The uronic acids assay: a method for the determination of chemical activity on biofilm EPS

In this work, the uronic acids assay was evaluated for its potential to function as a bioassay to screen for antagonistic activity against the production of microbial biofilm exopolysaccharide (EPS). The assay was first applied to biofilms produced in the presence of two universal disinfectants (sodium hypochlorite and sodium dodecyl sulfate) known to inhibit microbial growth and biofilm formation. The performance of the assay was then characterized through statistical assessment of threshold concentrations for disinfection efficiency and consistency relative to values reported in the literature. The assay was then evaluated for its utility in screening for enzymatic or chemical inhibitors of biofilm formation (eg glycosidases, halogenated furanones, and semi-crude fractions extracted from minimally fouled marine plants) and its ability to distinguish between true anti-biofilm activity and simple disinfection. Activity was characterized as (i) no effect, (ii) a true positive effect (ie increased biofilm EPS), (iii) anti-bacterial activity (ie decreased biofilm EPS and analogous decrease in planktonic growth), and (iv) anti-biofilm EPS activity (ie decreased biofilm EPS, without analogous decrease in planktonic growth). Results demonstrate that the uronic acids assay can augment existing biofilm characterization methods by providing a quantitative measure of biofilm EPS.     

Differences in carbohydrate profiles in batch culture grown planktonic and biofilm cells of Amphora rostrata Wm. Sm

Diatoms are abundant in biofilms developed on surfaces immersed in sunlit waters. In both the planktonic and the biofilm mode of growth, diatoms produce carbohydrate polymers which perform several functions including motility, protection, production of macro-aggregates and detoxification. However, little is known about the differences, if any, in the production and characterization at the molecular level of carbohydrates in planktonic and biofilm cells. In order to identify the differences in these two modes of growth, the concentration of total carbohydrates, carbohydrate fractions, neutral carbohydrates, uronic acids and amino sugars in planktonic and biofilm cells of Amphora rostrata were measured. The results showed that the distribution of carbohydrate fractions, uronic acids and amino sugars was different in biofilm and planktonic cells. Cell normalized concentrations of these components were two to five times greater in planktonic cells compared with biofilm cells. The concentrations of glucose and glucosamine decreased, whereas fucose increased in planktonic cells over the period of cultivation. Conversely, the concentrations of glucose and glucosamine increased while that of fucose decreased in attached cells. The study suggests that marked differences exist between the carbohydrates of the planktonic and the biofilm cells of A. rostrata.     

Gas chromatographic assay of iduronic and glucuronic acids as aldonic acid butaneboronates

A gas chromatographic procedure has been developed for the analysis of uronic acids as aldonic acid butaneboronates. With these derivatives, aldoses frequently accompanying uronic acids in polysaccharide hydrolyzates are readily separated and measured. The method has been applied to the assay of iduronic and glucuronic acid released by enzymes associated with various mucopolysaccharidoses.     

Estimations of Uronic Acids as Quantitative Measures of Extracellular and Cell Wall Polysaccharide Polymers from Environmental Samples

The extracellular polysaccharide polymers can bind microbes to surfaces and can cause physical modification of the microenvironment. Since uronic acids appear to be the components of these extracellular films that are most concentrated in a location outside the cell membrane, a quantitative assay for uronic acids was developed. Polymers containing uronic acids are resistant to quantitative hydrolysis, and the uronic acids, once released, form lactones irreproducibly and are difficult to separate from the neutral sugars. These problems were obviated by the methylation of the uronic acids and their subsequent reduction with sodium borodeuteride to the corresponding alcohol while they were in the polymer and could not form lactones. This caused the polymers to lose the ability to adhere to their substrates, so they could be quantitatively recovered. The hydrolysis of the dideuterated sugars was reproducible and could be performed under conditions that were mild enough that other cellular and extracellular polymers were not affected. The resulting neutral sugars were readily derivatized and then were separated and assayed by glass capillary gas-liquid chromatography. The dideuterated portion of each pentose, hexose, or heptose, identified by combined capillary gas-liquid chromatography and mass spectrometry, accurately provided the proportion of each uronic acid in each carbohydrate of the polymer. Examples of the applications of this methodology include the composition of extracellular polymers in marine bacteria, invertebrate feeding tubes and fecal structures, and the microfouling films formed on titanium and aluminum surfaces exposed to seawater.     

