Fluorescence Lectin-binding Analysis Research Articles

Combined pH ratiometry and fluorescence lectin-binding analysis (pH-FLBA) for microscopy-based analyses of biofilm pH and matrix carbohydrates.

Bacterial biofilms have a complex and heterogeneous three-dimensional architecture that is characterized by chemically and structurally distinct microenvironments. Confocal microscopy-based pH ratiometry and fluorescence lectin-binding analysis (FLBA) are well-established methods to characterize pH developments and the carbohydrate matrix architecture of biofilms at the microscale. Here, we developed a combined analysis, pH-FLBA, to concomitantly map biofilm pH and the distribution of matrix carbohydrates in bacterial biofilms while preserving the biofilm microarchitecture. As a proof of principle, the relationship between pH and the presence of galactose- and fucose-containing matrix components was investigated in dental biofilms grown with and without sucrose. The pH response to a sucrose challenge was monitored in different areas at the biofilm base using the ratiometric pH-sensitive dye C-SNARF-4. Thereafter, the fucose- and galactose-specific fluorescently labeled lectins Aleuria aurantia lectin (AAL) and Morus nigra agglutinin G (MNA-G) were used to visualize carbohydrate matrix components in the same biofilm areas and their immediate surroundings. Sucrose during growth significantly decreased biofilm pH (P < 0.05) and increased the amounts of both MNA-G- and AAL-targeted matrix carbohydrates (P < 0.05). Moreover, it modulated the biofilm composition towards a less diverse community dominated by streptococci, as determined by 16S rRNA gene sequencing. Altogether, these results suggest that the production of galactose- and fucose-containing matrix carbohydrates is related to streptococcal metabolism and, thereby, pH profiles in dental biofilms. In conclusion, pH-FLBA using lectins with different carbohydrate specificities is a useful method to investigate the association between biofilm pH and the complex carbohydrate architecture of bacterial biofilms.IMPORTANCEBiofilm pH is a key regulating factor in several biological and biochemical processes in environmental, industrial, and medical biofilms. At the microscale, microbial biofilms are characterized by steep pH gradients and an extracellular matrix rich in carbohydrate components with diffusion-modifying properties that contribute to bacterial acid-base metabolism. Here, we propose a combined analysis of pH ratiometry and fluorescence lectin-binding analysis, pH-FLBA, to concomitantly investigate the matrix architecture and pH developments in microbial biofilms, using complex saliva-derived biofilms as an example. Spatiotemporal changes in biofilm pH are monitored non-invasively over time by pH ratiometry, while FLBA with lectins of different carbohydrate specificities allows mapping the distribution of multiple relevant matrix components in the same biofilm areas. As the biofilm structure is preserved, pH-FLBA can be used to investigate the in situ relationship between the biofilm matrix architecture and biofilm pH in complex multispecies biofilms.

Unique architecture of microbial snottites from a methane driven biofilm revealed by confocal microscopy.

Microbial biofilms occur in many shapes and different dimensions. In natural and semi-artificial caves they are forming pendulous structures of 10 cm and more. In this study a methane driven microbial community of a former medicinal spring was investigated. The habitat was completely covered by massive biofilms and snottites with a wobbly, gelatinous appearance. By using fluorescence techniques in combination with confocal laser scanning microscopy the architecture of these so far unknown snottites was examined. The imaging approaches applied comprised reflection of geogenic and cellular origin, possible autofluorescence, nucleic acid staining for bacterial cells, protein staining for bacteria and extracellular fine structures, calcofluor white for β 1 → 3, β 1 → 4 polysaccharide staining for possible fungi as well as lectin staining for the extracellular biofilm matrix glycoconjugates. The results showed a highly complex, intricate structure with voluminous, globular, and tube-like glycoconjugates of different dimensions and densities. In addition, filamentous bacteria seem to provide additional strength to the snottites. After screening with all commercially available lectins, by means of fluorescence lectin barcoding and subsequent fluorescence lectin binding analysis, the AAL, PNA, LEA, and Ban lectins identified α-Fuc, β-Gal, β-GlcNAc, and α-Man with α-Fuc as a major component. Examination of the outer boundary with fluorescent beads revealed a potential outer layer which could not be stained by any of the fluorescent probes applied. Finally, suggestions are made to further elucidate the characteristics of these unusual microbial biofilms in form of snottites. RESEARCH HIGHLIGHTS: The gelatinous snottites revealed at the microscale a highly complex structure not seen before. The extracellular matrix of the snottite biofilm was identified as clusters of different shape and density. The matrix of snottites was examined by taking advantage of 78 fluorescently-labeled lectins. The extracellular matrix glycoconjugates of snottites identified comprised: α-Fuc, β-Gal, β-GlcNAc, and α-Man. Probing the snottite outer surface indicated an additional unknown stratum.

