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Examining substrate uptake patterns of Rhodocyclus-related PAO in full-scale EBPR plants by using the MAR-FISH technique.

While recognised as the important population responsible for enhanced biological phosphorus removal (EBPR), detailed knowledge on the physiology of Rhodocyclus-related polyphosphate accumulating organisms (PAO) has yet to be grasped. The objective of this study was to examine the in situ substrate uptake patterns of Rhodocyclus-related PAO present in full-scale EBPR plants by the combined technique of microautoradiography-fluorescent in situ hybridization (MAR-FISH). The presence of these PAO in the four investigated plants was confirmed by FISH and they constituted 17%, 9%, 8%, and 7% of the sludge community. By using MAR-FISH technique, Rhodocyclus-related PAO in all the plants demonstrated similar anaerobic substrate uptake patterns. They were capable of assimilating acetate, aspartate and glutamate under anaerobic condition but they showed negative uptake with palmitate. A significant fraction of the MAR-positive cells assimilated acetate, aspartate or glutamate was found to be Rhodocyclus-related PAO. Dual staining with DAPI and FISH showed that these PAO also accumulated polyphosphate aerobically with aspartate and glutamate as carbon source. The ability of assimilating amino acids besides acetate strongly indicates the versatile physiology of Rhodocyclus-related PAO, which could benefit them to achieve predominance in EBPR activated sludge.

Environmental Restoration and Remediation↗

Construction and analysis of a metagenomic library from an enhanced biological phosphorus removal biomass.

The enhanced biological phosphorus removal (EBPR) process is regularly used for the treatment of wastewater, but suffers from erratic performance. Successful EBPR relies on the growth of bacteria called polyphosphate-accumulating organisms (PAOs), which store phosphorus intracellularly as polyphosphate, thus removing it from wastewater. Metabolic models have been proposed which describe the measured chemical transformations, however genetic evidence is lacking to confirm these hypotheses. The aim of this research was to generate a metagenomic library from biomass enriched in PAOs as determined by phenotypic data and fluorescence in situ hybridisation (FISH) using probes specific for the only described PAO to date, "Candidatus Accumulibacter phosphatis". DNA extraction methods were optimised and two fosmid libraries were constructed which contained 93 million base pairs of metagenomic data. Initial screening of the library for 16S rRNA genes revealed fosmids originating from a range of nonpure-cultured wastewater bacteria. The metagenomic libraries constructed will provide the ability to link phylogenetic and metabolic data for bacteria involved in nutrient removal from wastewater.

Base Sequence↗

Calcium effect on enhanced biological phosphorus removal.

The role of calcium (Ca) in enhanced biological phosphorus removal and its possible implications on the metabolic pathway have been studied. The experience has been carried out in an SBR under anaerobic-aerobic conditions for biological phosphorus removal during 8 months. The variations of influent Ca concentration showed a clear influence on the EBPR process, detecting significant changes in Y(PO4). These Y(PO4) variations were not due to influent P/COD ratio, pH, denitrification and calcium phosphate formation. The Y(PO4) has been found to be highly dependent on the Ca concentration, increasing as Ca concentration decreases. The results suggest that high Ca concentrations produce "inert" granules of polyphosphate with Ca as a counterion that are not involved in P release and uptake. Furthermore, microbiological observations confirmed that appreciable changes in PAO and GAO populations were not observed. This behaviour could suggest a change in the bacterial metabolic pathway, with prevailing polyphosphate-accumulating metabolism (PAM) at low influent Ca concentration and glycogen-accumulating metabolism (GAM) at high concentration.

Aerobiosis↗

Enhanced biological phosphorus removal in an anaerobic-aerobic sequencing batch reactor: characteristics of carbon metabolism.