A colorimetric method for the quantitation of uronic acids and a specific assay for galacturonic acid.

A method of quantitating uronic acids and uronic acids from pectin in particular is described. The method uses carbazole in 80% sulfuric acid with borate ions added. The assay is carried out at 60 degrees C. This assay has some cross reactivity with aldose sugars and must be timed precisely. A further method that is specific for galacturonic acid is also described. This method uses concentrated sulfuric acid and carbazole only. Of the biological substances tested, only formaldehyde and glyceraldehyde showed a reactivity of more than 10% that of galacturonic acid on a weight to weight basis.     

Sugar composition of biofilms produced by paper mill bacteria

Biofilms of paper mill bacteria were cultivated in paper mill white water-simulating conditions on glass slides or stainless steel coupons in a laboratory culture system. The sugar content and composition of the biofilms were analysed and compared with the sugar composition of paper mill slimes. Acid methanolysis followed by gas chromatography revealed that Burkholderia was the major biofilm producer in pure culture, producing up to 50 microg of biofilm sugar cm(-2) in 5 days in rich medium and 10 microg in paper mill simulating medium. A mixture of simulated paper mill water with a culture medium yielded more biofilm (100 microg cm(-2)) than either of the media alone, so the biofilm accumulation was not proportional to the available substrate. More biofilm accumulated on stainless steel coupons than on glass slides, and the steel-coupon biofilms contained slightly more uronic acids. The biofilm sugars contained mainly galactose, glucose, mannose, and rhamnose. In paper mill medium, the Burkholderia biofilm contained more galactose and glucose, and less rhamnose, than in rich laboratory medium. The sugar composition of paper mill slimes was quite similar to those of steel-cultured Burkholderia cepacia biofilms. This suggests that Burkholderia cepacia is responsible for much of the slime in the paper mill.     

Measurement of uronic acids without interference from neutral sugars

Replacement of carbazole with meta-hydroxydiphenyl greatly improves the determination of uronic acids in the presence of neutral sugars by preventing substantially, but not completely, the browning that occurs during the heating of sugars in concentrated sulfuric acid and avoiding the formation of additional interference by the carbazole reagent (Blumenkrantz, N., and Asboe-Hansen, G. (1973) Anal. Biochem. 54, 484-489). However, interference is still substantial when uronic acids are determined in the presence of excess neutral sugar, particularly because of the browning that occurs during the first heating before addition of the diphenyl reagent. The browning can be essentially eliminated by addition of sulfamate to the reaction mixture (Galambos, J. T. (1967) Anal. Biochem. 19, 119-132). Although others have reported that sulfamate and the diphenyl reagent were incompatible, we find that a small amount of sulfamate suppresses color production by a 20-fold excess of some neutral sugars without substantial sacrifice of the sensitive detection of uronic acids by the diphenyl reagent. Sodium tetraborate is required for the detection of D-mannuronic acid and enhances color production by D-glucuronic acid. We propose this modified sulfamate/m-hydroxydiphenyl assay as a rapid and reliable means for the assay of uronic acids, particularly when present in much smaller amounts than neutral sugars.     

1H-NMR analysis of water mobility in cultured phototrophic biofilms

The present work reports on the first attempt to study water mobility in phototrophic biofilms, applying the (1)H-NMR relaxometry technique to closely monitored microbial communities grown in a microcosm under controlled ambient conditions. Longitudinal water proton relaxation times exhibited a bi-exponential behavior in all biofilm samples, indicating two types of water molecules with diverging dynamic properties, confined to different compartments of the biofilm. The fast-relaxing component can be attributed to water molecules tightly bound to the intracellular matrix, while the slow-relaxing component could reflect the behavior of water embedded in the biopolymer matrix, confined into matrix pores and channels. The results are discussed with respect to a possible key role of exopolysaccharides and uronic acids in water binding in phototrophic biofilms.     

Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa.

