Towards a nondestructive chemical characterization of biofilm matrix by Raman microscopy

In this study, the applicability of Raman microscopy (RM) for nondestructive chemical analysis of biofilm matrix, including microbial constituents and extracellular polymeric substances (EPS), has been assessed. The examination of a wide range of reference samples such as biofilm-specific polysaccharides, proteins, microorganisms, and encapsulated bacteria revealed characteristic frequency regions and specific marker bands for different biofilm constituents. Based on received data, the assignment of Raman bands in spectra of multispecies biofilms was performed. The study of different multispecies biofilms showed that RM can correlate various structural appearances within the biofilm to variations in their chemical composition and provide chemical information about a complex biofilm matrix. The results of RM analysis of biofilms are in good agreement with data obtained by confocal laser scanning microscopy (CLSM). Thus, RM is a promising tool for a label-free chemical characterization of different biofilm constituents. Moreover, the combination of RM with CLSM analysis for the study of biofilms grown under different environmental conditions can provide new insights into the complex structure/function correlations in biofilms.     

Chemical and physical methods for characterisation of biofilms

Research on biofilms has developed into interdisciplinary work, in which scientists from different fields are involved. This review summarizes the state- of-the-art including models due to different aspects of biofilms. Techniques for characterization of surfaces and interfaces, e.g. microscopic, spectroscopic and microsensoric methods will be presented. In addition, procedures that involve sampling of biofilms and subsequent separation of the components for their analysis are a part of this article. Analytical methods are summarized for the investigation of extracellular polymeric substances (EPS), mainly polysaccharides beside proteins in the microbial host. Finally, procedures for the analysis of minerals as components of biofilms are presented.     

Metal Complexes—A Promising Approach to Target Biofilm Associated Infections

Microbial biofilms are represented by sessile microbial communities with modified gene expression and phenotype, adhered to a surface and embedded in a matrix of self-produced extracellular polymeric substances (EPS). Microbial biofilms can develop on both prosthetic devices and tissues, generating chronic and persistent infections that cannot be eradicated with classical organic-based antimicrobials, because of their increased tolerance to antimicrobials and the host immune system. Several complexes based mostly on 3D ions have shown promising potential for fighting biofilm-associated infections, due to their large spectrum antimicrobial and anti-biofilm activity. The literature usually reports species containing Mn(II), Ni(II), Co(II), Cu(II) or Zn(II) and a large variety of multidentate ligands with chelating properties such as antibiotics, Schiff bases, biguanides, N-based macrocyclic and fused rings derivatives. This review presents the progress in the development of such species and their anti-biofilm activity, as well as the contribution of biomaterials science to incorporate these complexes in composite platforms for reducing the negative impact of medical biofilms.     

Atomic force microscopy of mechanically trapped bacterial cells.

This article presents a study on the influence of the protocol used for immobilization of bacterial cells onto surfaces by mechanically trapping them into a filter. In this sense, the surface and structure of trapped cells are analyzed. Bacteria can be present solely or with extracellular polymeric substances (EPS). To test the behavior of the EPS layer duing the filtering process, different strains of a well-known EPS-producer bacteria (Staphylococcus epidermidis), which produce an extracellular matrix clearly visible in AFM images, have been used. Results show that this immobilization method can cause severe structural and mechanical deformation to the cell membrane. This altered mechanical state may possibly influence the parameters derived from AFM force curves (which are micro/nano-mechanical tests). Also, our results suggest that the EPS layer might move during the filtering process and could accumulate at the upper part of the cell, thus favoring distorted data of adhesion/pull-off forces as measured by an AFM tip, especially in the case of submicron-sized microbial cells such as bacteria.     