Carbon metabolism of activated sludge performing enhanced biological phosphorus removal (EBPR) was investigated in a sequencing batch reactor (SBR), where glucose and acetate were supplied as carbon sources. There have been reports that EBPR often failed because of outgrowth of G-bacteria, which are able to use glycogen (or glucose) alone instead of glycogen and polyphosphate as an internal energy storage pool for the anaerobic uptake and storage of acetate. However, in this experiment, complete EBPR was achieved without proliferation of G-bacteria despite the supply of glucose in addition to the acetate. Additional one-cycle SBR operation was carried out to investigate the detailed metabolism of glucose and acetate. Acetate-using, phosphorus-accumulating organisms (PAOs) could use glucose as a source for the anaerobic polyhydroxyalkanoate (PHA) synthesis from acetate. However, glucose and acetate were metabolized separately through independent metabolic pathways by respective microorganisms: acetate-using PAOs, lactate-producing organisms (LPOs), and lactate-using PAOs. Glucose was rapidly converted to lactic acid by LPOs before acetate-using PAOs used the glucose as an energy source for the anaerobic PHA synthesis from acetate and the lactateusing PAOs anaerobically synthesized PHA from lactate at the expense of polyphosphate. The EBPR was accomplished by acetate-using PAOs and lactate-using PAOs independently when glucose acetate were supplied together.

Acetates↗

Diadenosine tetraphosphate (Ap4A) and triphosphate (Ap3A) signaling of human sperm motility.

The ubiquitous dinucleotide polyphosphate, diadenosine tetraphosphate (Ap4A), has been shown to be a signal molecule for DNA replication in mammalian cells. In this study, Ap4A and a related compound, diadenosine triphosphate (Ap3A), were tested for possible signaling functions in human spermatozoa. A computerized automated semen analyzer was used to detect changes in spermatozoa motility parameters. Cryopreserved-thawed donor spermatozoa were washed and incubated in 0.1 mM Ap4A, 0.1 mM Ap3A, or control medium. The data indicated that both Ap4A and Ap3A decreased the percentage of motile spermatozoa after 4 or more hours of incubation in vitro. The two dinucleotide polyphosphates caused an increase in the amplitude of lateral spermatozoa head displacement parameter only at the start of incubation. The other spermatozoa kinematic parameters were unaffected. No opposing ying-yang dual actions of Ap4A to Ap3A were seen. From the results, Ap4A and Ap3A were observed to be potential inhibitory signals of spermatozoa motility after prolonged exposure.

Dinucleoside Phosphates↗

Purification and characterization of a soluble polyphosphatase from mitochondria of Saccharomyces cerevisiae.

A polyphosphatase with the specific activity 2.2 U/mg was purified to apparent homogeneity from a soluble preparation of mitochondria of Saccharomyces cerevisiae. The polyphosphatase is a monomeric protein of approximately 41 kD. The purified enzyme hydrolyzes polyphosphates with an average chain length of 9 to 208 phosphate residues to the same extent, but its activity is approximately 2-fold higher with tripolyphosphate. ATP, PPi, and p-nitrophenyl phosphate are not substrates of this enzyme. The apparent Km values are 300, 18, and 0.25 microM obtained at hydrolysis of polyphosphates with a chain length of 3, 15, and 188 phosphate residues, respectively. Several divalent cations stimulated the enzyme activity 1.2-27-fold (Mg2+ = Co2+ = Mn2+ > Zn2+). Determination of the protein N-terminal sequence and its comparison with the EMBL data library indicates that the soluble polyphosphatase of mitochondria of S. cerevisiae is not encoded by the gene of the major yeast polyphosphatase PPX1.

Acid Anhydride Hydrolases↗

Contribution of Pseudomonas spp. to phosphorus uptake in the anoxic zone of an anaerobic-anoxic-aerobic continuous activated sludge system.

