An overview of different characterization methods and the possibilities for describing structural elements of humic substances from aqueous environments (HS, or refractory organic substances (ROS)) is given within this review. The application of different analytical methods for the qualitative and quantitative characterization of HS, including physical/chemical analysis (elemental analysis, acid/base titration), spectroscopic methods (UV/VIS, NMR, fluorescence, mass spectrometry), fractionation methods (gel chromatography, flow-fieldflow-fractionation), and degradation methods (oxidation, Aquatic Sciences pyrolysis, hydrolysis) are discussed. The paper focuses on the most commonly used chemical, spectroscopic and chromatographic methods used in the last few years. Aspects concerning information related to newer procedures are considered. Case studies representing results gained from reference samples isolated from brown water and wastewater effluents are given to cross-check results gained from several methods. The influence of different isolation procedures on the specific character of the fractions is also discussed.
Extracellular polymeric substances were extracted from the bacterial strain Pseudomonas putida and the fungal species Aureobasidium pullulans using three different methods (formaldehyde-NaOH, ethylenediaminetetraacetic acid (EDTA) and cation-exchange-resin). The composition of the extracellular polymeric substances (EPS) was analysed by biochemical and high-resolution solid state 13C nuclear magnetic resonance (NMR) spectroscopic methods. The EPS yield was strongly dependent on the extraction method, with the formaldehyde-NaOH method showing the best extraction efficiency. The NMR method revealed that when using the EDTA extraction method, about 40% of the EDTA accumulated in the EPS and that was responsible for the apparent high extraction yields. EPS protein content determined by the NMR method was up to 30% higher than the protein content determined using the biochemical (Lowry) method for P. putida and for A. pullulans. The average protein carbon content determined by the NMR method was approximately 70% of the total carbon content. NMR results could be supported by elemental analysis, which showed a high nitrogen content (approximately 10%) in the EPS. The carbohydrate carbon content detected with both methods in the cell aggregates and the EPS was approximately 20% in each. In this study, quantitative 13C cross-polarisation magic angle spinning NMR spectroscopy was conducted on unlabeled cell strains, and EPS and could be used to quantify protein and carbohydrate of different samples.
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