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M Hausner

Publications and source records attributed to M Hausner.

At least 19 recordsLinked to original sources
PubMed2005

Compatibility of the green fluorescent protein and a general nucleic acid stain for quantitative description of a Pseudomonas putida biofilm.

Better understanding of biofilm development is essential for making optimal use of beneficial biofilms as well as for devising effective control strategies for detrimental biofilms. Analysis of biofilm structure and quantification of biofilm parameters using optical (including confocal) microscopy and digital image analysis techniques are becoming routine in many laboratories. The purpose of this study was to evaluate a dual labeling technique based on fluorescence signals from the green fluorescent protein (GFP) and those resulting from staining with the general nucleic acid stain SYTO 60 for the quantitative description of a model biofilm. For this purpose, a Pseudomonas putida KT2442 derivative was genetically tagged with the green fluorescent protein gene. Biofilm formation by this strain was investigated using flow cells and confocal laser scanning microscopy (CLSM). Percentage surface coverage as well as microcolony size quantified using GFP and SYTO 60 signals showed significant correlation (R=0.99). The results indicated that intrinsic labelling of this model biofilm using constitutively expressed proteins such as GFP can be used for real-time biofilm observation and generation of reliable quantitative data, comparable to those obtained using conventional methods such as nucleic acid staining. Non-destructive time series observation of GFP-expressing biofilms in flow-cells can thus be confidently used for four-dimensional (x, y, z, t) analysis and quantification of biofilm development. The results also point to the possibility of using GFP and SYTO 60 to study dual species biofilms, as quantitative data generated using both fluorophore signals are comparable.

Biofilms↗
PubMed2005

Plasmid-mediated bioaugmentation of activated sludge bacteria in a sequencing batch moving bed reactor using pNB2.

AIMS: The applicability of plasmid pNB2 for bioaugmentation of bacteria in model wastewater treatment reactors receiving 3-chloroaniline (3-CA) was investigated. METHODS AND RESULTS: A setup of three biofilm reactors was studied, all initially inoculated with bacteria from activated sludge. Reactor PB received a Pseudomonas putida pNB2 donor strain not able to degrade 3-CA. Positive control reactor P received a 3-CA degrading Comamonas testosteroni pNB2-transconjugant. The negative control reactor N remained unchanged. Reactor P showed 3-CA degradation from the beginning of the experiment whereas in reactor PB, degradation started after an initial lag period. No degradation was observed in reactor N. PCR analysis showed that the P. putida donor abundance dropped in reactor PB, whereas the plasmid abundance did not, indicating transfer to other bacteria. A number of different 3-CA degrading C. testosteroni strains carrying pNB2 could be isolated from reactor PB. CONCLUSIONS: A successful plasmid-mediated bioaugmentation was achieved with C. testosteroni being the dominant 3-CA degrading pNB2 transconjugant species active in reactor PB. SIGNIFICANCE AND IMPACT OF THE STUDY: The study underlines the potential of gene transfer to contribute to establishment and spread of genetic information in general, particularly emphasizing the spread of xenobiotic degrading potential by dissemination of catabolic genes.

Aniline Compounds↗
PubMed2005

Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge.

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-mum cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 +/- 12 ml g(-1), and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 +/- 2 ml g(-1). EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.

Aerobiosis↗
PubMed2005

Architecture of a nascent Sphingomonas sp. biofilm under varied hydrodynamic conditions.

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 x 10(5) microm(2)) or microcolony size (1 x 10(6) microm(2)) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 mum above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-a-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.

Biofilms↗
PubMed2004

Microbial communities and their interactions in biofilm systems: an overview.

Several important advances have been made in the study of biofilm microbial populations relating to their spatial structure (or architecture), their community structure, and their dependence on physicochemical parameters. With the knowledge that hydrodynamic forces influence biofilm architecture came the realization that metabolic processes may be enhanced if certain spatial structures can be forced. An example is the extent of plasmid-mediated horizontal gene transfer in biofilms. Recent in situ work in defined model systems has shown that the biofilm architecture plays a role for genetic transfer by bacterial conjugation in determining how far the donor cells can penetrate the biofilm. Open channels and pores allow for more efficient donor transport and hence more frequent cell collisions leading to rapid spread of the genes by horizontal gene transfer. Such insight into the physical environment of biofilms can be utilized for bioenhancement of catabolic processes by introduction of mobile genetic elements into an existing microbial community. If the donor organisms themselves persist, bioaugmentation can lead to successful establishment of newly introduced species and may be a more successful strategy than biostimulation (the addition of nutrients or specific carbon sources to stimulate the authochthonous population) as shown for an enrichment culture of nitrifying bacteria added to rotating disk biofilm reactors using fluorescent in situ hybridization (FISH) and microelectrode measurements of NH4+, NO2-, NO3-, and O2. However, few studies have been carried out on full-scale systems. Bioaugmentation and bioenhancement are most successful if a constant selective pressure can be maintained favoring the promulgation of the added enrichment culture. Overall, knowledge gain about microbial community interactions in biofilms continues to be driven by the availability of methods for the rapid analysis of microbial communities and their activities. Molecular tools can be grouped into those suitable for ex situ and in situ community analysis. Non-spatial community analysis, in the sense of assessing changes in microbial populations as a function of time or environmental conditions, relies on general fingerprinting methods, like DGGE and T-RFLP, performed on nucleic acids extracted from biofilm. These approaches have been most useful when combined with gene amplification, cloning and sequencing to assemble a phylogenetic inventory of microbial species. It is expected that the use of oligonucleotide microarrays will greatly facilitate the analysis of microbial communities and their activities in biofilms. Structure-activity relationships can be explored using incorporation of 13C-labeled substrates into microbial DNA and RNA to identify metabolically active community members. Finally, based on the DNA sequences in a biofilm, FISH probes can be designed to verify the abundance and spatial location of microbial community members. This in turn allows for in situ structure/function analysis when FISH is combined with microsensors, microautoradiography, and confocal laser scanning microscopy with advanced image analysis.