溶解有机质对光照自然水体生物膜体系中H2O2生成的影响

通过模拟实验研究了生物膜胞外聚合物(EPS)和乙二胺四乙酸(EDTA) 2种典型溶解有机质(DOM)成分对自然水体生物膜体系中过氧化氢(H₂O₂)生成特征的影响, 并研究了体系初始pH值、 DOM浓度、 溶解氧(DO)等因素的影响. 结果表明, DOM的存在对自然水体生物膜体系中H₂O₂的生成有明显影响. 光照能促使EPS产生H₂O₂, 而EPS的存在对生物膜产生H₂O₂的直接影响不显著, EPS与生物膜共存体系中的H₂O₂由二者共同产生; EDTA本身不产生H₂O₂, 且对H₂O₂分解影响很小, 但会显著抑制生物膜产生H₂O₂, 且浓度越高抑制作用越明显. 体系pH值、 DOM浓度和DO均能不同程度影响EPS产生H₂O₂及EDTA抑制生物膜产生H₂O₂的作用.     

Analysis of microbial extracellular polysaccharides in biofilms by HPLC. Part I: development of the analytical method using two complementary stationary phases

The investigation of microbial extracellular polymeric substances (EPS) is helpful for the implementation of analytical methods which are suitable for biofilm analysis in order to understand the architecture and function of biofilms. A procedure for the qualitative and quantitative determination of various monosaccharides, oligosaccharides and uronic acids as important components of the carbohydrate fraction of microbial EPS by high-performance liquid chromatography (HPLC) and refractive index (RI)/UV detection is presented. Porous graphitic carbon and lead-form cation-exchanger have been examined as stationary phases. Therefore, two complementary HPLC methods are presented. To simulate the conditions of hydrolysis, the influences of various salts, acids and alkalis as matrix components have been investigated. Furthermore, the dependencies on the pH value and temperature of the mobile phase have been thoroughly studied. The results showed that the lead-form cation-exchanger is suitable for the separation of the neutral monosaccharides. However, for direct analysis after acidic hydrolysis with H₂SO₄, HCl or trifluoroacetic acid, an additional purification step, e.g., precipitation or lyophilization, is necessary when the cation-exchanger is used. With the exception of hydrolysis with HCl, the porous graphitic carbon stationary phase can be used without any further purification step and is appropriate for the separation of uronic acids and their γ-lactones. Additionally, the separation of a single monosaccharide and its derivatives is possible. Analytical parameters including the sensitivities, repeatabilities, limits of detection and limits of quantification of both HPLC methods using the RI detector are presented. The optimized method has been applied for the characterization of alginates and is also suitable for other extracellular polysaccharides in biofilms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Analysis of Pathogenic Bacterial and Yeast Biofilms Using the Combination of Synchrotron ATR-FTIR Microspectroscopy and Chemometric Approaches

Biofilms are assemblages of microbial cells, extracellular polymeric substances (EPS), and other components extracted from the environment in which they develop. Within biofilms, the spatial distribution of these components can vary. Here we present a fundamental characterization study to show differences between biofilms formed by Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative Pseudomonas aeruginosa, and the yeast-type Candida albicans using synchrotron macro attenuated total reflectance-Fourier transform infrared (ATR-FTIR) microspectroscopy. We were able to characterise the pathogenic biofilms’ heterogeneous distribution, which is challenging to do using traditional techniques. Multivariate analyses revealed that the polysaccharides area (1200–950 cm⁻¹) accounted for the most significant variance between biofilm samples, and other spectral regions corresponding to amides, lipids, and polysaccharides all contributed to sample variation. In general, this study will advance our understanding of microbial biofilms and serve as a model for future research on how to use synchrotron source ATR-FTIR microspectroscopy to analyse their variations and spatial arrangements.     

Funktionelle Lebensmittel: Mehr als ein Trend?

Food is considered to be functional if it shows a beneficial health effect beyond the supply of nutrients with proven functions. Regarding the prevention of a variety of chronic diseases carotinoides, polyphenoles and phytosterols seem to be of particular interest. However, it has not been sufficiently investigated to what extent single substances, for example given as a food supplement, can effectively be used for this purpose. More importance should be assigned to natural food which guarantees the uptake even of those nutrients of which biological functions are currently still unknown.     

A study on the isotope composition of carbon <Superscript>13</Superscript>C in poppy seeds and narcotic poppy straw present in the same package aimed at the revelation of the possible artificial superinducements of a narcotic substance

The rise in crime associated with the illicit trafficking in narcotics and psychotropic substances is one of the most severe problems in Russia today. In recent years, cases of using seeds of poppy plant “masked as food,” the bulk of which contains macroparticles of poppy straw visible to an unaided eye and which can be used as raw materials for the preparation of narcotic substances have become more frequent. In his work, using a Delta V Advantage isotopic mass spectrometer, we proposed criteria based on which tone can reveal artificial superinducements of poppy straw into “food” poppy seeds or their absence.     