Characterization of Extracellular Polymeric Substances Produced by an Acidianus Species and Their Relevance to Bioleaching

Extracellular polymeric substances (EPS) produced by microorganisms play a crucial role in various bioprocesses, including bioleaching. The microbial leaching of metal sulfides requires an initial cell attachment, which is facilitated by EPS. These substances are mixtures of polysaccharides, proteins, lipids, and other compounds, and their composition and properties can vary depending on the species, growth conditions, and environmental factors. Despite the significance of iron/sulfur oxidizing species in biomining processes, the knowledge of the interfacial processes between thermoacidophilic archaeal species and mineral surfaces is limited. This study examines the cell surface characteristics and EPS produced by an Acidianus strain. The research was conducted using microscopic techniques, Zeta-potential measurements, spectrophotometric methods, Fourier transform infrared spectroscopy, and fluorescence lectin-binding analysis. The results suggest that non-soluble substrates, such as sulfur or pyrite, induce changes in cell surface structures, including the presence of cell appendages, wider cell envelopes, higher hydrophobicities, and increased EPS production, compared to cells grown with soluble substrates such as tetrathionate or ferrous iron. The EPS mainly consist of proteins and carbohydrates, including glucose, manose, N-acetylgalactosamine, and N-acetylglucosamine residues. This study contributes to a better understanding of the relationship between thermophilic archaea and mineral surfaces in biomining processes.

Fluorescence lectin binding analysis of carbohydrate components in dental biofilms grown in situ in the presence or absence of sucrose.

Carbohydrate components, such as glycoconjugates and polysaccharides, are constituents of the dental biofilm matrix that play an important role in biofilm stability and virulence. Exopolysaccharides in Streptococcus mutans biofilms have been characterized extensively, but comparably little is known about the matrix carbohydrates in complex, in situ-grown dental biofilms. The present study employed fluorescence lectin binding analysis (FLBA) to investigate the abundance and spatial distribution of glycoconjugates/polysaccharides in biofilms (n=306) from 10 participants, grown in situ with (SUC) and without (H2O) exposure to sucrose. Biofilms were stained with 10 fluorescently labeled lectins with different carbohydrate specificities (AAL, ABA, ASA, HPA, LEA, MNA-G, MPA, PSA, VGA and WGA) and analyzed by confocal microscopy and digital image analysis. Microbial composition was determined by 16S rRNA gene sequencing. With the exception of ABA, all lectins targeted considerable matrix biovolumes, ranging from 19.3% to 194.0% of the microbial biovolume in the biofilms, which illustrates a remarkable variety of carbohydrate compounds in in situ-grown dental biofilms. MNA-G, AAL, and ASA, specific for galactose, fucose, and mannose, respectively, stained the largest biovolumes. AAL and ASA biovolumes were increased in SUC biofilms, but the difference was not significant due to considerable biological variation. SUC biofilms were enriched in streptococci and showed reduced abundances of Neisseria and Haemophilus spp., but no significant correlations between lectin-stained biovolumes and bacterial abundance were observed. In conclusion, FLBA demonstrates the presence of a voluminous biofilm matrix comprising a variety of different carbohydrate components in complex, in situ-grown dental biofilms.

Matrix glycoconjugate characterization in multispecies biofilms and bioaggregates from the environment by means of fluorescently-labeled lectins.