Bacteria↗
PubMed2004

Bioaugmentation of a sequencing batch biofilm reactor by horizontal gene transfer.

Bioaugmentation by introduction of catabolic genes residing on mobile genetic elements into the microbial community of a soil or wastewater environment might be an alternative to bioaugmentation by addition of bacterial cells with chromosomally encoded catabolic genes. This study investigates the possibility to enhance degradation of the xenobiotic model compound 2,4-dichlorophenoxyacetic acid in a sequencing batch biofilm reactor (SBBR) by using the conjugative plasmid pJP4 carrying genes for 2,4-D degradation. After introduction of a plasmid donor strain to a lab-scale SBBR operated without 2,4-D, the number of plasmid-carrying cells first dropped, and then increased after switching to 2,4-D as the sole carbon source. The donor cells were unable to grow in the applied synthetic wastewater with 2,4-D as the sole carbon source. Transconjugants could be detected both by culture-dependent and culture-independent methods in the 2,4-D degrading biofilm. In contrast to 90% 2,4-D degradation in the bioaugmented reactor within 40 h, a control reactor which had not received the plasmid still contained 60% of the initial 2,4-D concentration after 90 h. This experiment clearly demonstrates the introduction of 2,4-D degradative genes into a microbial biofilm and indicates that horizontal gene transfer is a promising tool for bioaugmentation of reactors treating wastewater.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2003

Population dynamics of rumen microbes using modern techniques in rumen enhanced solid incubation.

The microbial ecology of the rumen is very complex. Different species of bacteria, protozoa, and fungi are involved in digestion of plant material in ruminants. In spite of complicated interrelationships among the various groups of microorganisms in the rumen ecosystem, Bacteria and Archaea are believed to play a major role because of their numerical predominance and metabolic diversity. In this work we are presenting the results for microbial population dynamics of rumen microbes during two-stage anaerobic digestion of grass. The reactors were inoculated with fresh rumen content. Fluorescent in situ hybridization, confocal laser scanning microscopy and epifluorescence microscopy were employed for microbial investigation. It was observed that Bacteria dominated in the hydrolytic reactor (1st stage) whereas Archaea were predominant in the methanogenic reactor (2nd stage). The stability of the methanogenic reactor was result of the dominance of Methanosaeta species (mainly the filamentous type).

Animals↗
PubMed2002

A photoacoustic technique for depth-resolved in situ monitoring of biofilms.

Biofilms occur in natural and engineered water systems. Biofouling in technical processes lowers the water quality and increases the frictional resistance in tubes. In wastewater treatment plants, biofilms are used for removal of organic an inorganic pollutants. For improvement of antifouling strategies and for process optimization in wastewater treatments plants, an analytical technique for online monitoring of biofilms is needed. In this article, a new setup for in situ monitoring of biofilms by photoacoustic spectroscopy is presented. To produce a biofilm, a mixture of microorganisms was grown in a nutrient solution inside a tube reactor. The content of the tube reactor was pumped through a flow channel, and biofilms were generated at the inner surfaces. Three photoacoustic sensor heads were integrated at different positions into the base plate of the flow channel. By photoacoustic spectroscopy, growth, thickness, and detachment of biofilms can be monitored on-line and nondestructively. Experiments presented in this article showed that the flow conditions influence the structure and thickness of biofilms. By changing the pH value, electrostatic interactions inside the biofilm matrix were influenced, and the subsequent detachment processes were observed online. The interaction of iron(III) oxide particles with biofilms led to particle adsorption on the outer and inner surfaces of the biofilm. Afterwards, biofilm flocs were sloughed off from the base biofilm.

Acoustics↗
PubMed2001

Time-resolved study of biofilm architecture and transport processes using experimental and simulation techniques: the role of EPS.