Groups of Organic Molecules That Operate Collectively

A “chemical system” is defined as an assemblage of molecules that collectively does something interesting or useful. The key word here is “collectively”, a word that implies an interdependency and a group behavior that can be quite different from that of individual molecules. Batteries, computer chips, concrete, mayonnaise, shampoo, paint, liquid crystal displays, composites, and viruses are all examples of commonly encountered systems. A host–guest or “supramolecular” complex, on the other hand, would not be considered a system (as defined here), because only two species are involved. A chemical system is multimolecular, a collection of molecules interlocked in a tangle of dependencies. The review delves into a variety of chemical systems investigated by the author, including micelles, water pools, films, vesicles, and polymers. All of them can be categorized as “self-assembling” or “self-organizing” in the sense that defined structures arise spontaneously owing to noncovalent forces among the component molecules. Such chemical systems are useful for many purposes, including decontamination of environmentally dangerous substances, drug delivery, and separation of organic compounds.     

Period four metal nanoparticles on the inhibition of biofouling

Biofilms present operational problems to a variety of industrial areas including but not limited to, medicine, water treatment, sensor sensitivity and shipping. Bacterial adhesion resides as a tiny monolayer and builds-up over time with the production of protective slimes known as extracellular polymeric substances (EPS) forming the ‘biofilm’. Infection, inefficiency and diminution of quality are caused by biofilms, which have the potential to be prohibitively expensive to repair.The value of an effective coating that prevents the adhesion of bacteria and subsequent fouling is paramount in preserving sensitivity and longevity of a subjected operational substrate. Polymer and sol–gel (SG) based coatings tender a matrix for the introduction of biocides and antimicrobial agents that offer this prevention. They present a relatively cheap and optically clear platform that can then be doped with the antimicrobial agent. This proves useful in transferring across a range of industries that may require a transparent function to the coating.Nanoparticles offer a means of new line research in combating biofouling and biocorrosion with interest stemming from silver metal nanoparticles (MNPs) that already offer antimicrobial property. The aim of this work is to investigate period four metal nanoparticles for any antimicrobial potential they offer, in the prevention of fouling in the early stages. The research presented herein uses a range of period four MNPs synthesised through an adapted polyol reduction, which have then been doped into SG coatings and tested for their efficacy in preventing levels of biofouling. After a 7-day freshwater study results showed that MNPs prevent levels of biofouling upto 125% compared to the SG blank. The work uses bacterial enumeration, minimum inhibitory concentration (MIC), surface characterisation and slime and biomass analysis to complete a range of studies in assessing the level of fouling observed on the test substrates.     

The perfect slime

The biofilm mode of life is only possible because biofilm organisms are transiently immobilized in a matrix of extracellular polymeric substances (EPS), also known as "slime". The matrix can be considered an emerging property of microorganisms, allowing them to form stable synergistic consortia, supporting interaction by signaling molecules and horizontal gene transfer and, eventually being activated by extracellular enzymes which turn the matrix into an external digestion system. As unsightly it is, this matrix is--although seemingly stochastic--a highly differentiated and functional immediate environment for biofilm cells.     

A New Kinetic Model for Water Sorption Isotherms of Cellulosic Materials

Summary: Some natural fibres like flax, hemp and others show excellent mechanical properties which make them a promising choice for the reinforcement of polymers. For natural fibre reinforced composites, hydrophilicity is a problem with respect to dimensional changes, fibre to matrix adhesion and long term stability. The interaction of differently prepared and modified fibres with water vapour has been investigated by dynamic vapour sorption. It has been found that the sorption and desorption kinetics of cellulosic fibres can be excellently fitted by assuming two parallel, independent first order processes. This empirical model, defined here as the “Parallel Exponential Kinetics” model (PEK-model), reveals two distinct mechanisms with slow and fast exchange of water vapour respectively, related to different sorption sites. The specific sorption mechanisms are represented by individual sorption-desorption isotherms as components of the total isotherms. The results suggest a relation to the differing types of amorphous regions in the fibres and/or to the different states of “bound” or “free” water, discussed for hydrophilic materials. The PEK-model proved to be consistently applicable for sorption and desorption over the whole humidity range, and also for all tested cellulose fibres. It is especially useful for a clearer distinction of different fibre types or modifications and can be successfully used for an extended fibre characterization.     