A continuously operated laboratory-scale (32 L) nitrification denitrification biological excess phosphorus removal (NDBEPR) activated sludge system (modeled on the 3-stage Phoredox configuration) was maintained for 140 d. The transition from a non-biological excess phosphorus removal (BEPR) sludge to one exhibiting a strong BEPR mechanism was monitored. Mixed liquor seed inoculum was obtained from a full-scale single aerobic activated sludge installation and subjected to conditions conducive to BEPR, i.e. increasing influent acetate (HAc) concentrations. At a sludge age of 10 d with 100% HAc feed, the system was capable of removing a maximum of ca. 40 mgPO4-P/L from the bulk liquid; P/VSS of ca. 0.27 (mgP/mgVSS); and VSS/TSS of 0.53 (mgVSS/mgTSS) in the aerobic zone was attained. Although typical BEPR phosphorus transformation patterns were routinely observed, i.e. anaerobic phosphate release and aerobic phosphate uptake, phosphate uptake in the anoxic zone was also recorded indicating the presence of denitrifying phosphorus accumulating organisms (DPAOs) in the sludge community. The microbial community was screened (using both isolation and direct methods of analysis) for the presence of Pseudomonas spp. as this genus is known to perform both polyphosphate accumulation and denitrification processes. Isolation of anoxic mixed liquor bacteria on solid media and identification using the APl 20NE system resulted in the total dominance of the Pseudomonads (> 50%). However, direct fluorescent in situ hybridizations (FISH) revealed that Pseudomonas spp. only constituted ca. 3% of the total bacterial community indicating that other bacterial genera are contributing to simultaneous polyphosphate accumulation and denitrification processes in the anoxic zones of NDBEPR systems.

DNA, Bacterial↗

Selective agonism of group I P2X receptors by dinucleotides dependent on a single adenine moiety.

We have investigated the activity of naturally occurring high-performance liquid chromatography-purified diadenosine polyphosphates (Ap(n)A, n = 5-6), adenosine polyphospho guanosines (Ap(n)G, n = 5-6), and diguanosine polyphosphates (Gp(n)G, n = 5-6) under voltage-clamp conditions at recombinant rat P2X1-4 purinoceptor subtypes expressed in Xenopus laevis oocytes. At rP2X1 and rP2X3 receptors, Ap(n)As and Ap(n)Gs evoked concentration-dependent inward currents. Gp(n)Gs were not active at these receptors. At rP2X2 and rP2X4 receptors, dinucleotides did not show significant activity. For the rP2X1 receptor, Ap(n)As and Ap(n)Gs were partial agonists; for the P2X3 receptor, only Ap5G was full agonist, whereas the other tested substances were partial agonists. The rank order of potency at rP2X1 was ATP > or = Ap6A > or = Ap5A > or = Ap6G > or = Ap5G, and rank order of efficacy was ATP > or = Ap5A > or = Ap6A > Ap5G > Ap6G, whereas at rP2X3 the rank order of potency was ATP > Ap5G > or = Ap5A > or = Ap6A > or = Ap6G and the rank order of efficacy was ATP approximately Ap5G > or = Ap5A approximately Ap6A > or = Ap6G. For rP2X1 and rP2X3 it is evident that receptor agonism depended on the presence of at least one adenine moiety in the dinucleotide, while the presence of a guanine moiety had a significant impact and decreased agonist efficacy. The data suggest that naturally occurring Ap(n)As and Ap(n)Gs may play an important physiological role in different human tissues and systems by activating group I P2X receptors.

Adenine↗

Presence of Rhodocyclus in a full-scale wastewater treatment plant and their participation in enhanced biological phosphorus removal.

The objective of this research was to assess the relevance of organisms related to Rhodocyclus in enhanced biological phosphorus removal in full-scale wastewater treatment plants. The presence of these organisms in full-scale plants was first confirmed by fluorescent in situ hybridization. To address which organisms were involved in phosphorus removal, a method was developed which selected polyphosphate-accumulating organisms from activated sludge samples by DAPI staining and flow cytometry. Sorted samples were characterized using fluorescent in situ hybridization. The results of these analyses confirmed the presence of organisms related to Rhodocyclus in full-scale wastewater treatment plants and supported the involvement of these organisms in enhanced biological phosphorus removal. However, a significant fraction of the polyphosphate-accumulating organisms were not related to Rhodocyclus.

