Ulrich Lankes

Structural characterization of aquatic humic substances: The need for a multiple method approach

Abstract.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-field-flow-fractionation), and degradation methods (oxidation, 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.

Comparison of the information gained by pyrolytic techniques and NMR spectroscopy on the structural features of aquatic humic substances

Abstract The molecular composition of fulvic acid (FA) and humic acid (HA) fractions from a German natural lake have been characterized by solid state 13 C-NMR spectroscopy and on-line Curie-point pyrolysis at 510°C both in the presence and the absence of tetramethylammonium hydroxide (TMAH) followed by the separation and identification of the compounds released by gas chromatography-mass spectrometry (GC-MS). The two humic fractions released similar assemblages of aliphatic and aromatic pyrolysis compounds, arising most probably from carbohydrate, polyaromatic, and aliphatic structural subunits. After thermochemolysis with TMAH, most of the products released were found as methyl esters. These products also included typical lignin markers. The same structural components responsible for the release of pyrolysis products were apparent in the NMR spectra of the FA and HA, showing a similar distribution in both fractions. The reliable quantitative distribution of C atoms pertaining to alkyl and aromatic structures obtained from NMR data was not in total agreement with the relative quantification of pyrolysis products, which seems to be biased towards an ‘enhancement’ of aliphatic moieties. However, the two techniques give similar information on the overall structural make-up of the humic fractions, and thus agree that the FA fraction does contain less aromatics and more aliphatics than the HA fraction, whereas the content in O -alkyl structures is similar in the two fractions.

Reconsidering the quantitative analysis of organic carbon concentrations in size exclusion chromatography.

The evaluation of the molecular size distribution of natural organic matter (NOM) in aquatic environments via size exclusion chromatography (SEC) is important for the understanding of environmental processes such as nutrient cycling and pollutant transport as well as of technical water treatment processes. The use of organic carbon (OC) detectors has become popular in recent studies due to improved availability and quantification possibilities, which supposedly are superior to those of ultraviolet (UV) detectors. A set of 12 NOM samples was used to demonstrate the limitations of online OC detection (OCD) when analyzing complex aquatic organic matter. A novel evaluation approach for SEC data is introduced by combining the information from UV absorbance (UVA) and OCD chromatograms as well as offline total OC (t-OC) and dissolved OC-specific UVA (SUVA) measurements. It could be shown that about 70% of certain OC components were not detected with the OCD system used in this study. For the investigated samples, these types of carbon accounted for up to 72% of the t-OC, i.e. for such NOM samples quantification by OCD is not possible or at least highly questionable. The addition of an oxidant improved the overall oxidation efficiency only slightly. Most likely NOM that predominantly consists of polysaccharides and features a nominal molecular weight of 150kg/mol or more was responsible for low OCD yields. For future applications, a further improvement of the OCD system would be worthwhile so that quantitative analytical data on the molecular size distribution of NOM and its structural characteristics such as the SUVA distribution can be obtained.

The concentration of polysaccharides and proteins in EPS of Pseudomonas putida and Aureobasidum pullulans as revealed by 13C CPMAS NMR spectroscopy

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 (~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.

Monitoring the formation of an Aureobasidium pullulans biofilm in a bead-packed reactor via flow-weighted magnetic resonance imaging

The biofilm-forming fungus, Aureobasidium pullulans DSM 2404, was grown in a bead-packed reactor. Alterations within the reactor were analysed in several cross-sectional slices by magnetic resonance imaging (MRI) with flow contrast. For the first time, biofilm accumulation could be continuously elucidated without using any contrast agents, and the non-stationary flow through the fixed-bed reactor could be visualized. The results indicate that the non-stationary flow through the biofilm reactor changes significantly due to the changing reactor morphology. Preferential flow lines arise during biofilm formation. The accumulation of the biomass was determined and compared to gravimetrical biomass data. The described technique can be used to monitor hydrodynamic transport, and to combine flow-field characteristics with morphological data for the prediction of undesirable reactor processes, e.g. clogging.