FG Biotechnologie der Wasseraufbereitung
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The data presented in this article provide supporting information to the related research article “Comparison of ten different DNA extraction procedures with respect to their suitability for environmental samples” (revised manuscript submitted to J. Microbiol. Methods). In that article, we compared the suitability of ten selected DNA extraction methods based on DNA quality, purity, quantity and applicability to universal PCR. Here we provide the data on the specific DNA gel sample load, all unreported gel images of crude DNA and PCR results, and the complete cost analysis for all tested extraction procedures and in addition two commercial DNA extraction kits for soil and water.
The FISH (fluorescence in situ hybridization) method is widely used to identify various types of cells. In comparison to cultivation-dependent methods, identification of microbes by FISH is easier
and generally takes several hours. This paper presents a review of improvements to the FISH method, its advantages and disadvantages, as well as examples of applications. Particular consideration was
given to the efficiency of microbes identification in samples taken from sewerage. The effectiveness of the method was confirmed by the results obtained in samples from the membrane bioreactor (MBR).
DNA extraction for molecular biological applications usually requires target optimized extraction procedures depending on the origin of the samples. For environmental samples, a range of different procedures has been developed. We compared the applicability and efficiency of ten selected DNA extraction methods published in recent literature using four different environmental samples namely: activated sludge from a domestic wastewater treatment plant, river sediment, anaerobic digestion sludge and nitrifying enrichment culture. We assessed the suitability of the extraction procedures based on both DNA yield and quality. DNA quantification was performed by both ultra violet (UV) spectrophotometry and fluorescence spectrophotometry after staining with PicoGreen. In our study, DNA yields based on UV measurement were overestimated in most cases while DNA yields from fluorescence measurements correlated well with the sample load on agarose gels of crude DNA. The quality of the DNA extracts was determined by gel electrophoresis of crude DNA and PCR products from 16S rDNA with the universal primer set 27f/1525r. It was observed that gel electrophoresis of crude DNA was not always suitable to evaluate DNA integrity and purity since interfering background substances (e.g. humic substances) were not visible. Therefore, we strongly recommend examining the DNA quality of both crude DNA and 16S rDNA PCR products by gel electrophoresis when a new extraction method is established.
Summarizing, we found four out of ten extraction procedures being applicable to all tested samples without noticeable restrictions. The procedure G (according to the standard method 432_10401 of the Lower Saxony State Office for Consumer Protection and Food Safety) had the broadest application range over procedure J
(published by Wilson, 2001). These were followed by procedures F (Singka et al., 2012) and A (Bourrain et al., 1999). All four extraction procedures delivered reliable and reproducible crude DNA and PCR products. From an economical point of view, all procedures tested during this study were cheaper compared to commercial DNA extraction kits.
This study aims at investigating the abiotic degradation pathway of
ethylenediaminetetra(methylenephosphonic acid) (EDTMP) simulated applying UV
irradiation. The degradation of EDTMP and formation of degradation products was
determined using LC-MS and ³¹P-NMR. In the laboratory scale experiments, EDTMP was
degraded within 30 min and the degradation products, iminodi(methylenephosphonic
acid) (IDMP), ethylaminobis(methylenephosphonic acid) (EABMP), and amino-
(methylenephosphonic acid) (AMPA), were simultaneously released. IDMP was the
main degradation product of EDTMP. Therefore, we conclude that the initial cleavage of
EDTMP is a heterolytically driven process, which starts the degradation process at the
intramolecular C-N bond. In contrast, the main product of a possible homolytic C-C
cleavage of methylaminobis(methylenephosphonic acid) could not be confirmed with
either LC-MS or ³¹P-NMR. Additionally, there was no evidence for a primary attack on
the C-P bond. All identified degradation products of EDTMP have been mineralized to
carbon dioxide (CO₂). Three additional degradation products (M1, M2, and M3) have
been found using the ³¹P-NMR analysis but have not yet been quantified using LC-MS.
We assume that the unidentified degradation product M1 is related to m/z 312, M2
to m/z 341, and M3 to m/z 409. Thus we concluded that EDTMP undergoes photochemical
conversion to IDMP, the main degradation product. EABMP and AMPA also accumulate,
but in smaller amounts. All intermediates are further mineralized to CO₂.
The anammox process is a potential alternative to the conventional nitrogen removal from wastewater. However, due to large generation times of anammox bacteria, the start-up of treatment reactors may be impeded. An efficient storage technique can handle this drawback and may be also suitable for seasonally operated treatment plants like in touristic areas. In the current study, several storage techniques were investigated with respect to its suitability for the
preservation of the specific anammox activity after long-term storage. Storing conditions differed in terms of temperature, redox buffer and nutrient supplementation. The specific activity of immobilized anammox bacteria (Candidatus Kuenenia stuttgartiensis) was determined three times during a long-term preservation of 78 days and 106 days, respectively. The highest activity was ensured at a storing temperature of 4 WC, providing nitrate as redox buffer and a nutrient
supplement every 23 days. Thus, 91.4% of the initial anammox activity could be preserved after a storage of 106 days. Superiority of the presented treatment condition was confirmed by a calculated nitrate-ammonium consumption rate close to the optimal ratio of 1.32. This technique
provided an economical and simple method suitable for long-term storage of immobilized anammox biomass.
The precision of cell number quantification in environmental samples depends on the complexity of the sample and on the applied technique. We compared fluorescence microscopy after filtration, quantification of gene copies and the cultivation based most probable number technique for their precision. We further analyzed the effect of increasing complexity of the sample material on the precision of the different methods by using pure cultures of Pseudomonas aeruginosa, fresh water samples and sediment slurries with and without ultrasonic treatment for analyses. Microscopy reached the highest precision, which was similar between pure cultures and water samples, but lower for sediment samples due to a higher percentage of cells in clusters and flocks. The PCR based quantification was most precise for pure cultures. Water and sediment samples were similar but less precise, which might be caused by the applied DNA extraction techniques. MPN measurements were equally precise for pure cultures and water samples. For sediment slurries the precision was slightly lower. The applied ultrasonic treatment of the slurries dispersed the cell clusters and flocks, increased the precision of microscopical and MPN measurements and also increased the number of potential colony forming units. However, the culturable cell number decreased by half. For MPN quantification of viable cells in samples with a high proportion of clustered cells we therefore recommend an optimization of ultrasonic treatment and a confirmation by microscopy and cultivation to reach highest possible dispersion of the cells with a minimum of inactivation. As a result of these observations we suggest a correction factor for MPN measurements to consider the effect of sonication on complex samples. The results are most likely applicable to other complex samples such as soil or biofilms.
Unerwünschte Bewohner : Wasserasseln und andere Makroorganismen in Trinkwasserversorgungsnetzen
(2013)