Bioreactors↗

[Magnesium orthophosphate, a new form of reserve phosphates in the halophilic archaeon Halobacterium salinarium].

The accumulation and utilization of reserve phosphates in the extremely halophilic archaeon Halobacterium salinarium were studied. The growth of H. salinarium was found to depend on the initial concentration of inorganic phosphate (Pi) in the culture medium and its content in the inoculum. Growing cells consumed 85-95% of Pi from the medium. Unlike the reserve phosphates of many other microorganisms, which are mainly polyphosphates, the reserve phosphates of H. salinarium cells contain no more than 15% polyphosphates, the rest being magnesium orthophosphate. The excessive consumption of Pi from the medium changed cell morphology and caused the death of part of the cell population. The cells that remained viable could grow in a Pi-deficient medium, utilizing about 70% of reserve magnesium phosphate as the phosphorus source.

Culture Media↗

[The efficacy on natural extrinsic stain of Colgate whitening dentifrice--eight-week study].

OBJECTIVE: To assess the effect on natural extrinsic dental stain by use of a dentifrice containing 1% tetrasodium polyphosphate (TSPP), 7% sodium tripolyphosphate (STPP), 0.243% sodium fluoride and 10% high cleaning silica (HCS) over an eight-week period was compared to a sodium fluoride/silica placebo dentifrice without stain removal ingredients. METHODS: Ninety one subjects were selected and stratified into two balanced groups randomly assigned to use the test dentifrice or the placebo dentifrice. The two groups were well balanced with regard to their mean baseline stain index scores, gender and tobacco habits. Subjects were instructed to brush their teeth twice daily (morning and evening) for one minute with their assigned dentifrice and a commercially available soft bristled toothbrush. Extrinsic dental stain examinations, which included mean stain area and mean stain intensity, were conducted at baseline, four and eight weeks. The same dental examiner conducted examinations on the subjects at each examination. RESULTS: After eight-week use of their assigned products, those subjects in the TSPP/STPP/HCS dentifrice group demonstrated a statistically significant (P = 0.0001) stain reduction of 37.1%, as compared to the fluoride placebo dentifrice group without stain removal ingredients. CONCLUSION: The use of a dentifrice containing 1% tetrasodium polyphosphate, 7% sodium tripolyphosphate, 0.243% sodium fluoride and 10% high cleaning silica is effective in reducing natural extrinsic dental stain, when compared to a placebo fluoride dentifrice.

Adolescent↗

[The distribution of bacterioplankton and mesozooplankton biomass in the White and Barents Seas coastal water].

The total population density and the biomass of bacterioplankton, mesozooplankton, and phosphate-accumulating bacteria (PAB) were estimated during the 2000-2001 summer-autumn seasons in the coastal waters of the White and Barents Seas, which are subjects to the action of tidal and sea currents, the inflow of riverine waters, and anthropogenic impact. In the shallow estuarine waters with salinities of 6.5-32@1000 near the Chernaya, Pesha, and Pechora River mouths, the population of PAB fluctuated from 0.1 to 9.1 million cells/ml (0-36% of the total bacterial population). In pelagic seawaters, which are low in phosphates (12-50 micrograms/l) and are characterized by an increased iron/phosphorus ratio (2.0-3.6), bacterioplankton amounted to 0.1-1.6 million cells/ml and was mainly represented by small organisms with a volume of 0.08-0.15 micron 3, commonly lacking intracellular polyphosphates. In the pelagic zone of the Barents Sea, the biomass of mesozooplankton (Bz) was comparable with that of bacterioplankton (Bb = 39-175 mg/m3), the Bb/Bz ratio being 1.4-4.6. Off the Varandeiskii, Pechora, and Kolguyev oil terminals, Bb increased to 155-300 mg/m3 and the Bb/Bz ratio rose to 1.4 to 50.3 (with an average value of 20.9), presumably due to the severe anthropogenic impact on these waters. In this case, the dense population of bacterioplankton (0.9-7.6 million cells/ml) was mainly represented by large cells (0.12-0.76 micron 3 in volume), most of which (3-43% of the total bacterioplankton population) contained polyphosphates. The chemical composition of these waters was characterized by an elevated content of the total phosphorus (65-128 micrograms/l) and by a low iron/phosphorus ratio (0.9-1.2).

