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.

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.

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.

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.