FIGURE 6.7 (A) CLSM image of mixed species river biofilm stained with nucleic acid sensitive stain Syto9; (B) STXM image of the same location showing the location of nucleic acids as detected by fitting models based on spectra in Figure 6.3; (C) localization of fucose containing polysaccharide using the fucose sensitive lectin Tetragonolobuspurpureas; and (D) the same area imaged using STXM and fitting of general polysaccharide.

identification of polysaccharide by CLSM and the probe independent detection based on soft x-ray spectroscopy. Lectin binding could be shown to identify subsets of the total polysaccharide regions detected using STXM (Figure 6.7). Significant questions remain however regarding the precise chemical interpretation of the binding of a specific lectin. Antibodies

Antibodies have been suggested as potential probes for sugars and carbohydrates, however there are limitations to their application in complex microbial communities. For example, (i) the production of antibodies against carbohydrates is in general difficult relative to proteins, (ii) it requires the isolation of pure polysaccharide material from the complex polysaccharide matrix of a complex microbial biofilm community, and (iii) if the antibody could be produced its specificity would allow only the detection of a very limited fraction of the carbohydrates present in a complex biofilm community. Thus the application of antibodies presents significant technical and interpretative barriers for in situ characterization procedures.

6.3.2 Proteins-Lipids

Proteins are major constituents of the exopolymeric matrix of floc and biofilm systems. Particularly in activated sludge flocs, protein can be the most important contributor representing 50% or more of the extractable EPS.14,54 Both extractive analysis and in situ detection of protein is complicated by the presence of lipoproteins and gly-coproteins, molecules that have a chemistry representative of more than one class of biomolecule.

Neu and Marshall55 applied a "protein specific" probe Hoechst 2495 to detect bacterial footprints on surfaces. In this case these remnant structures were readily detected with this dye, consistent with the presence of a high level of protein in the EPS. The SYPRO series of protein stains, although developed for protein in gels and solutions have been proposed for application in situ. Lawrence et al.25 applied SYPRO orange alone and in combination with other macromolecular stains. These SYPRO stains bound extensively in the biofilm system, both in a cell associated and matrix distributed pattern. They found strong colocalization of protein, lipid, and polysaccharide. Parallel studies using STXM verified that colocalization was a valid interpretation and representative of conditions in the biofilm matrix. Again this may reflect the lipoprotein, glycoprotein distribution in the matrix polymer.

The hydrophobic lipid stain Nile Red has also been used extensively to detect lipids in algal and bacterial cells and associated materials. Wolfaardt et al.56 reported using Nile Red to detect hydrophobic cell surfaces within a degradative biofilm community, while Lamont et al.57 indicated that lipid deposits associated with Frankia could be localized.

6.3.3 Nucleic Acids

Nucleic acids are also abundant in biofilms both as cell associated DNA-RNA and within the extracellular matrix of the biofilm or floc. Although noncellular binding of nucleic acid stains has often been classified as nonspecific staining, it has become apparent that DNA may be a structural component of biofilms. Indeed extractive studies have often indicated that a considerable fraction of the biofilm EPS is DNA. Some reports reviewed in Nielsen and Jahn14 indicate that nucleic acids may comprise 5 to 15% of the extracellular materials in pure culture biofilms and activated sludge.

Recently, Whitchurch et al.58 indicated using pure cultures that this extracellular DNA may be structural in nature and required for biofilm development. Similarly, Lawrence et al.25 detected extracellular nucleic acids in biofilm materials using STXM. Correlation of fluorescent nucleic acid staining with the results of soft x-ray analyses indicated that both detected regions of non-cellular nucleic acids within a biofilm.

6.3.4 Charge/Hydrophobicity

The essential approach to in situ determination of surface charge involves the application of probes with known characteristics with assessment of their binding patterns in the floc matrix. The use of fluorescent beads with sulfated or carboxylated surface chemistry has been used for determination of hydrophobicity and hydrophilicity of bacterial cells and may be used for flocs. Zita and Hermannson59 describe the essential method using beads obtained from Molecular Probes Inc. (Molecular Probes, Eugene, OR). Fluorescent beads have also been used to analyze under in situ conditions the surface properties of filamentous bacteria in activated sludge flocs.60 As noted above the binding of the hydrophobic dye Nile Red a lipophilic compound may also be interpreted as recognition of hydrophobic regions. Similarly, dextrans may be obtained with anionic, polyanionic, neutral, or positive charges, these may also be reacted with microbial EPS to assess charge and charge distribution. This approach has been applied by Wolfaardt et al.56 Figure 6.8 are three-dimensional (3D) stereo pairs of the binding of 100 nm carboxylate and 20 nm sulfate modified beads at the same location in a river biofilm. The image shows differential binding based on hydrophobicity and penetration of the biofilm material based on hydrated radius of the probe.

FIGURE 6.8 CLSM stereo pairs A and B showing the differential sorption of the 100 nm carboxylate modified beads and 20 nm sulfate modified beads in river biofilm.

6.3.5 Permeability

There are a number of fluor conjugated probes that may be used to assess permeability and diffusion coefficients of bacterial cells and polymer, these include ficols, size fractionated dextrans, and a range of fluorescent beads (10 nm to 15 ^m diameter) (see, e.g., Molecular Probes, Eugene, OR). Lawrence et al.61 used 1P-LSM to monitor the migration of fluor conjugated dextrans to determine effective diffusion coefficients for biofilm systems. Microinjection and 1P-LSM has been developed by De Beer et al.62 to determine diffusion coefficients in biofilm materials. The standard FRAP (fluorescence recovery after photobleaching) approach may also be applied to bacteria, aggregates, and biofilms. Figure 6.9 (see also Section 6.3.4) illustrates the penetration of biofilm microcolonies by 100 and 20 nm diameter fluorescent beads.

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