FG Biotechnologie der Wasseraufbereitung
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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.
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.
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.
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.
Aminophosphonates such as hexamethylenediaminetreta(methylene phosphonic acid) (HDTMP) are categorised as persistent substances. They are commonly used as scale inhibitors in cooling water systems and desalination processes. After utilisation, they are often discharged into aquatic environment without pre-treatment. Advanced oxidation processes (AOP) are promising pre-treatments for industrial wastewater treatments. We investigated the photodegradation of HDTMP with or without addition of
manganese (Mn2þ) and/or H2O2. Similar to results of our former photodegradation studies, we found that HDTMP also undergoes conversion with or without additives during the ultra violet (UV) irradiation. The reaction rate was most affected by the addition of H2O2, i.e. the HDTMP degradation was accelerated
by a factor 3.85 compared with UV treatment without additives. The addition of Mn2þ accelerated the degradation of HDTMP only by a factor 1.53 compared with the UV treatment without additives. The combined addition of Mn2þ and H2O2 accelerated the HDTMP degradation by a factor 2.81. Interestingly, the initial cleavage is not initiated as expected at the CeN bond but at the CeP bond of the methyl carbon and the phosphorus of the methylenephosphonic acid group of HDTMP. This initial
cleavage was independent whether the UV treatment was performed with or without additives. Therefore, we conclude that the degradation mechanism is similar independent of the four tested
treatment conditions. We identified amino(methylenephosphonic acid) AMPA, dimethylamino(methylenephosphonic acid) DAMP and iminodi(methylenephosphonic acid) IDMP as the major breakdown
products by performing LC/MS analyses. The major mineralisation products were ortho-phosphate, ammonium and carbon dioxide. The mass balances of unknown breakdown products
Photodegradation of aminophosphonates such as ethylenediaminetetra(methylenephosphonic acid) (EDTMP) is recently assumed being the major degradation pathway in aquatic environments. Several photolysis studies were reported about EDTMP and possible breakdown products occurring in natural ecosystems. Reliable prediction of environmental photolysis of parent compounds and possible release of breakdown products requires different
set-up conditions and varying the parameters influencing the photodegradation. We studied the influence of three different system configurations during UV degradation of EDTMP. These three configurations differed either in geometry and/or treated sample volumes. System 1 was equipped with a direct cooling jacket at the UV lamp. System 2 had the geometry of system 1 but there was no usage of a direct cooling jacket. System 3 was a gas-tight system with a larger sample volume. Using the chemical actinometer potassium ferrioxalate, we determined the highest photon flux for system 3 followed by system 2 and 1. In addition, we performed scavenger experiments with methanol and ascorbic acid in order to prove the dominating radical species. In system 1, the addition of methanol showed almost no effect while the ascorbic acid resulted in a reduction of 57.1% orthophosphate released. Therefore we conclude that in system 1 the radical-drive degradation of EDTMP is mainly
based on superoxide radicals. In system 2 and 3 both radical species, i.e., hydroxyl radicals and superoxide radicals, contribute to the photodegradation of EDTMP. We determined different half-lives for EDTMP for the three different systems configurations. For system 1, the estimated half-life achieved was 14.09 ± 0.15 min. For
system 2 and 3, the half-lives were almost similar and averaged 4.75 ± 0.05 min and 5.02 ± 0.20 min, respectively. Contrary to our assumption to also find the highest degradation rate for system 3, we found the highest degradation rate for system configuration 2 as a result of the differences in the construction and geometry
of the three systems. Our findings lead us to recommend the three system configuration for different research purposes. Thus, we recommend system 1 for detailed studies on the degradation pathway of the parent compound and their breakdown products. System 2 is recommended as a suitable configuration for kinetic studies of the parent compound. And finally, we recommend the system configuration 3 for complete mass balances. The gas-tight system allows determining all soluble and gaseous compounds.
Aminophosphonate sind synthetisch hergestellte Komplexbildner, die kovalente C-P Bindungen aufweisen. Durch zusätzlich eingefügte Aminogruppen (NH2) komplexieren Amino-phosphonate besonders gut Metallionen, weshalb sie in einem sehr breiten Anwendungsspektum zum Einsatz kommen. So werden Phosphonate vielseitig als Haushalts- und Industriereiniger, aber auch als Komponenten von Kosmetika und in der Medizin verwendet. Des weiteren werden sie zur Kühlwasserkonditionierung, Stabilisierung von Peroxiden oder Bleichbädern und als Korrosionsinhibiter eingesetzt. Entsprechend der vielseitigen Anwendungsgebiete lag bereits in den früher 90igern des vergangenen Jahrhunderts die Gesamtproduktion an Phosphonaten in Europa bei über 11.000 Tonnen pro Jahr (Jaworska et al., 2002). Weltweit zeigen die Nutzung und der damit verbundene Bedarf an Phosphonaten einen immer weiter steigenden Trend. Dem entgegen steht der noch immer wenig untersuchte Verbleib und das chemische Verhalten von Phosphonaten in der Umwelt.