Element determination in natural biofilms of mine drainage water by total reflection X-ray fluorescence spectrometry

Human impacts like mining activities lead to higher element concentration in surface waters. For different pollution levels, the consequences for aquatic organisms are not yet investigated in detail. Therefore, the aim of this investigation is to determine the influence of mining affected surface waters on biofilms.Elements like heavy metals can be absorbed on cell walls and on polymeric substances or enter the cytoplasm of the cells. Thus, they are important for the optimization of industrial biotechnological processes and the environmental biotechnology. Beyond this, biofilms can also play an important role in wastewater treatment processes and serve as bioindicators in the aquatic environment.The presented total reflection X-ray fluorescence spectroscopic investigation was performed to compare the element accumulation behavior of biofilms grown on natural or on artificial materials of drainage water affected by former copper mining activities. A high salt and heavy metal pollution is characteristic for the drainage water. For an assessment of these results, samples from stream Schlenze upstream the confluence with the drainage water, a small tributary of the Saale River in central Germany, were analyzed, too.     

Towards chemical analysis of nanostructures in biofilms I: imaging of biological nanostructures

Due to their direct influence on the stability of bacterial biofilms, a better insight into the nanoscopic spatial arrangement of the different extracellular polymeric substances (EPS), e.g., polysaccharides and proteins, is important for the improvement of biocides and for process optimization in wastewater treatment and biofiltration. Here, the first application of a combination of confocal laser-scanning microscopy (CLSM) and atomic force microscopy (AFM) to the investigation of river-water biofilms and related biopolymers is presented. AFM images collected at selected areas of CLS micrographs dramatically demonstrate the heterogeneity of biofilms at the nanometer scale and the need for a chemical imaging method with nanoscale resolution. The nanostructures (e.g., pili, flagella, hydrocolloids, and EPS) found in the extracellular matrix are classified according to shape and size, which is typically 50–150 nm in width and 1–10 nm in thickness, and sets the demands regarding spatial resolution of a potential chemical imaging method. Additionally, thin layers of the polysaccharide alginate were investigated. We demonstrate that calcium alginate is a good model for the EPS architecture at the nanometer scale, because of its similar network-like structure. Figure CLSM-AFM allows imaging of nanometer-sized extracellular structures     

Adhesion of bacterial exopolymers to alpha-FeOOH: inner-sphere complexation of phosphodiester groups

Extracellular polymeric substances (EPS) constitute a heterogeneous mixture of polyelectrolytes that mediate biomineralization and bacterial adhesion and stabilize biofilm matrixes in natural and artificial environments. Although nucleic acids are exuded extracellularly and are purported to be required for biofilm formation, direct evidence of the active mechanism is lacking. EPS were extracted from both Bacillus subtilis (a gram-positive bacterium) and Pseudomonas aeruginosa (a gram-negative bacterium) and their interaction with the goethite (alpha-FeOOH) surface was studied using attenuated total internal reflection infrared spectroscopy. Correspondence between spectral data and quantum chemical calculations demonstrate that phosphodiester groups of nucleic acids mediate the binding of EPS to mineral surfaces. Our data indicate that these groups emerge from the EPS mixture to form monodentate complexes with Fe centers on the goethite (alpha-FeOOH) surface, providing an energetically stable bond for further EPS or cell adhesion.     