Environmental biofilms represent a complex mixture of different microorganisms. Their identity is usually analyzed by means of nucleic acid-based techniques. However, these biofilms are also composed of a highly complex extracellular matrix produced by the microbes within a particular biofilm system. The biochemical identity of this extracellular matrix remains in many cases an intractable part of biofilms and bioaggregates. Consequently, there is a need for an approach that will give access to the fully hydrated structure of the extracellular matrix or at least a major part of it. A crucial compound of the matrix identified as carbohydrate-based polymers represents major structural and functional constituents. These glycoconjugates can be characterized by using fluorescently-labeled lectins in combination with confocal laser scanning microscopy. The lectin approach is defined previously, as fluorescence lectin barcoding (FLBC) and fluorescence lectin-binding analysis (FLBA), where FLBC is equal to the screening of a particular sample with all the commercially available lectins and FLBA is the actual analysis of the matrix throughout an experiment with a selected panel of lectins. As the application of immune-based techniques in environmental biofilm systems is impossible, the lectin approach is currently the only option for probing lectin-specific glycoconjugates in complex biofilms and bioaggregates. From all the commercially available lectins tested, the lectins such as AAL, HAA, WGA, ConA, IAA, HPA, and LEA showed the highest binding efficiency. Furthermore, 20 of the overall lectins tested showed an intermediate signal intensity, nevertheless very useful for the assessment of matrix glycoconjugates. With the data compiled, we shall virtually shed more light on the dark matter of the extracellular matrix and their 3-dimensional distribution in environmental biofilm systems. The results will be helpful in future studies with a focus on the extracellular matrix glycoconjugates present in environmental microbial communities.

Production of nonulosonic acids in the extracellular polymeric substances of \u201cCandidatus Accumulibacter phosphatis\u201d

Nonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as an important compound in the extracellular matrix of virtually all microbial life and in “Candidatus Accumulibacter phosphatis”, a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of “Ca. Accumulibacter” to produce different types of NulOs. Proteomic analysis showed the ability of “Ca. Accumulibacter” to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of “Ca. Accumulibacter” in particular, and biofilms in general.Key Points•“Ca. Accumulibacter” has the potential to produce a range of nonulosonic acids.•Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids.•The role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.

EPS Characterization of a Cell Wall-Lacking Archaeon &lt;i&gt;Ferroplasma&lt;/i&gt; &lt;i&gt;acidiphilum&lt;/i&gt;

We studied the surface properties of F. acidiphilum DSM 28986 by attenuated total reflection-Fourier transformed infra-red (ATR-FTIR) spectroscopy and microbial adhesion to hydrocarbon (MATH) techniques. In addition, extracellular polymeric substances (EPS) were extracted and characterized by conventional colorimetric analysis and fluorescence lectin-binding analysis (FLBA). Results showed that: 1) cells selectively adhered to mineral surfaces and showed maximum attachment to pyrite of approx. 50% within 30 min; 2) EPS synthesis by F. acidiphilum DSM 28986 was influenced by growth substrates; and 3) tightly-bound EPS (capsular EPS) were composed of carbohydrates and proteins. In contrast, loosely-bound EPS (colloidal EPS) were mainly characterized as carbohydrates. Monosaccharides like glucose, fucose, arabinose, galactose, mannose, and sialic acid were detected in the EPS of F. acidiphilum DSM 28986. This study provides first insight into surface characterization of the cell wall-lacking archaeon F. acidiphilum and facilitates the understanding of interactions of this organism with other acidophiles and metal sulfides.

The acid soluble extracellular polymeric substance of aerobic granular sludge dominated by Defluviicoccus sp.

A new acid soluble extracellular polymeric substance (acid soluble EPS) was extracted from an acetate fed aerobic granular sludge reactor operated at 35 °C. Acid soluble EPS dominated granules exhibited a remarkable and distinctive tangled tubular morphology. These granules are dominated by Defluviicoccus Cluster II organisms. Acetic acid instead of the usually required alkaline extraction medium was needed to dissolve the granules and solubilise the polymeric matrix. The extracted acid soluble EPS was analysed and identified using various instrumental analysis including 1H and 13C Nuclear Magnetic Resonance, Fourier Transform Infrared Spectroscopy and Raman spectroscopy. In addition, the glycoconjugates were characterized by fluorescence lectin-binding analysis. The acid soluble EPS is α-(1 → 4) linked polysaccharide, containing both glucose and galactose as monomers. There are OCH3 groups connected to the glucose monomer. Transmission and scanning electron microscopy (TEM, SEM) as well as confocal laser scanning microscopy (CLSM) showed that the acid soluble EPS was present as a tightly bound capsular EPS around bacterial cells ordered into a sarcinae-like growth pattern. The special granule morphology is decided by the acid soluble EPS produced by Defluviicoccus Cluster II organisms. This work shows that no single one method can be used to extract all possible extracellular polymeric substances. Results obtained here can support the elucidation of biofilm formation and structure in future research.

Fluorescence Lectin Bar-Coding of Glycoconjugates in the Extracellular Matrix of Biofilm and Bioaggregate Forming Microorganisms.

