Seasonal Variability of Cultivable Nitrate-Reducing and Denitrifying Bacteria and Functional Gene Copy Number in Fresh Water Lake.

This study describes the seasonal course of denitrifying and nitrate-reducing bacteria in a dimictic mesotrophic lake (Lake Scharmützelsee, Brandenburg, Germany) within a three-year period from 2011 to 2013. The bacterial cell numbers were quantified by the fluorescence microscopy, most probable number (MPN) and PCR-dependent quantification of the chromosomal 16S rDNA and of the nirS and nirK gene copy number. The highest seasonal differences (up to three orders of magnitudes) have been measured using MPN in the epilimnion. This variation was not reflected by PCR-dependent approaches or direct microscopical enumeration. At adverse conditions (low temperature and/or low nitrate concentrations), the differences between MPN and gene copy numbers increased by up to five orders of magnitudes and decreased to one magnitude at favourable environmental conditions. These results can be explained best by an increasing ratio of viable but not cultivable (VBNC) cells or dead cells at impairing conditions. In the hypolimnion, the courses of MPN and nir gene copy numbers were similar. This can be explained by a higher feeding pressure and therefore smaller amounts of dormant cells. In the pelagial in general, the total cell numbers enumerated by either microscopical or molecular approaches were similar. In the sediment, more than 99% of the DNA was obviously not related to viable bacteria but was rather DNA in dead cells or adsorbed to particle surfaces.

Biodegradation of selected aminophosphonates by the bacterial isolate Ochrobactrum sp. BTU1.

Aminophosphonates, like glyphosate (GS) or metal chelators such as ethylenediaminetetra(methylenephosphonic acid) (EDTMP), are released on a large scale worldwide. Here, we have characterized a bacterial strain capable of degrading synthetic aminophosphonates. The strain was isolated from LC/MS standard solution. Genome sequencing indicated that the strain belongs to the genus Ochrobactrum. Whole-genome classification using pyANI software to compute a pairwise ANI and other metrics between Brucella assemblies and Ochrobactrum contigs revealed that the bacterial strain is designated as Ochrobactrum sp. BTU1. Degradation batch tests with Ochrobactrum sp. BTU1 and the selected aminophosphonates GS, EDTMP, aminomethylphosphonic acid (AMPA), iminodi(methylene-phosphonic) (IDMP) and ethylaminobis(methylenephosphonic) acid (EABMP) showed that the strain can use all phosphonates as sole phosphorus source during phosphorus starvation. The highest growth rate was achieved with AMPA, while EDTMP and GS were least supportive for growth. Proteome analysis revealed that GS degradation is promoted by C-P lyase via the sarcosine pathway, i.e., initial cleavage at the C-P bond. We also identified C-P lyase to be responsible for degradation of EDTMP, EABMP, IDMP and AMPA. However, the identification of the metabolite ethylenediaminetri(methylenephosphonic acid) via LC/MS analysis in the test medium during EDTMP degradation indicates a different initial cleavage step as compared to GS. For EDTMP, it is evident that the initial cleavage occurs at the C-N bond. The detection of different key enzymes at regulated levels, form the bacterial proteoms during EDTMP exposure, further supports this finding. This study illustrates that widely used and structurally more complex aminophosphonates can be degraded by Ochrobactrum sp. BTU1 via the well-known degradation pathways but with different initial cleavage strategy compared to GS.

Novel standard biodegradation test for synthetic phosphonates.

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.

A novel biomethane (BMP) and composting (CMP) potential framework for determining biogas and composting potential of urban organic waste.

Without treatment, urban market-generated organic waste is discarded in landfills. This could cause environmental contamination due to leachate. This study aims to develop a novel bio-methane and composting potential framework for evaluating the biogas and compostability of organic waste from the Bantama market in Kumasi. Using novel dashboards, the bio-methane and composting potentials were evaluated, and the Theoretical Biomethane Potential (TBMP) was reported to be between 331.52 and 457.93 l CH4/kg VS for carrot leaves and banana peels, respectively, assuming a biodegradability of 80% for the substrates. Only one of the four conditions for compostability was met by the substrates, indicating that they are not suitable for direct composting. In order to optimize the potential of the waste, an integrated system of anaerobic digestion (AD) and composting was utilized. The proposed plant was profitable since the payback period was less than two years, the Net Present Value (NPV) was greater than one, and the Benefit Cost Ratio (BCR) was greater than one. The anaerobic digester and composting plant are capable of producing 12269392 kWh of electricity and 19585 kg of compost per year, respectively. Lastly, the AD and composting technologies at the Bantama market are cost-effective. The government and municipalities may therefore assist private investors in constructing a waste processing plant.