Die gegenwärtig technisch eingesetzten Phosphonate ähneln in ihrer Struktur den bekannteren Aminocarboxylaten wie dem EDTA und NTA. Chemisch synthetisierte Phosphonate sind nur schwer biologisch abbaubar, weshalb zu einer kontinuierlichen Anreicherung in der aquatischen Umwelt kommen kann (Jaworska et al., 2002). Dies liegt unter anderem auch daran, dass Phosphonate als Phosphatersatz in großen Mengen in Waschmitteln enthalten sind und über das Abwasser in die Kläranlagen gelangen, wo sie nicht weiter biologisch abgebaut werden. Sie werden hauptsächlich durch Absorption an die Oberfläche vom Belebtschlamm aus den Abwasser entfernt. Nowack (2004) beschrieb die Toxizität von Phosphonaten in Kläranlagen als gering, verwies aber gleichzeitig darauf, dass aufgrund ihrer hohen Stabilität Phosphonate hemmend auf Proteasen und Syntheasen (Enzyme) von Mikroorganismen wirken.
Der photochemische und biologische Abbau von dem Phosphonat EDTMP wurde eingehend untersucht, um dessen Umweltverhalten besser abschätzen zu können. Ziel der Untersuchungen war es den Abbauprozess zu entschlüsseln als Grundlage für die langfristige Entwicklung umweltverträglicher Aminophosphonatstrukturen. Der photochemische Abbau von EDTMP und die Bildung von Metaboliten konnte mittels LC-MS und 31P-NMR bereits aufgeklärt werden. So zeigt sich das EDTMP bereits nach wenigen Minuten im UV-Licht zerfällt und nicht mehr nachgewiesen werden kann. Als Hauptprodukt konnte das Phosphonat IDMP identifiziert werden. Als weitere Abbauprodukte konnten die Phosphonate EABMP und AMPA identifiziert werden. In allen Versuchen konnte nach 300 min UV-Behandlung mindestens 75% des Endprodukts CO2 nachgewiesen werden. Weitere drei maßgebliche Abbauprodukte wurden mit Hilfe der LC-MS gefunden. Deren chemische Struktur konnte bisher jedoch noch nicht eindeutig beschrieben werden. Im Anschluss an die UV-Experimente wurde ein Versuch mit Sonnenlicht simuliert. Insgesamt verlief der EDTMP-Abbau im Sonnenlicht wesentlich langsamer. Auch hier konnten wieder dieselben Abbauprodukte IDMP, EABMP und AMPA identifiziert werden. Zusätzlich traten auch die drei noch nicht eindeutig identifizierten Metaboliten von EDTMP auf. Aus den gewonnen Daten kann somit geschlussfolgert werden, dass EDTMP unter Einwirkung von UV-Licht abbaubar ist. Die Annahme, dass EDTMP und dessen Abbauprodukte langfristig zur Schädigung des aquatischen Ökosystems führt, kann somit nicht bestätigt werden.
In weiteren Untersuchungen wurde der biologische Abbau von EDTMP und seiner photochemischen Abbauprodukten eingehend studiert. Für die Abbauversuche wurden die beiden Stämme Pseudomonas aeruginosa und Ochrobactrum sp. aus Boden isoliert. Es konnte gezeigt werden, dass sie 1mM EDTMP innerhalb von 35 Tagen zu mindestens 94% abbauen. Zusätzlich zum biologischen Abbau von EDTMP wurden auch die Verdopplungszeitung der beiden Stämme für EDTMP, IDMP, EABMP und AMAP ermittelt. Es zeigte sich, dass der Stamm P. aeruginosa für alle vier Phosphonate ein besseres wachstum zeigte als Ochrobactrum sp. Der Stamm P. aeruginosa erreichte für die Verstoffwechselung von EDTMP eine Verdopplungszeit von 10,1 Tagen, für IDMP 7,4 Tage, für EABMP 7,4 Tage und für AMPA 13,7 Tage. Der Stamm Ochrobactrum sp. erreichte für das Substrat EDTMP eine Verdopplungszeit von 11,8 Tagen, für IDMP 18,9 Tage, für EABMP 7,7 Tage und für AMPA 22,0 Tage. Offensichtlich war für beide Stämme der Abbau von AMPA der limitierende, aber nicht hemmende Schritt im biologischen Abbau. Somit kann geschlussfolgert werden, dass die Abbauprodukte der Photolyse auch biologisch abgebaut werden können. In weiteren Untersuchungen soll der biologische Abbau auf enzymatischer Ebene entschlüsselt werden. Die Identifizierung der Schüsselenzyme spielt hierbei eine wesentliche Rolle, um neuartige Phosphonatstrukturen herzuleiten, die auf der einen Seite ihre Funktionalität und Eigenschaften behalten, aber auf der anderen Seite eine erheblich verbesserte Bioverfügbarkeit aufweisen, so dass eine dauerhafte Akkumulation in der Umwelt und Folgebeeinträchtigungen minimiert ggf. ausgeschlossen werden können.
