Hans-Curt Flemming

The biofilm matrix

The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mechanical stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix acts as an external digestive system by keeping extracellular enzymes close to the cells, enabling them to metabolize dissolved, colloidal and solid biopolymers. Here we describe the functions, properties and constituents of the EPS matrix that make biofilms the most successful forms of life on earth.

The EPS Matrix: The “House of Biofilm Cells”

In response to a suggestion by the Biofilms 2007 organizing committee to hold an evening session on biofilm extracellular polymeric substances (EPS), an exceptionally inspiring event followed contributions by Ken Bayles, Alan Decho, Martina Hausner, Jan Kreft, Thomas Neu, Per Nielsen, Ute Romling,

Biofouling in water systems - cases, causes and countermeasures

Abstract. Biofouling is referred to as the unwanted deposition and growth of biofilms. This phenomenon can occur in an extremely wide range of situations, from the colonisation of medical devices to the production of ultra-pure, drinking and process water and the fouling of ship hulls, pipelines and reservoirs. Although biofouling occurs in such different areas, it has a common cause, which is the biofilm. Biofilms are the most successful form of life on Earth and tolerate high amounts of biocides. For a sustainable anti-fouling strategy, an integrated approach is suggested which includes the analysis of the fouling situation, a selection of suitable components from the anti-fouling menu and an effective and representative monitoring of biofilm development.

Biofouling—the Achilles heel of membrane processes☆

Microorganisms in membrane systems tend to adhere to surfaces and to form a gel layer called biofilm, which participates in the separation process as a secondary membrane. On the raw water side, it causes an increase of fluid friction resistance which increases Δpfeed/brine. Also, overall hydraulic resistance of the membrane Δpmembrane can increase due to the biofilm. If these effects exceed a certain threshold of interference, they are addressed as biofouling. Countermeasures require a three step protocol: (1) detection, (2) sanitation, and (3) prevention. Detection has to be performed on the surface as planctonic cell numbers released randomly from the biofilm do reflect neither site nor extent of biofilm growth. The analysis includes microbiological and biochemical parameters; the differentiation between other kinds of fouling such as scaling or organic fouling can be performed by FTIR-ATR spectroscopical analysis. Sanitation should be focused on removal of the biomass rather than on killing the microorganisms attached to the surface. First, the slime matrix, consisting mainly of polysaccharides and proteins, must be weakened. This requires interference with the binding forces, which are weak physico-chemical interactions such as hydrogen bonds, van der Waals and electrostatical interactions. Then, increased shear forces can remove the biomass. A preventive concept should acknowledge the fact that biodegradable substances in the water represent the biofouling potential. Biofouling can be regarded as a “biofilm reactor in the wrong place”. Reduction of the nutrient content of the raw water can be achieved by a “biofilm reactor in the right place”, i.e., a biofilter on which microorganisms form biofilms and sequester the nutrients from the water phase. Mandatory for any optimized antifouling strategy is monitoring of biofilm development; a fiber optical device which provides real-time, on-line, in situ information non-destructively is proposed which can be adjusted to membrane modules.

FTIR-spectroscopy in microbial and material analysis

The investigation of the development and the properties of biofilms is difficult because classical microbiology does not offer non-destructive methods other than microscopical observations. This paper discusses the use of different Fourier transform infrared spectroscopy (FTIR-spectroscopy) techniques as a means to investigate microorganisms in biofilms. FTIR-spectroscopy is suitable for the identification of microorganisms and presents a new addition to taxonomic and genetic methods. The FTIR analysis of bacterial isolates provides fingerprint spectra, allowing the rapid characterization of microbial strains. Secondly, the FTIR-attenuated total reflection (ATR) technique can be used for the observation of biofilms forming directly on the interface of an ATR crystals such as germanium. These crystals can be coated to obtain a surface more relevant to study interfacial processes. Spectra can be acquired non-destructively, in situ and in real time. This method is suitable for fundamental biofilm research, as well as for monitoring of biofilm formation, e.g., in an ultrapure or drinking water systems. Furthermore, FTIR—ATR also allows the rapid analysis of deposits on surfaces, e.g., filtration membranes. The analytical discrimination between microorganisms, inorganic material or other foulants can be obtained. Thirdly, with the diffuse reflectance technique (DRIFT) it is possible to investigate reflecting surfaces like metals or very small samples. The composition of surface coatings like biomass or other surface contaminants can be detected. These different measurement techniques demonstrate that FTIR-spectroscopy is suitable for biofilm and surface analysis and can be applied in many different ways.