Animals↗

[The signal transduction of serotonin secretion involves protein tyrosine phosphorylation in rat basophilic leukemia cells].

We investigated the signal transduction of serotonin secretion by stimulation with DNP-Ascaris antigen or ionomycin in rat basophilic leukemia cells (RBL-2H3). The modes of action of antigen and ionomycin for serotonin secretion were shown to be similar. The treatment of cells with antigen resulted in increased tyrosine phosphorylation of 105 and 72 KDa proteins, in particular, the tyrosine phosphorylation of 72 KDa protein seemed to correlate with serotonin secretion. Furthermore, we observed that antigen stimulation caused a marked increase in inositol polyphosphates production, which derived from the tyrosine phosphorylation of phospholipase C-gamma in RBL-2H3 cells. On the other hand, treatment with ionomycin also resulted in an increase in tyrosine phosphorylation of 72 KDa protein, but did not induce inositol polyphosphates production. These results suggested that the activation of tyrosine kinase may be related to serotonin secretion, and that intracellular Ca2+ increase may also play an important role in this activation.

Animals↗

Quantitative estimation of the role of denitrifying phosphate accumulating organisms in nutrient removal.

It has been reported that a proportion of polyphosphate-accumulating organisms (PAOs) can denitrify or utilize nitrate as an electron acceptor. The usage of denitrifying-PAO (DN-PAO) can relieve the competition for COD between denitrification and phosphorus removal because they can treat nitrate and phosphate by using the same carbon source. To effectively use DN-PAO for biological nutrient removal (BNR), a new system was proposed in which an anaerobic phase is placed at the influent end, followed by he anoxic and external nitrification phase. In this study, the lab-scale proposed system (A2N system) was operated with a municipal wastewater 1) to confirm stable settlement of DN-PAO in the proposed system, 2) to quantitatively estimate the fraction of different groups of organisms like denitrifiers without polyphosphate accumulating capability, aerobic-PAO and DN-PAO and 3) to estimate the advantages of DN-PAO's presence in the system. Moreover, batch experiments in which anoxic and aerobic phosphate uptake rates (PUR) were measured were also carried out. The activity of DN-PAO was observed throughout the experimental period by the batch experiment. From the results of the calculation of COD utilized by each group of organisms, it was concluded that the proposed system could accumulate much more PAO (as DN-PAO) than conventional BNR systems. Moreover, they were responsible for both EBPR and denitrification.

Bacteria, Anaerobic↗

Characterization of an inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain.

Some of the properties of a high affinity Ins(1,3,4,5)P4 3-phosphatase (Km approximately 400 nM) from the soluble fraction of pig brain are presented. Several inositol polyphosphates reduced the activity of the Ins-(1,3,4,5)P4 3-phosphatase. The most effective inhibitors were Ins(1,3,4, 5,6)P5 and InsP6 with Ki-values of about 60 nM and 3 nM, respectively. We could show that at least InsP6 is a likely substrate of the Ins(1,3,4,5)P4 3-phosphatase, which degraded InsP6 with a very low reaction velocity. This 3-phosphatase may be important for the metabolism of higher phos-phorylated inositol polyphosphates.

Animals↗

GTP gamma S effects on phosphatidylinositol phospholipase C alpha isoenzyme activity isolated from guinea pig uterine smooth muscle at different stages of pregnancy.