Aminophosphonates are commonly utilised for complexing bivalent ions such as calcium and magnesium. With regard to environmental samples, the analysis of these highly polar compounds is still challenging due to matrix effects and lacking analytical standard methods. We have recently developed a LC/MS method for common aminophosphonates without derivatisation. This LC/MS method delivers precise and accurate measurement for standard samples with very low concentration of cations disturbing the analysis. However, due to matrix effects this LC/MS method requires a sample clean-up being also applicable to natural water or wastewater samples.
We developed and optimised a sample clean-up procedure applying strong cation exchange resin Dowex 50WX8. This clean-up allows a single LC/MS analysis of hydroxyethelidene(diphosphonic acid) (HEDP), aminotris(methylenephosphonic acid) (ATMP), ethylenediaminetetra(methyloenephosphonic acid) (EDTMP), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP) and its major intermediates amino(methylphosphonic acid) (AMPA) and iminodi(methylenephosphonic acid) (IDMP). We compared different test conditions with six aminophosphonates in either ultra pure water or tap water. The latter was used to simulate cation concentrations typical for natural waters.
To elute all aminophosphonate including AMPA, ammonium acetate addition was necessary. The addition of ammonium acetate was combinable with all tested aminophosphonates and provides high sample quality for LC/MS analysis. For acceptable recovery, the smallest aminophosphonate AMPA required the highest addition of ammonium acetate (1000 mg L-1) during the sample clean-up. Finally, the optimised clean-up procedure was successfully applied to identify and quantify phosphonates from an industrial wastewater sample. The sample clean-up procedure is simple, cheap, rapid and precise and can be further combined with solid phase extraction and more sensitive LC/MS methods.
Durch anthropogene Aktivität hat sich der Nährstoffeintrag in die Umwelt stark erhöht. Die Hauptquellen sind die Landwirtschaft, kommunales und industrielles Abwasser. Erhöhte Stickstofffrachten haben einen starken Einfluss auf den Stickstoffkreislauf. Das Überangebot an Stickstoff führt zu Eutrophierung in Gewässern und somit zu einem weniger diversen, instabileren Ökosystem. Die Stickstoffeliminierung aus Abwässern ist essentiell für den Schutz der Ökosysteme. Das etablierte System der Nitrifikation/Denitrifikation ist aufgrund des hohen Sauerstoffbedarfs sehr energie- und somit auch kostenintensiv. Der Anammox-Prozess (anaerobe Ammonium Oxidation) ist eine potentielle Alternative zur konventionellen Stickstoffelimination. Anammox-Bakterien nutzen Ammonium und Nitrit als Elektronenakzeptor. Durch Nutzung der partiellen Nitrifikation in Kombination mit Anammox (PN/A) kann bis zu 60 % der Belüftungsenergie eingespart werden. Trotz Erfolgen in der Teilstrombehandlung ist der Prozess noch nicht erfolgreich in der Hauptstrombehandlung eingesetzt worden. Die größten Herausforderungen sind die niedrigen Temperaturen, geringe Zulaufkonzentrationen und hohe Konzentrationen organischen Kohlenstoffs. Die vorliegende Arbeit konnte die Wirksamkeit von membranbelüfteten Biofilm Reaktoren unter Verwendung von synthetischem Abwasser bei niedrigen Temperaturen (18 - 8 °C) belegen. Die Innovation in diesem System ist die räumliche Trennung von nitrifizierenden Bakterien und Anammox Bakterien. Die regelmäßige mechanische Entfernung des Nitrifikanten-Biofilms ermöglichte einen langzeitstabilen Reaktorbetrieb. Diese Reaktorkonfiguration konnte auch erfolgreich für hochsaline Abwässer (5 -30 g NaCl*L-1) bei einer Reaktortemperatur von 30 °C und auch unter Zugabe von TOC (40-100 mg TOC*L-1) genutzt werden. Weiterhin wurden Untersuchungen zur Aktivität von salzadaptierter Anammox-Biomasse und nicht adaptierter Biomasse in einem Bereich von 0 bis 50 g NaCl*L-1 durchgeführt. Bei Konzentrationen unter 30 g NaCl*L-1 konnten keine signifikanten Unterschiede im Abbauverhalten festgestellt werden. Allerdings war adaptierte Biomasse bei höheren Salzkonzentrationen konkurrenzstärker. Außerdem wurden Langzeitlagerungsversuche durchgeführt. Die Lagerungsbedingungen variierten in der Lagerungstemperatur, im Redoxpuffer und der Nährstoffversorgung. Nach 106 Tagen bei 4 °C konnte die Anammox-Biomasse noch 91 % ihrer ursprünglichen Aktivität erhalten. Somit konnte eine einfache und kostengünstige Lagerungsmethode für Anammo-Bakterien entwickelt werden.