What are Bacterial Extracellular Polymeric Substances

The vast majority of microorganisms live and grow in aggregated forms such as biofilms and flocs (“planktonic biofilms”). This mode of existence is lumped in the somewhat inexact but generally accepted expression “biofilm”. The common feature of all these phenomena is that the microorganisms are embedded in a matrix of extracellular polymeric substances (EPS). The production of EPS is a general property of microorganisms in natural environments and has been shown to occur both in prokaryotic (Bacteria, Archaea) and in eukaryotic (algae, fungi) microorganisms. Biofilms containing mixed populations of these organisms are ubiquitously distributed in natural soil and aquatic environments, on tissues of plants, animals and man as well as in technical systems such as filters and other porous materials, reservoirs, plumbing systems, pipelines, ship hulls, heat exchangers, separation membranes, etc. (Costerton et al. 1987; 1995; Flemming and Schaule 1996). Biofilms develop adherent to a solid surface (substratum) at solid-water interfaces, but can also be found at water-oil, water-air and solid-air interfaces. Biofilms are accumulations of microorganisms (prokaryotic and eukaryotic unicellular organisms), EPS, multivalent cations, biogenic and inorganic particles as well as colloidal and dissolved compounds. EPS are mainly responsible for the structural and functional integrity of biofilms and are considered as the key components that determine the physicochemical and biological properties of biofilms.

The role of intermolecular interactions: studies on model systems for bacterial biofilms.

The mechanical stability of biofilms and other microbial aggregates is of great importance for both the maintenance of biofilm processes and the removal of undesired biofilms. The binding forces are weak interactions such as London dispersion forces, electrostatic interactions and hydrogen bonds. In a first attempt to rank their contribution, the viscosity of solutions of extracellular polymeric substances (EPS) from a mucoid strain of Pseudomonas aeruginosa is measured. In order to distinguish the binding forces, substances are chosen which individually address the different types of bonds. Polyacrylic acid is identified as a suitable model system for EPS when molecular interactions are studied. Electrostatic interactions and hydrogen bonds are found to be the dominating forces among macromolecules within the biofilm.

Reverse osmosis membrane biofouling

The development of biofouling and its effects on membrane processes are reviewed, emphasizing the microbial attack on the membrane material and its costs. Detection and monitoring techniques are compared, and different types of countermeasures are described. Discussion is also focused on the use of biocides and the cleaning strategies to be followed in practical cases.

Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa.

Fluorescently labelled lectins were used in combination with epifluorescence microscopy and confocal laser scanning microscopy to allow the visualization and characterization of carbohydrate-containing extracellular polymeric substances (EPS) in biofilms of Pseudomonas aeruginosa. A mucoid strain characterized by an overproduction of the exopolysaccharide alginate, and an isogenic, non-mucoid strain were used. Model biofilms grown on polycarbonate filters were treated with lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) that were fluorescently labelled with fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate. Fluorescently labelled ConA yielded cloud-like regions that were heterogeneously distributed within mucoid biofilms, whereas these structures were only rarely present in biofilms of the non-mucoid strain. The bacteria visualized with the fluorochrome SYTO 9 were localized both within and between the ConA-stained regions. In WGA-treated biofilms, the lectin was predominantly associated with bacterial cells. Alginate seemed to be involved in the interaction of ConA with the EPS matrix, since (i) pre-treatment of biofilms with an alginate lyase resulted in a loss of ConA biofilm staining, and (ii) using an enzyme-linked lectinsorbent assay (ELLA), ConA was shown to bind to purified alginate, but not to alginate that was degraded by alginate lyase. The application of fluorescently labelled lectins in combination with ELLA was found to be useful for the visualization and characterization of extracellular polysaccharide structures in P. aeruginosa biofilms.

Metagenome survey of biofilms in drinking-water networks

ABSTRACT Most naturally occurring biofilms contain a vast majority of microorganisms which have not yet been cultured, and therefore we have little information on the genetic information content of these communities. Therefore, we initiated work to characterize the complex metagenome of model drinking water biofilms grown on rubber-coated valves by employing three different strategies. First, a sequence analysis of 650 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to the Proteobacteria. Only a small fraction of the 16S rRNA sequences were highly similar to rRNA sequences from Actinobacteria, low-G+C gram-positives and the Cytophaga-Flavobacterium-Bacteroides group. Our second strategy included a snapshot genome sequencing approach. Homology searches in public databases with 5,000 random sequence clones from a small insert library resulted in the identification of 2,200 putative protein-coding sequences, of which 1,026 could be classified into functional groups. Similarity analyses indicated that significant fractions of the genes and proteins identified were highly similar to known proteins observed in the genera Rhizobium, Pseudomonas, and Escherichia. Finally, we report 144 kb of DNA sequence information from four selected cosmid clones, of which two formed a 75-kb overlapping contig. The majority of the proteins identified by whole-cosmid sequencing probably originated from microbes closely related to the alpha-, beta-, and gamma-Proteobacteria. The sequence information was used to set up a database containing the phylogenetic and genomic information on this model microbial community. Concerning the potential health risk of the microbial community studied, no DNA or protein sequences directly linked to pathogenic traits were identified.