Characterization of glyphosate-resistant Burkholderia anthina and Burkholderia cenocepacia isolates from a commercial Roundup® solution.

Roundup® is the brand name for herbicide solutions containing glyphosate, which specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase of the shikimate pathway. The inhibition of the EPSP synthase causes plant death because EPSP is required for biosynthesis of aromatic amino acids. Glyphosate also inhibits the growth of archaea, bacteria, Apicomplexa, algae and fungi possessing an EPSP synthase. Here, we have characterized two glyphosate-resistant bacteria from a Roundup solution. Taxonomic classification revealed that the isolates 1CH1 and 2CH1 are Burkholderia anthina and Burkholderia cenocepacia strains respectively. Both isolates cannot utilize glyphosate as a source of phosphorus and synthesize glyphosate-sensitive EPSP synthase variants. Burkholderia. anthina 1CH1 and B. cenocepacia 2CH1 tolerate high levels of glyphosate because the herbicide is not taken up by the bacteria. Previously, it has been observed that the exposure of soil bacteria to herbicides like glyphosate promotes the development of antibiotic resistances. Antibiotic sensitivity testing revealed that the only the B. cenocepacia 2CH1 isolate showed increased resistance to a variety of antibiotics. Thus, the adaptation of B. anthina 1CH1 and B. cenocepacia 2CH1 to glyphosate did not generally increase the antibiotic resistance of both bacteria. However, our study confirms the genomic adaptability of bacteria belonging to the genus Burkholderia.

Impact of pH conditions and the characteristics of two electrodialysis membranes on biofilm development under semi-realistic conditions.

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.

Aminophosphonates in Nanofiltration and Reverse Osmosis Permeates.

Aminophosphonates such as aminotris(methylenephosphonic acid) (ATMP) are common constituents of antiscalants. In nanofiltration (NF) and reverse osmosis (RO) processes, ATMP prevents inorganic scaling leading to more stable membrane performance. So far, little attention has been paid to the possible permeation of aminophosphonates through NF and RO membranes. We have investigated the permeability of these membrane types for ATMP and its potential metabolites iminodi(methylenephosphonic acid) (IDMP) and amino(methylenephosphonic acid) (AMPA) with two different NF membranes (TS40 and TS80) and one RO membrane (ACM2) and three different water compositions (ultra-pure water, synthetic tap water and local tap water). We found traces of phosphonates in all investigated permeates. The highest phosphonate rejection occurred with local tap water for all three membranes investigated. Filtration experiments with a technical antiscalant formulation containing ATMP indicated similar trends of phosphonate permeability through all three membranes. We assume that the separation mechanisms of the membranes are the results of a very complex relationship between physico-chemical properties such as Donnan exclusion, feed pH, feed ionic strength and feed concentration, as well as solute-solute interactions.

Molecular mechanisms underlying glyphosate resistance in bacteria.

Glyphosate is a nonselective herbicide that kills weeds and other plants competing with crops. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, thereby depleting the cell of EPSP serving as a precursor for biosynthesis of aromatic amino acids. Glyphosate is considered to be toxicologically safe for animals and humans. Therefore, it became the most-important herbicide in agriculture. However, its intensive application in agriculture is a serious environmental issue because it may negatively affect the biodiversity. A few years after the discovery of the mode of action of glyphosate, it has been observed that bacteria evolve glyphosate resistance by acquiring mutations in the EPSP synthase gene, rendering the encoded enzyme less sensitive to the herbicide. The identification of glyphosate-resistant EPSP synthase variants paved the way for engineering crops tolerating increased amounts of the herbicide. This review intends to summarize the molecular mechanisms underlying glyphosate resistance in bacteria. Bacteria can evolve glyphosate resistance by (i) reducing glyphosate sensitivity or elevating production of the EPSP synthase, by (ii) degrading or (iii) detoxifying glyphosate and by (iv) decreasing the uptake or increasing the export of the herbicide. The variety of glyphosate resistance mechanisms illustrates the adaptability of bacteria to anthropogenic substances due to genomic alterations.

Supplementary data on rapid sample clean-up procedure of aminophosphonates for LC/MS analysis.