Reporter gene technology was employed to detect the activity of an alginate promoter of Pseudomonas aeruginosa when the organism was grown as a biofilm on a Teflon mesh substratum and as planktonic cells in liquid medium. Alginate biosynthetic activity was determined with a mucoid cell line derived from a cystic fibrosis isolate and containing an alginate algC promoter fused to a lacZ reporter gene. Reporter activity was demonstrated with chromogenic and fluorogenic substrates for beta-galactosidase. Expression of algC was shown to be upregulated in biofilm cells compared with planktonic cells in liquid medium. Gene up-expression correlated with alginate biosynthesis as measured by Fourier transform infrared spectroscopy, uronic acid accumulation, and alginate-specific enzyme-linked immunosorbent assay. The algC promoter was shown to have maximum activity in planktonic cultures during the late lag and early log phases of the cell growth cycle. During a time course experiment, biofilm algC activity exceeded planktonic activity except during the period immediately following inoculation into fresh medium. In continuous-culture experiments, conversion of lacZ substrate was demonstrated microscopically in individual cells by epifluorescence microscopy.     

A natural terrestrial biofilm

Material recovered from an extensive and viscous biofilm found on areas of rough hill pasture in Southern Scotland proved to consist of a thick mucilaginous deposit of polysaccharide in which species of green algae, withGleocystis spp as the dominant microorganism and lesser numbers ofOocystis spp. Cyanobacteria were also entrapped. On laboratory culture other green algal and cyanobacterial species were detected. Analysis of the native polysaccharide and of the exopolysaccharide from a mixed culture of the dominant algal species derived from the original material, revealed the major components as glucose, galactose, mannose and rhamnose. The content of uronic acids was very low. The viscosity of the polysaccharide preparations was determined and compared with bacterial biofilm material; viscosity was lost following phenol extraction indicating that the original material was probably closely associated with proteins.     

Uronic acid analysis by automated anion exchange chromatography

A rapid and sensitive assay for individual uronic acids has been developed based on their separation on a 0.5 × 22-cm column of Aminex A-25 in 0.12 m Tris-acetate buffer, pH 7.4. Quantification of these sugars is accomplished by coupling the column to the analytical portion of a Technicon sugar analyzer. Each determination is complete in 3 hr, and as little as 25 nmol of uronic acid can be measured with accuracy.     

1H-NMR analysis of water mobility in cultured phototrophic biofilms

The present work reports on the first attempt to study water mobility in phototrophic biofilms, applying the ¹H-NMR relaxometry technique to closely monitored microbial communities grown in a microcosm under controlled ambient conditions. Longitudinal water proton relaxation times exhibited a bi-exponential behavior in all biofilm samples, indicating two types of water molecules with diverging dynamic properties, confined to different compartments of the biofilm. The fast-relaxing component can be attributed to water molecules tightly bound to the intracellular matrix, while the slow-relaxing component could reflect the behavior of water embedded in the biopolymer matrix, confined into matrix pores and channels. The results are discussed with respect to a possible key role of exopolysaccharides and uronic acids in water binding in phototrophic biofilms.     

Biofilms of As(III)-oxidising bacteria: formation and activity studies for bioremediation process development

The formation and activity of an As(III)-oxidising biofilm in a bioreactor, using pozzolana as bacterial growth support, was studied for the purpose of optimising fixed-bed bioreactors for bioremediation. After 60 days of continuous functioning with an As(III)-contaminated effluent, the active biofilm was found to be located mainly near the inflow rather than homogeneously distributed. Biofilm development by the CAsO1 bacterial consortium and by Thiomonas arsenivorans was then studied both on polystyrene microplates and on pozzolana. Extra-cellular polymeric substances (EPS) and yeast extract were found to enhance bacteria attachment, and yeast extract also appears to increase the kinetics of biofilm formation. Analysis of proteins, sugars, lipids and uronic acids indicate that sugars were the main EPS components. The specific As(III)-oxidase activity of T. arsenivorans was higher (by ninefold) for planktonic cells than for sessile ones and was induced by As(III). All the results suggest that the biofilm structure is a physical barrier decreasing As(III) access to sessile cells and thus to As(III)-oxidase activity induction. The efficiency of fixed-bed reactors for the bioremediation of arsenic-contaminated waters can be thus optimised by controlling different factors such as temperature and EPS addition and/or synthesis to increase biofilm density and activity.     

