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Comparative Study of Different Methods for Analyzing Denitrifying Bacteria in Fresh Water Ecosystems
(2014)
Bacteria capable of denitrification play a significant role in the nitrogen cycle of freshwater ecosystems.
By metabolizing nitrogen compounds they e.g. counteract the eutrophication of natural
waters. To get detailed insights into the in situ turnover rates of nitrogen a reliable tool of quantification
for active microorganisms is essential. In the present investigation, quantification capabilities
of a molecular tool (Polymerase Chain Reaction—PCR) and a cultivation based tool (Most
probable number—MPN) were investigated and compared. The total bacterial concentration
yielded by the molecular PCR approach was up to 6-fold higher compared to the results of the MPN
approach. However, the portion of culturable denitrifying bacteria compared to the number of
specific gene copies (nirS) was much lower. Depending on the environmental conditions, the difference
between the PCR and the MPN approach was up to three orders of magnitude. From lab
scale experiments with a pure P. aeroginosa strain it can be concludes, that these differences are
not the result of inappropriate culture conditions but rather reflect the portion of so called viable
but not culturable bacteria (VBNC). Low nitrate concentrations as found in many fresh water ecosystems
induced a significant increase in the portion of non culturable denitrifying bacteria. Referred
to the investigation of dynamic populations, the number of metabolic active bacteria is
represented by the MPN rather than by the PCR approach.
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.
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.