Cellular material and extracellular polymeric substances are the basic structural elements in biofilm systems. The structure and role of EPS for biofilm development and metabolic processes have not been precisely determined and, therefore, have not yet been included as a necessary element in modelling and simulation studies. This is due to the difficulty of experimentally detecting the extracellular polymeric substances in situ and differentiating them from cellular material on the one hand, and to the subsequent uncertainty about appropriate models--e.g. rigid hindrances, porous microstructure or visco-elastic structure--on the other hand. In this work, we report on the use of confocal laser scanning microscopy to monitor the development of a monoculture biofilm of Sphingomonas sp. grown in a flow cell. The bacterial strain was genetically labelled resulting in strong constitutive expression of the green fluorescent protein. The development of extracellular polymeric substances was followed by binding of the lectin concavalin A to cell exopolysaccharides. The growth of the resulting strain was digitally recorded by automated confocal laser scanning microscopy. In addition, local velocity profiles of fluorescent carboxylate-modified microspheres were observed on pathlines throughout the biofilm. The CLSM image stacks were used as direct input for the explicit modelling and three-dimensional numerical simulation of flow fields and solute transport processes based on the conservation laws of continuum mechanics. At present, a strongly simplifying EPS-model is applied for numerical simulations. The EPSs are preliminarily assumed to behave like a rigid and dense hindrance with diffusive-reactive solute transport.

Biofilms↗
PubMed2000

Evaluation of fluorescently labeled lectins for noninvasive localization of extracellular polymeric substances in Sphingomonas biofilms.

Three strains of Sphingomonas were grown as biofilms and tested for binding of five fluorescently labeled lectins (Con A-type IV-TRITC or -Cy5, Pha-E-TRITC, PNA-TRITC, UEA 1-TRITC, and WGA-Texas red). Only ConA and WGA were significantly bound by the biofilms. Binding of the five lectins to artificial biofilms made of the commercially available Sphingomonas extracellular polysaccharides was similar to binding to living biofilms. Staining of the living and artificial biofilms by ConA might be explained as binding of the lectin to the terminal mannosyl and terminal glucosyl residues in the polysaccharides secreted by Sphingomonas as well as to the terminal mannosyl residue in glycosphingolipids. Staining of the biofilms by WGA could only be explained as binding to the Sphingomonas glycosphingolipid membrane, binding to the cell wall, or nonspecific binding. Glycoconjugation of ConA and WGA with the target sugars glucose and N-acetylglucosamine, respectively, was used as a method for evaluation of the specificity of the lectins towards Sphingomonas biofilms and Sphingomonas polysaccharides. Our results show that the binding of lectins to biofilms does not necessarily prove the presence of specific target sugars in the extracellular polymeric substances (EPS) in biofilms. The lectins may bind to non-EPS targets or adhere nonspecifically to components of the biofilm matrix.

Biofilms↗
PubMed1999

High rates of conjugation in bacterial biofilms as determined by quantitative in situ analysis.

Quantitative in situ determination of conjugative gene transfer in defined bacterial biofilms using automated confocal laser scanning microscopy followed by three-dimensional analysis of cellular biovolumes revealed conjugation rates 1,000-fold higher than those determined by classical plating techniques. Conjugation events were not affected by nutrient concentration alone but were influenced by time and biofilm structure.

Bacteria↗
PubMed1998

Automated confocal laser scanning microscopy and semiautomated image processing for analysis of biofilms.

The purpose of this study was to develop and apply a quantitative optical method suitable for routine measurements of biofilm structures under in situ conditions. A computer program was designed to perform automated investigations of biofilms by using image acquisition and image analysis techniques. To obtain a representative profile of a growing biofilm, a nondestructive procedure was created to study and quantify undisturbed microbial populations within the physical environment of a glass flow cell. Key components of the computer-controlled processing described in this paper are the on-line collection of confocal two-dimensional (2D) cross-sectional images from a preset 3D domain of interest followed by the off-line analysis of these 2D images. With the quantitative extraction of information contained in each image, a three-dimensional reconstruction of the principal biological events can be achieved. The program is convenient to handle and was generated to determine biovolumes and thus facilitate the examination of dynamic processes within biofilms. In the present study, Pseudomonas fluorescens or a green fluorescent protein-expressing Escherichia coli strain, EC12, was inoculated into glass flow cells and the respective monoculture biofilms were analyzed in three dimensions. In this paper we describe a method for the routine measurements of biofilms by using automated image acquisition and semiautomated image analysis.

Automation↗
PubMed1997

Direct examination of subgingival plaque from a diseased periodontal site using confocal laser scanning microscopy (CLSM).

Material taken directly from a periodontal site was investigated using immunofluorescence, acridine orange staining and confocal laser scanning microscopy (CLSM). Porphyromonas gingivalis was tracked by a specific polyclonal antibody and its pronounced occurrence in inflamed as compared to non-inflamed areas was demonstrated. Further accompanying microorganisms were counterstained with acridine orange which could provide information on the viability of individual cells. Optical sections by laser microscopy revealed the spatial arrangement of the investigated material. The combination of specific staining and CLSM allows a detailed microbiological investigation of clinical material obtained directly without cultivation.

Antibodies, Bacterial↗