Microbial biofilm systems are defined as interface-associated microorganisms embedded into a self-produced matrix. The extracellular matrix represents a continuous challenge in terms of characterization and analysis. The tools applied in more detailed studies comprise extraction/chemical analysis, molecular characterization, and visualisation using various techniques. Imaging by laser microscopy became a standard tool for biofilm analysis, and, in combination with fluorescently labelled lectins, the glycoconjugates of the matrix can be assessed. By employing this approach a wide range of pure culture biofilms from different habitats were examined using the commercially available lectins. From the results, a binary barcode pattern of lectin binding can be generated. Furthermore, the results can be fine-tuned and transferred into a heat map according to signal intensity. The lectin barcode approach is suggested as a useful tool for investigating the biofilm matrix characteristics and dynamics at various levels, e.g. bacterial cell surfaces, adhesive footprints, individual microcolonies, and the gross biofilm or bio-aggregate. Hence fluorescence lectin bar-coding (FLBC) serves as a basis for a subsequent tailor-made fluorescence lectin-binding analysis (FLBA) of a particular biofilm. So far, the lectin approach represents the only tool for in situ characterization of the glycoconjugate makeup in biofilm systems. Furthermore, lectin staining lends itself to other fluorescence techniques in order to correlate it with cellular biofilm constituents in general and glycoconjugate producers in particular.

Visualizing the dental biofilm matrix by means of fluorescence lectin-binding analysis

ABSTRACTThe extracellular matrix is a poorly studied, yet important component of dental biofilms. Fluorescence lectin-binding analysis (FLBA) is a powerful tool to characterize glycoconjugates in the biofilm matrix. This study aimed to systematically investigate the ability of 75 fluorescently labeled lectins to visualize and quantify extracellular glycoconjugates in dental biofilms. Lectin binding was screened on pooled supragingival biofilm samples collected from 76 subjects using confocal microscopy. FLBA was then performed with 10 selected lectins on biofilms grown in situ for 48 h in the absence of sucrose. For five lectins that proved particularly suitable, stained biovolumes were quantified and correlated to the bacterial composition of the biofilms. Additionally, combinations of up to three differently labeled lectins were tested. Of the 10 lectins, five bound particularly well in 48-h-biofilms: Aleuria aurantia (AAL), Calystega sepiem (Calsepa), Lycopersicon esculentum (LEA), Morniga-G (MNA-G) and Helix pomatia (HPA). No significant correlation between the binding of specific lectins and bacterial composition was found. Fluorescently labeled lectins enable the visualization of glycoconjugates in the dental biofilm matrix. The characterization and quantification of glycoconjugates in dental biofilms require a combination of several lectins. For 48-h-biofilms grown in absence of sucrose, AAL, Calsepa, HPA, LEA, and MNA-G are recommendable.

Osteopontin adsorption to Gram-positive cells reduces adhesion forces and attachment to surfaces under flow

ABSTRACTThe bovine milk protein osteopontin (OPN) may be an efficient means to prevent bacterial adhesion to dental tissues and control biofilm formation. This study sought to determine to what extent OPN impacts adhesion forces and surface attachment of different bacterial strains involved in dental caries or medical device–related infections. It further investigated if OPN’s effect on adhesion is caused by blocking the accessibility of glycoconjugates on bacterial surfaces. Bacterial adhesion was determined in a shear-controlled flow cell system in the presence of different concentrations of OPN, and interaction forces of single bacteria were quantified using single-cell force spectroscopy before and after OPN exposure. Moreover, the study investigated OPN’s effect on the accessibility of cell surface glycoconjugates through fluorescence lectin-binding analysis. OPN strongly affected bacterial adhesion in a dose-dependent manner for all investigated species (Actinomyces naeslundii, Actinomyces viscosus, Lactobacillus paracasei subsp. paracasei, Staphylococcus epidermidis, Streptococcus mitis, and Streptococcus oralis). Likewise, adhesion forces decreased after OPN treatment. No effect of OPN on the lectin-accessibility to glycoconjugates was found. OPN reduces the adhesion and adhesion force/energy of a variety of bacteria and has a potential therapeutic use for biofilm control. OPN acts upon bacterial adhesion without blocking cell surface glycoconjugates.