Effect of exopolymeric substances on the kinetics of sorption and desorption of trivalent chromium in soil

Laboratory batch sorption-desorption and column experiments were performed to better understand the effects of microbial exopolymeric substances (EPS) on Cr(III) sorption/desorption rates in the soil-water system. The experiments were carried out in two different modes: one mode (sorption) in which Cr(III) and EPS were applied simultaneously, and the other (desorption) included the sequential application of Cr(III) and EPS to the soil-water system. The batch sorption and desorption experiments showed that, while chromium(III) desorption was significantly enhanced in the presence of EPS relative to non-EPS-containing systems, the desorption rates were much smaller than the sorption rates, and the fraction dissolved by EPS accounted for only a small portion of the total chromium initially sorbed onto soil minerals. Similarly, the column experiments suggested that, while the microbial EPS led to an increase in Cr dissolution relative to non-EPS-containing systems, only a small portion of the total chromium initially added to the soil was mobilised. The differences observed in Cr sorption and desorption rates can be explained through the very low solubility and strong interactions of chromium species with soil minerals as well as the mass transfer effects associated with low diffusion rates. The overall results suggest that, while microbial EPS may play an important role in microbial Cr(VI) treatment in sub-surface systems due to the formation of soluble Cr-EPS complexes, the extent and degree of Cr mobilisation are highly dependent on the type of initial Cr sorption.     

Acoustic sensing of the bacterium-substratum interface using QCM-D and the influence of extracellular polymeric substances

It is commonly assumed that bacterial presence on a QCM sensor-surface is associated with a negative frequency shift according to conventional mass-loading theory. Here, we demonstrate that bacteria adhering to QCM sensor-surface may yield positive frequency shifts up to 1.9×10(-6) Hz per bacterium according to a coupled-oscillator theory. Furthermore, it is demonstrated that the excretion of extracellular polymeric substances (EPS) by adhering bacteria can change the frequency shift in the negative direction by 1.7×10(-6) Hz per bacterium, according to conventional mass-loading theory. The difference in frequency shifts between an EPS-producing and a non-EPS producing staphylococcal strain correlated with the excretion of 3×10(-14) g EPS per bacterium, representing only a few percent of the weight of a bacterium. Thus an adsorbed molecular mass as low as a few percent of the mass of an adhering bacterium significantly alters the QCM-signal. Since adhesion of many different bacterial strains is accompanied by molecular adsorption of EPS, with potentially opposite effects on the QCM-signal, a combination of the coupled-oscillator and normal mass-loading theory has to be applied for proper interpretation of QCM-frequency shifts in bacterial detection.     

Forces between surfaces in liquids

The results and implications of direct force measurements between molecularly smooth mica surfaces in liquids are reviewed. These discussions include four interactions fundamental to colloid science: van der Waals forces, double layer forces, adhesion forces and structural or solvation forces (e.g. hydration forces). Also considered are the effects of preferential surface adsorption of solute molecules on these interactions, e.g. surfactant adsorptions from aqueous solutions and water condensation from non-aqueous solvents.In aqueous media it is apparent that the DLVO theory is valid at all surface separations down to the “force barrier”, but that under certain conditions hydration forces can become significant at distances below 30 Å.The measured adhesion force between two solid surfaces can be simply related to their surface energies and where meniscus forces are also present due to “capillary condensation” from vapor solvent, their effect on adhesion can be understood in terms of straightforward bulk thermodynamic principles. Here, too, it is concluded that structural forces cannot be ignored.Our results suggest that structural forces may either very monotonically with distance or be oscillatory with a periodicity equal to the molecular size. Their origin, nature, mode of action and importance for particle interactions will no doubt take many years to sort out.     

Physicochemical Characterization of Acidiphilium sp. Biofilms

The biofilm formation of a strain of the extremophile bacterium Acidiphilium sp., capable of donating electrons directly to electrodes, was studied by different surface characterization techniques. We develop a method that allows the simultaneous study of bacterial biofilms by means of fluorescence microscopy and atomic force microscopy (AFM), in which transparent graphitic flakes deposited on a glass substrate are used as a support for the biofilm. The majority of the cells present on the surface were viable, and the growth of the biofilms over time showed a critical increase of the extracellular polymeric substances (EPS) as well as the formation of nanosized particles inside the biofilm. Also, the presence of Fe in Acidiphilium biofilms was determined by X‐ray photoelectron spectroscopy (XPS), whereas surface‐enhanced infrared absorption spectroscopy indicated the presence of redox‐active proteins.