Calculation of hypolimnic denitrification in a dimictic freshwater lake during summer stratification
(2019)
Phosphonates comprise a very large group of organophosphonates including aminophosphonates. Their main chemical feature leads to complexation of earth alkaline metals and transition metals (under stoichiometric). Therefore they are very often applied as complexing agents of detergents, as compounds of industrial cleaning products, or as antiscalants in cooling water systems and desalination processes. The high consumption of phosphonates within the past two decades leads to an increased discharge into the aquatic environment, of which the environmental risk is still uncertain. To date, there are several analytical methods published to determine phosphonates from environmental samples. However, no standard method has been defined until now. Different treatment technologies to break down phosphonates have been studied extensively such as photochemical degradation, chemical degradation and other advanced oxidation processes. The results obtained from those studies gained our chemical understanding with respect to possible implementation of such pre-treatment approaches in wastewater treatment, cooling water treatment and desalination process. The biological degradation of phosphonates has been studied for more than forty years. It was demonstrated that microorganisms are capable to breakdown the C-P bond with at least three different degradation pathways.
Rapid urbanisation in Ghana has resulted in individuals expanding the cities for abodes without considerations of the negative externalities these may have on the environment. One of the major challenges with rapid urbanisation is the formation of urban slums associated with lack of basic sanitation facilities. This has led to recurrent outbreak of cholera and typhoid fever. The use of a single-stage solar-supported hyper-thermophilic anaerobic biogas digester for the treatment of black water has not been investigated, hence this study. The performance of three seeding sludge under three different hyper-thermophilic temperatures (60°C, 65°C and 70°C) were tested in batch tests. The three seeding sludge were sewage sludge, sludge from maize silage and cow manure. The results from the batch tests showed cow manure at 65°C as the preferred seeding sludge and optimal hyper-thermophilic temperature.
A 50 L single-stage laboratory-scale hyper-thermophilic continuous stirred tank reactor (HT-CSTR) was operated to treat only black water for 10 weeks using cow manure at 65°C as the seeding sludge and optimal hyper-thermophilic temperature. Afterwards, co-digestion of blended kitchen food waste and black water was also practised for 12 weeks. With a mean hydraulic retention time (HRT) of 23.3 days, a mean total COD removal of 86.3 % was achieved. The reactor had an average COD volumetric loading rate of 6.22 kgCOD/(m3.d) and remained uninhibited. It also had organic loading rate of 0.3 kgVS/(m3.d) and a degradation performance (R) of 5.43 kgCOD/(m3.d). Treatment of only black water produced biogas with less methane content of 34.9 % even though a stable pH of 6.9 was recorded both in the reactor and in the effluent. Co-digestion with kitchen food waste increased the percentage content of methane in the biogas by 77 % from 34.9 % to 61.8 %.
The effectiveness of the HT-CSTR to hygienise the effluent for agricultural purpose was assessed by spiking the reactor with 200 ml each of 2 x 109 CFU/ml Salmonella senftenbergensis and 8 x 108 CFU/ml Escherichia coli. The HT-CSTR was able to hygienise all bacteria of Salmonella senftenbergensis and E. coli. A simulation test confirmed that between 30 minutes and 1 hour, all the cells of Salmonella senftenbergensis and E. coli in the treatment system were killed at 65 °C. Eubacteria, Methanosarcina spp., Methanomicrobium spp. and Methanococcus spp. were identified in the seeding sludge at the hyper-thermophilic temperature of 65°C. The design, construction and performance of a pilot-scale reactor in Terterkessim slum in Elmina, Ghana was based on results from the laboratory-scale HT-CSTR. It achieved 97 % removal of influent total COD and could produce about 2.52 Nm³CH₄/(kgCOD.d) which could be burned for at least 8 hours. The effluent cannot be used for cultivation of leafy vegetables such as cabbage since it had some concentrations of pathogens like Salmonella spp. and E. coli but can be used for cotton crop.
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.