The biofilm matrix of Campylobacter jejuni determined by fluorescence lectin-binding analysis

Campylobacter jejuni is responsible for the most common bacterial foodborne gastroenteritis. Despite its fastidious growth, it can survive harsh conditions through biofilm formation. In this work, fluorescence lectin-binding analysis was used to determine the glycoconjugates present in the biofilm matrix of two well-described strains. Screening of 72 lectins revealed strain-specific patterns with six lectins interacting with the biofilm matrix of both strains. The most common sugar moiety contained galactose and N-acetylgalactosamine. Several lectins interacted with N-acetylglucosamine and sialic acid, probably originated from the capsular polysaccharides, lipooligosaccharides and N-glycans of C. jejuni. In addition, glycoconjugates containing mannose and fucose were detected within the biofilm, which have not previously been found in the C. jejuni envelope. Detection of thioflavin T and curcumin highlighted the presence of amyloids in the cell envelope without association with specific cell appendages. The lectins ECA, GS-I, HMA and LEA constitute a reliable cocktail to detect the biofilm matrix of C. jejuni.

Visualization and analysis of EPS glycoconjugates of the thermoacidophilic archaeon Sulfolobus metallicus.

Biofilms are surface-associated colonies of microorganisms embedded in a matrix of extracellular polymeric substances (EPS). As EPS mediate the contact between cells and surfaces, an understanding of their composition and production is of particular interest. In this study, the EPS components of Sulfolobus metallicus DSM 6482(T) forming biofilms on elemental sulfur (S(0)) were investigated by confocal laser scanning microscopy (CLSM). In order to visualize cell and EPS distributions, biofilm cells were stained with various dyes specific for glycoconjugates, proteins, nucleic acids and lipids. Biofilm cells on S(0) were heterogeneously distributed and characterized as individual cells, microcolonies, and large clusters up to a hundred micrometers in diameter. The glycoconjugates in biofilms were detected by fluorescence lectin-binding analysis (FLBA). Screening of 72 commercially available lectins resulted in the selection of 21 lectins useful for staining biofilms of S. metallicus (T). Capsular EPS from planktonic cells were mainly composed of carbohydrates and proteins. In contrast, colloidal EPS from planktonic cells were dominated by carbohydrates. Proteins were found to be major components in EPS from biofilms on S(0). Using specific probes combined with CLSM, we showed that extracellular proteins and nucleic acids were present in the EPS matrix. Finally, we showed that S. metallicus (T) cells were embedded in a flexible EPS matrix. This study provides new insights into archaeal biofilms and EPS composition and properties with respect to their interactions with S(0).

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 6482T 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.

Assessment of bacterial and structural dynamics in aerobic granular biofilms.

Aerobic granular sludge (AGS) is based on self-granulated flocs forming mobile biofilms with a gel-like consistence. Bacterial and structural dynamics from flocs to granules were followed in anaerobic-aerobic sequencing batch reactors (SBR) fed with synthetic wastewater, namely a bubble column (BC-SBR) operated under wash-out conditions for fast granulation, and two stirred-tank enrichments of Accumulibacter (PAO-SBR) and Competibacter (GAO-SBR) operated at steady-state. In the BC-SBR, granules formed within 2 weeks by swelling of Zoogloea colonies around flocs, developing subsequently smooth zoogloeal biofilms. However, Zoogloea predominance (37–79%) led to deteriorated nutrient removal during the first months of reactor operation. Upon maturation, improved nitrification (80–100%), nitrogen removal (43–83%), and high but unstable dephosphatation (75–100%) were obtained. Proliferation of dense clusters of nitrifiers, Accumulibacter, and Competibacter from granule cores outwards resulted in heterogeneous bioaggregates, inside which only low abundance Zoogloea (<5%) were detected in biofilm interstices. The presence of different extracellular glycoconjugates detected by fluorescence lectin-binding analysis showed the complex nature of the intracellular matrix of these granules. In the PAO-SBR, granulation occurred within two months with abundant and active Accumulibacter populations (56 ± 10%) that were selected under full anaerobic uptake of volatile fatty acids and that aggregated as dense clusters within heterogeneous granules. Flocs self-granulated in the GAO-SBR after 480 days during a period of over-aeration caused by biofilm growth on the oxygen sensor. Granules were dominated by heterogeneous clusters of Competibacter (37 ± 11%). Zoogloea were never abundant in biomass of both PAO- and GAO-SBRs. This study showed that Zoogloea, Accumulibacter, and Competibacter affiliates can form granules, and that the granulation mechanisms rely on the dominant population involved.

