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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.
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 Anammox process is an efficient low energy alternative for the elimination of nitrogen from wastewater. The process is already in use for side stream applications. However, some industrial wastewaters, e.g. from textile industry are highly saline. This may be a limit for the application of the Anammox process. The aim of this study was to evaluate the effects of different NaCl concentrations on the efficiency of adapted and non-adapted Anammox biofilms. The tested NaCl concentrations ranged from 0 to 50 g NaCl*L⁻¹. Concentrations below 30 g NaCl*L⁻¹did not significantly result in different nitrogen removal rates between adapted and non-adapted bacteria. However, adapted bacteria were significantly more resilient to salt at higher concentrations (40 and 50g NaCl*L⁻¹). The IC50 for adapted and non-adapted Anammox bacteria were 19.99 and 20.30 g NaCl*L⁻¹, respectively. Whereas adapted biomass depletes the nitrogen in ratios of NO-₂/NH+₄ around 1.20 indicating a mainly Anammox-driven consumption of the nitrogen, the ratio increases to 2.21 at 40 g NaCl*L⁻¹ for non-adapted biomass. This indicates an increase of other processes like denitrification. At lower NaCL concentrations up to 10 g NaCl*L⁻¹, a stimulating effect of NaCl to the Anammox process has been observed.
An enrichment culture of Candidatus Brocadia fulgida was identified by three independent methods: analysis of autofluorescence using different microscope filter blocks and a fluorescence spectrometer, fluorescence in situ hybridization (FISH) with anammox-specific probes and partial sequencing of the 16S rDNA, hydrazine synthase hzsA and hydrazine oxidoreductase hzo. The filter block BV-2A (400–440, 470 LP, Nikon) was suitable for preliminary detection of Ca. B. fulgida. An excitation-emission matrix revealed three pairs of excitation-emission maxima: 288–330 nm, 288–478 nm and 417–478 nm. Several autofluorescent cell clusters could not be stained with DAPI or by FISH, suggesting empty but intact cells (ghost cells) or inhibited permeability. Successful staining of autofluorescent cells with the FISH probes Ban162 and Bfu613, even at higher formamide concentrations, suggested insufficient specificity of Ban162. Under certain conditions, Ca. B. fulgida lost its autofluorescence, which reduced the reliability of autofluorescence for identification and detection. Non-fluorescent Ca. Brocadia cells could not be stained with Ban162, but with Bfu613 at higher formamide concentrations, suggesting a dependency between both parameters. The phylogenetic analysis showed only good taxonomical clustering of the 16S rDNA and hzsA. In conclusion, careful consideration of autofluorescent characteristics is recommended when analysing and presenting FISH observations of Ca. B. fulgida to avoid misinterpretations and misidentifications.
Calculation of hypolimnic denitrification in a dimictic freshwater lake during summer stratification
(2019)
The cultivation based characterization of microbial communities and the quantification of certain functional bacterial groups is still an essential part of microbiology and microbial ecology. For plate count methods meanwhile low strength media are recommended, since they cover a broader range of different species and result in higher counts compared to established high strength media. For liquid media, as they are used for most probable number (MPN) quantifications, comparisons between high and low strength media are rare. In this study we compare the performance of different high and low strength media for the MPN quantification of nitrate reducing and denitrifying bacteria in two different fresh water environments. We also calculated the cell specific turnover rates of several denitrifying cultures previously enriched in high and low strength media from three different fresh water environments and a waste water treatment plant. For fresh water samples, our results indicate that high strength media detect higher MPN of denitrifying bacteria and in equal MPN of nitrate reducing bacteria compared to low strength media, which is in contrary to plate count techniques. For sediment samples, high and low strength media performed equal. The cell specific turnover rate was independent from the enrichment media and the media of the performance test. The cause of the lower denitrifyer MPN in low strength media remains, however, unclear. The results are important for further MPN quantifications of bacteria in nutrient poor environments and for calculations of nitrogen turnover rates by kinetical models using the number of metabolic active cells as one parameter.
The reuse of treated wastewater for irrigation is of increasing importance. The Ecosave farming project developed a new photocatalytic electrodialysis process for desalination and hygienization. However, membrane scaling significantly reduces filtration efficiency. This study investigated biofilm development on anion and cation exchange membranes at a wide pH range in pre-treated wastewater. Epifluorescence microscopic quantification of the biofilm by cell counts and surface coverage together with 16S rDNA gene copy numbers showed stronger biofilm development on the anion exchange membrane (AEM) compared with the cation exchange membrane (CEM) with up to 105 cells mm−2 and 20% surface coverage after three weeks. As the AEM biofilm developed best in neutral and a slightly alkaline pH, the CEM was colonized preferably at alkaline conditions. Extreme pH conditions strongly inhibited biofilm growth, which might help to minimize the maintenance effort by creating those conditions during the operation of the dialysis cell itself.
Today, nanotechnologies (NTs) are well established in both private households and commercial markets. NTs are fully accepted in several sectors, such as medicine and pharmacy, and in industries, such as chemistry, electricity, food production, military, and other commercial branches, due to their unique properties. With regard to the growing demands for environmental resources caused by the still-growing global population, the application of NTs is an extremely important new branch in the environmental sector, delivering several advantages. Our review provides a comprehensive overview of the current developments in environmental remediation, wastewater treatment, drinking water treatment, and agriculture. More specifically, in the section on environmental remediation, we review the application of NTs towards enhanced reductive dechlorination, removal of heavy metals and remediation of oil spills. In the section on wastewater treatment, we highlight developments in the adsorption of heavy metals and persistent substances, advanced photocatalytic
degradation of common wastewater pollutants, and improvements in membrane filtration processes.
In the section on drinking water treatment, we discuss applications for the enhanced disinfection of pathogens, removal of heavy metals, point-of-use treatments, and the removal of organic matter. In the final section, on agriculture, we provide an overview of precision farming and the current state of the art concerning nanofertilisers, nanopesticides, nanoherbicides, and nano(bio)sensors.
Determination of biodegradation of synthetic phosphonates such as aminotris(methylenephosphonic acid) (ATMP), ethylenediamine tetra(methylenephosphonic acid) (EDTMP), or diethylenetriamine penta(methylenephosphonic acid) (DTPMP) is a great challenge. Commonly, ready biodegradability of organic substances is
assessed by OECD 301 standard tests. However, due to the chemical imbalance of carbon to phosphorus synthetic phosphonates do not promote microbial growth and, thus, limiting its biodegradation. Therefore, standard OECD test methods are not always reliable to predict the real biodegradability of phosphonates. In the presented study, we report the development of a standardized batch system suitable to synthetic phosphonates such as ATMP, EDTMP, DTPMP and others. The novel standard batch test is applicable with pure
strains, activated sludge from different wastewater treatment plants (i.e., municipal and industrial), and with tap water as inoculum. We optimized the required calcium and magnesium exposure levels as well as the amount of the start inoculum biomass. We demonstrated that our test also allows to determine several parameters including
ortho-phosphate (o-PO43 ), total phosphorus (TP), ammonium (NH4+) and total organic carbon (TOC). In addition, also LC/MS analyses of cell-free medium is applicable for determining the mother compounds and metabolites. We applied our optimized standardized batch with selected phosphonates and evidenced that the chemical structure has a major influence of the microbial growth rates. Thus, our novel batch test overcomes drawbacks of the OECD 301 test series for determination of easy biodegradability for stoichiometric imbalanced organic
compounds such as phosphonates.