Capsular polysaccharides of cultured phototrophic biofilms

Phototrophic biofilm samples from an Italian wastewater treatment plant were studied in microcosm experiments under varying irradiances, temperatures and flow regimes to assess the effects of environmental variables and phototrophic biomass on capsular exopolysaccharides (CPS). The results, obtained from circular dichroism spectroscopy and High Performance Liquid Chromatography, suggest that CPS have a stable spatial conformation and a complex monosaccharide composition. The total amount present was positively correlated with the biomass of cyanobacteria and diatoms, and negatively with the biovolume of green algae. The proportion of uronic acids showed the same correlation with these taxon groups, indicating a potential role of cyanobacteria and diatoms in the removal of residual nutrients and noxious cations in wastewater treatment. While overall biofilm growth was limited by low irradiance, high temperature (30°C) and low flow velocity (25 l h^?1) yielded the highest phototrophic biomass, the largest amount of CPS produced, and the highest proportion of carboxylic acids present.     

Characterization of extracellular polymers synthesized by tropical intertidal biofilm bacteria

AIM: This study was performed to determine the potential of tropical intertidal biofilm bacteria as a source of novel exopolymers (EPS). METHODS AND RESULTS: A screening procedure was implemented to detect EPS-producing biofilm bacteria. Isolates MC3B-10 and MC6B-22, identified respectively as a Microbacterium species and Bacillus species by 16S rDNA and cellular fatty acids analyses, produced different EPS, as evidenced by colorimetric and gas chromatographic analyses. The polymer produced by isolate MC3B-10 displays significant surfactant activity, and may chelate calcium as evidenced by spectroscopic analysis. CONCLUSIONS: Polymer MC3B-10 appears to be a glycoprotein, while EPS MC6B-22 seems to be a true polysaccharide dominated by neutral sugars but with significant concentrations of uronic acids and hexosamines. EPS MC3B-10 possesses a higher surfactant activity than that of commercial surfactants, and given its anionic nature, may chelate cations thus proving useful in bioremediation. The chemical composition of polymer MC6B-22 suggests its potential biomedical application in tissue regeneration. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a Microbacterium species producing EPS with surfactant properties, which expands our knowledge of the micro-organisms capable of producing these biomolecules. Furthermore, this work shows that tropical intertidal environments are a nonpreviously recognized habitat for bioprospecting EPS-producing bacteria, and that these molecules might be involved in ecological roles protecting the cells against dessication.     

An automated version of the periodate-thiobarbituric acid assay for analysis of delta-4,5 unsaturated uronic acids and its application to the assay of hyaluronic acids and chondroitin sulfates

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μm) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.     

L-Altruronic acid formed by epimerization of D-galacturonic acid methyl esters during saponification of citrus pectin

While searching for oligosaccharides containing rhamnose residues in the endopolygalacturonase (EPG) digest of saponified citrus pectin, we found several oligomers containing, in addition to galacturonic acid, a sugar previously unreported in pectin. The 1- and 2-D 1H NMR spectra of the oligosaccharides were consistent with the sugar being a uronic acid with its 2- and 3-hydroxyls being axial and 4-hydroxyl being equatorial. MALDI-TOF mass spectrometry indicated that the oligomers consisted solely of uronic acids. Reduction of the uronic acids in the oligosaccharides converted them to galactose and altrose. The altrose was found to be the L enantiomer by comparison of its trimethylsilyl (-)-2-butyl glycosides to those of authentic D-altrose and a racemic mixture. The sugar was not found in oligosaccharides prepared from EPG digestion of citrus pectin deesterified with pectin methylesterase rather than saponification. Thus, it appears that during saponification, a small proportion of the methylesterified galacturonic acid residues in pectins is epimerized at C-5 leading to formation of L-altruronic acid residues.     