Characterization of Glycoconjugates of Extracellular Polymeric Substances in Tufa-Associated Biofilms by Using Fluorescence Lectin-Binding Analysis

Freshwater tufa deposits are the result of calcification associated with biofilms dominated by cyanobacteria. Recent investigations highlighted the fact that the formation of microbial calcium carbonates is mainly dependent on the saturation index, which is determined by pH, the ion activity of Ca(2+) and CO(3)(2-), and the occurrence of extracellular polymeric substances (EPS) produced by microorganisms. EPS, which contain carboxyl and/or hydroxyl groups, can strongly bind cations. This may result in inhibition of CaCO(3) precipitation. In contrast, the formation of templates for crystal nucleation was reported by many previous investigations. The purposes of this study were (i) to characterize the in situ distribution of EPS glycoconjugates in tufa-associated biofilms of two German hard-water creeks by employing fluorescence lectin-binding analysis (FLBA), (ii) to verify the specific lectin-binding pattern by competitive-inhibition assays, and (iii) to assess whether carbonates are associated with structural EPS domains. Three major in situ EPS domains (cyanobacterial, network-like, and cloud-like structures) were detected by FLBA in combination with laser scanning microscopy (LSM). Based on lectin specificity, the EPS glycoconjugates produced by cyanobacteria contained mainly fucose, amino sugars (N-acetyl-glucosamine and N-acetyl-galactosamine), and sialic acid. Tufa deposits were irregularly covered by network-like EPS structures, which may originate from cyanobacterial EPS secretions. Cloud-like EPS glycoconjugates were dominated by sialic acid, amino sugars, and galactose. In some cases calcium carbonate crystals were associated with cyanobacterial EPS glycoconjugates. The detection of amino sugars and calcium carbonate in close association with decaying sheath material indicated that microbially mediated processes might be important for calcium carbonate precipitation in freshwater tufa systems.

Tufa-forming biofilms of German karstwater streams: microorganisms, exopolymers, hydrochemistry and calcification

Abstract To understand mechanisms of tufa biofilm calcification, selected karstwater stream stromatolites in Germany have been investigated with regard to their hydrochemistry, biofilm community, exopolymers, physicochemical microgradients, calcification pattern and lamination. In stream waters, CO 2 degassing drives the increase in calcite saturation to maximum values of approximately 10-fold, independent from the initial Ca 2+ /alkalinity ratio. For the cyanobacteria of tufa biofilms, a culture-independent molecular approach showed that microscopy of resin-embedded biofilm thin sections underestimated the actual diversity of cyanobacteria, i.e. the six cyanobacteria morphotypes were opposed to nine different lineages of the 16S rDNA phylogeny. The same morphotype may even represent two genetically distant cyanobacteria and the closest relatives of tufa biofilm cyanobacteria may be from quite different habitats. Diatom diversity was even higher in the biofilm at the studied exemplar site than that of the cyanobacteria, i.e. 13 diatom species opposed to 9 cyanobacterial lineages. The non-phototrophic prokaryotic biofilm community is clearly different from the soil-derived community of the stream waters, and largely composed of flavobacteria, firmicutes, proteobacteria and actinobacteria. The exopolymeric biofilm matrix can be divided into three structural domains by fluorescence lectin-binding analysis. Seasonal and spatial variability of these structural EPS domains is low in the investigated streams. As indicated by microsensor data, biofilm photosynthesis is the driving mechanism in tufa stromatolite formation. However, photosynthesis-induced biofilm calcification accounts for only 10–20% of the total Ca 2+ loss in the streams, and occurs in parallel to inorganic precipitation driven by CO 2 -degassing within the water column and on biofilm-free surfaces. Annual stromatolite laminae reflect seasonal changes in temperature and light supply. The stable carbon isotope composition of the laminae is not affected by photosynthesis-induced microgradients, but mirrors that of the bulk water body only reflecting climate fluctuations. Tufa stromatolites with their cyanobacterial–photosynthesis-related calcification fabrics form an analogue to porostromate cyanobacterial stromatolites in fossil settings high in CaCO 3 mineral supersaturation but comparatively low in dissolved inorganic carbon. Here, the sum-effect of heterotrophic exopolymer-degradation and secondary Ca 2+ -release rather decreases calcite saturation, contrary to settings high in dissolved inorganic carbon such as soda lakes.