complex environmental biofilms (7,75). In conjunction with confocal microscopy, Neu used the full range of commercially available lectins to evaluate the presence of biofilm-specific glycoconjugates as well as the volume of biofilm cellular and polymeric constituents.…”

Section: Emerging Technologies For Studying Heterogenous Communitiesmentioning

confidence: 99%

Biofilms 2003: Emerging Themes and Challenges in Studies of Surface-Associated Microbial Life

2004

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Investigation of lotic microbial aggregates by a combined technique of fluorescent in situ hybridization and lectin-binding-analysis

Cited by 77 publications

References 33 publications

Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

Microbial Population Dynamics and Community Structure during the Formation of Nitrifying Granules to Treat Ammonia-Rich Inorganic Wastewater

Biofilms 2003: Emerging Themes and Challenges in Studies of Surface-Associated Microbial Life

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