The ability of GTP gamma S to activate release of inositol polyphosphates from isolated permeabilised guinea pig uterine smooth muscle cells and from partially purified PI-PLC alpha has been studied. Streptolysin O permeabilised and [3H]inositol prelabelled cells show a time dependent release of inositol polyphosphates, predominantly inositol 4-phosphate. Ca2+ stimulated IP release with a Ka of 161 +/- 1.1 nM and this was further enhanced in an additive manner by GTP gamma S between 1-100 microM; the Ka for Ca2+ in the presence of 0.1 mM GTP gamma S was 117 +/- 0.7 nM. GTP gamma S activation of IP production did not require Ca2+ in the medium. Permeabilisation of the uterine smooth muscle cells with Streptolysin O readily released PI-PLC activity into the medium. However, unlike studies with isolated membranes 63.4 +/- 6.4% of the enzyme activity remained associated with membranes and/or particulate fractions of the cell. Studies were undertaken with PI-PLC alpha, the predominant isoenzyme form, partially purified from uterine smooth muscle at different stages of pregnancy by Q-Sepharose and Heparin-Agarose chromatography. The enzyme co-purifies with firmly associated GTP-binding activity. Enzyme prepared from near-term uterus is activated by 0.1 mM GTP gamma S, up to 100% when Ca2+ is between 0.1-1 microM, while 10 microM AlF4- under those conditions caused complete inhibition of the enzymes. Responses for enzymes prepared from non-pregnant uteri were broadly similar. In contrast enzyme preparations from guinea pig uteri at 20-60 days of pregnancy show an inhibition of activity in response to 0.1 mM GTP gamma S addition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Role of nucleoside triphosphatase in the formation of factor-X in frog myocardium following exposure to acetylcholine].

Under the effect of acetylcholine and vagal stimulation on the donor frog myocardium, a uridine polyphosphate-like substance (X-factor) is released. It intensifies contractions of the isolated heart-recipient and decreases the heart's sensitivity to acetylcholine and vagal stimulation. Acetylcholine (1-10(-4)-1-10(-5 g/ml) decreases UTPase activity (by 20-25%) and ATPase activity (by 15%) in isolated ventricle of the frog heart. When acetylcholine is washed away, UTPase activity is almost completely restored. Suppression of UTPase activity by acetylcholine seems to be one of the mechanisms responsible for the accumulation and release and uridine polyphosphates in the heart muscle.

Acetylcholine↗

Optimizing phosphorus characterization in animal manures by solution phosphorus-31 nuclear magnetic resonance spectroscopy.

A procedure involving alkaline extraction and solution 31P nuclear magnetic resonance (NMR) spectroscopy was developed and optimized for the characterization of P in animal manures (broiler, swine, beef cattle). Inclusion of ethylenediaminetetraacetic acid (EDTA) in the alkaline extraction solution recovered between 82 and 97% of the total P from the three manures, which represented a significant improvement on recovery in NaOH alone. Low concentrations of paramagnetic ions in all manure extracts meant that relatively long delay times (> 5 s) were required for quantitative analysis by solution 31P NMR spectroscopy. The manures contained inorganic orthophosphate, orthophosphate monoesters, orthophosphate diesters, and inorganic polyphosphates, but results were markedly influenced by the concentration of NaOH in the extractant, which affected both spectral resolution and the apparent P composition of the extracts. For example, extraction of swine manure and broiler litter with 0.5 M NaOH + 50 mM EDTA produced remarkable spectral resolution that allowed accurate quantification of the four signals from phytic acid, the major organic P compound in these manures. In contrast, more dilute NaOH concentrations produced considerable line broadening that obscured individual signals in the orthophosphate monoester region of the spectra. Spectral resolution of cattle manure extracts was relatively unaffected by NaOH concentration. Improvements in spectral resolution of more concentrated NaOH extracts were, however, compromised by the disappearance of phospholipids and inorganic polyphosphates, notably in swine and cattle manure extracts, which indicated either degradation or a change in solubility. The optimum extraction conditions will therefore vary depending on the manure type and the objectives of the study. Phytic acid can be accurately quantified in swine manure and broiler litter by extraction with 0.5 M NaOH + 50 mM EDTA, while a more dilute NaOH concentration should be used for complete P characterization or comparison among different manure types.

Animals↗