Automated analysis of uronic acids by high-performance liquid chromatography with photometric and fluorimetric postcolumn labelling using 2-cyanoacetamide

A convenient method for the rapid and sensitive automated analysis of uronic acids, based on high-performance anion-exchange chromatography on a Hitachi 2633 column and photometric as well as fluorimetric postcolumn labeling with 2-cyanoacetamide, has been developed. This method allows the simultaneous determination of 1-1000 nmol of D-mannuronic and D-galacturonic acids and 5-1000 nmol of L-iduronic and D-glucuronic acids in approx 70 min with high precision by photometric monitoring. In fluorimetric monitoring the linearity range was 1-1000 nmol for all these uronic acids, but reproducibility was rather low at the lowest limit of linearity. Application of this method to the analysis of component uronic acids in some polyuronides has suggested the inadequacy of generally accepted conditions for hydrolysis.     

Increase of Free Space Solutes in Tobacco Leaves in Relation to the Localized Cellular Response Following Injections of a Bacterial Protein-Lipopolysaccharide Complex*

Variations in pH, cell ultrastructure, conductivity, calcium ion molarity, reducing sugars, uronic acids and in protein of the intercellular washing fluid were studied at 0, 12, 24 and 48 h in tobacco leaf halves intercellularly injected with a bacterial pr-LPS complex (250 μg-ml⁻¹). These injections induced a localized cellular reaction (LCR) in points opposite to intercellular structured deposits on the plant cell walls. The intercellular fluid pH was higher at 24 h than in the control, but not at 12 and 48 h. The conductivity, the calcium ion molarity, the free and hydrolyzed sugar content were higher at 12, 24 and 48 h than in the control tissue; the uronic acid content was higher at 24 and 48 h, but not at 12 h. There was a peak for, all 5 parameters at around 24 h, when LCR showed its highest activity. The protein content was significantly higher at 24 and 48 h than in the control intercellular fluid. The increase in conductivity, calcium ions, sugar, uronic acids and proteins in the intercellular fluids of the pr-LPS injected tissue were interpreted as direct or indirect effects of the LCR, i.e. as an exocytosis induced by pr-LPS injections. The associated high sugar and uronic acid content of the intercellular fluid at 12 and 24 h was not correlated to its capacity to prevent the hypersensitive confluent necrosis when injected intercellularly into tobacco tissue.     

Linking Sediment Biofilms, Hydrodynamics, and River Bed Clogging: Evidence from a Large River

Abstract We investigated possible effects of the hydrodynamics at the water/sediment interface on river bed biofilms within the reservoir Freudenau (Vienna, Austria) of the Danube River during the period 1996/97. Two study sites (OBB and SSF) that differed in the magnitude of surface/subsurface water exchange were selected and intersite comparisons revealed higher organic matter, bacterial cell numbers, and esterase activity in SSF with lower horizontal outflow. Concentrations of colloidal carbohydrates and uronic acids were unaffected by hydrodynamics. The relative contribution of uronic acids to bulk colloidal carbohydrates was higher in the low-flow site SSF. The distribution patterns of this relative contribution generally matched the subsurface flow pattern. Shortly after impoundment in March 1996 and along with decreased surface flow velocity, maximal biofilm carbohydrate exopolymers concurred with minimal esterase activity in OBB. We hypothesize that this inverse relationship is due to increased diffusional resistance within the exopolymer biofilm matrix that reduces mass transfer and hydrolytic activity. These results, to our knowledge, are the first evidence for microbial participation in the clogging of a large river bed. Biofilm-associated organic carbon increased significantly by a factor of ∼3.3 to 4.4 with progressive clogging as determined by the sediment leakage coefficient, which increased ∼3.8 times. Concomitantly, with ongoing clogging, esterase activity exhibited increasingly higher values at the interface relatively to deeper sediment layers, which translates into steeper depth gradients. Furthermore, minimal inflow from the surface water into the river bed along with steepest esterase gradients concurred with a senescent benthic algal bloom. This suggests an important role for algae in clogging. Either algae obstruct voids mechanically, or their exudates fuel heterotrophic bacteria that in turn are involved in clogging processes. However, our data do not allow unequivocal differentiation between biogenic and physical clogging mechanisms. Received: 14 September 1998; Accepted: 31 December 1998