Minimising matrix effects through high sample purity is of major importance for LC/MS analysis. Here we provide supplementary data and protocols related to the article "Rapid sample clean-up procedure of aminophosphonates for LC/MS analysis" (revised article submitted to Talanta) [1]. It is demonstrated that the tested phosphonates iminodi(methylenephosphonic acid) (IDMP), hydroxyethelidene(diphosphonic acid) (HEDP), aminotris(methylenephosphonic acid) (ATMP), ethylenediaminetetra(methyloenephosphonic acid) (EDTMP) and diethylenetriaminepenta(methylenephosphonic acid) (DTPMP) dissolved in tap water are not detectable by LC/MS without sample clean-up. Only the smallest aminophosphonate amino(methylenephosphonic acid) (AMPA) was detectable but the recovery is decreased drastically. The optimised sample clean-up with cation exchange resin (CER) Dowex 50WX8 is described in detail and illustrated. The protocol is provided. The influence of the incubation time, addition of different ammonium acetate concentrations, different samples pHs and different water qualities is demonstrated and preferred clean-up conditions are recommended. Calibration results of all tested aminophosphonates are validated regarding limit of detection, limit of quantification, lower limit of quantification, absolute and relative process standard deviation. A final recommendation for the best clean-up condition for all six tested aminophosphonates is provided.•AMPA analysis without derivatisation is possible with optimised clean-up procedure•Clean-up procedure is combinable with derivatisation method of [2]•Procedure is simple, rapid and highly reproducible.

Comparison of different media for the detection of denitrifying and nitrate reducing bacteria in mesotrophic aquatic environments by the most probable number method.

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.

Autofluorescent characteristics of Candidatus Brocadia fulgida and the consequences for FISH and microscopic detection.

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.

Effect of increasing salinity to adapted and non-adapted Anammox biofilms.

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-1 did 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 50 g 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 NO2- / NH4+ 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.

The influence of selected bivalent metal ions on the photolysis of diethylenetriamine penta(methylenephosphonic acid).

DTPMP is predominantly utilized as scale inhibitor. We investigated the reaction rates and degradation mechanism of DTPMP with and without addition of Fe2+, Mg2+ and Ca2+ by performing LC/MS and 31P NMR analyses. DTPMP undergoes conversion with and without addition of bivalent metal ions. The initial cleavage of DTPMP is initiated at the CN bond leading to release of IDMP as its major breakdown product. The release of smaller quantities of EABMP and AMPA confirmed the nucleophilic attack on the DTPMP amines. Oxidation of Fe2+ to Fe3+ during the initial 30 min indicated an intramolecular electron transfer changing the electron density distribution at the nitrogen centre, which increased the radical attack during UV irradiation. Independent of the fact that Fe2+ acted as catalyst and Mg2+ and Ca2+ acted as reactants, we found no significant differences in their degradation mechanisms. However, the reaction rates were strongly affected by the addition of the bivalent metal ions as Fe2+ accelerated most DTPMP degradation followed by Mg2+ and Ca2+. The UV treatment without metal ion addition was four times slower compared with Fe2+ addition. We conclude that in environments rich in ferrous iron and/or at reduced redox potential, photolysis of DTPMP will be catalysed by iron and will lead to accumulation of IDMP, EABMP and AMPA and several other none-quantifiable breakdown products.

Data on DNA gel sample load, gel electrophoresis, PCR and cost analysis.

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" (Kuhn et al., 2017) [1]. 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.

Comparison of ten different DNA extraction procedures with respect to their suitability for environmental samples.

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.

Highly efficient long-term storage of carrier-bound anammox biomass.

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 °C, 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 bacterial quantification techniques on different kinds of environmental samples and the effect of ultrasonic treatment.

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.

Nitrogen transforming community in a horizontal subsurface-flow constructed wetland.

Constructed wetlands are important ecosystems with respect to nitrogen cycling. Here we studied the activity and abundance of nitrogen transforming bacteria as well as the spatial distribution of nitrification, anaerobic ammonium oxidation (anammox), and denitrification processes in a horizontal subsurface-flow constructed wetland. The functional genes of the nitrogen cycle were evenly distributed in a linear way along the flow path with prevalence at the superficial points. The same trend was observed for the nitrification and denitrification turnover rates using isotope labeling techniques. It was also shown that only short-term incubations should be used to measure denitrification turnover rates. Significant nitrate consumption under aerobic conditions diminishes nitrification rates and should therefore be taken into account when estimating nitrification turnover rates. This nitrate consumption was due to aerobic denitrification, the rate of which was comparable to that for anaerobic denitrification. Consequently, denitrification should not be considered as an exclusively anaerobic process. Phylogenetic analysis of hydrazine synthase (hzsA) gene clones indicated the presence of Brocadia and Kuenenia anammox species in the constructed wetland. Although anammox bacteria were detected by molecular methods, anammox activity could not be measured and hence this process appears to be of low importance in nitrogen transformations in these freshwater ecosystems.