Polyphosphate Accumulation Research Articles

The feasibility of citric acid as external carbon source for biological phosphorus removal in a sequencing batch biofilm reactor (SBBR)

The aim of this study was to evaluate the feasibility of citric acid as external carbon source for biological phosphorus removal. Citric acid was the only source of organic carbon and biomass occurred only as a biofilm. The effect of varying the duration of the mixing and aeration phases on the process of dephosphatation and consumption of organic substrate was examined. The operation consisted of filling (0.5h), mixing (2.75–8.25h), aeration (2.75–8.25h) and draw (0.5h) phases. Total phosphorus and nitrogen influent concentrations were 7.02mgPL−1 and 2.05mgNL−1, respectively. Enhanced intracellular accumulation of polyphosphates (poly-P) and their release in reactors with immobilized biomass (SBBR) was stated. The highest rates of phosphates release (0.059±0.004gP–PO43−m−2h−1) and phosphates uptake (0.028±0.002gP–PO43−m−2h−1) were observed for mixing to aeration phase duration ratio of 1:3. The percentage of phosphorus in dry matter of biofilm reached 4.6%.

Inorganic phosphate is a trigger factor for Microbispora sp. ATCC-PTA-5024 growth and NAI-107 production

BackgroundNAI-107, produced by the actinomycete Microbispora sp. ATCC-PTA-5024, is a promising lantibiotic active against Gram-positive bacteria and currently in late preclinical-phase. Lantibiotics (lanthionine-containing antibiotics) are ribosomally synthesized and post-translationally modified peptides (RiPPs), encoded by structural genes as precursor peptides.The biosynthesis of biologically active compounds is developmentally controlled and it depends upon a variety of environmental stimuli and conditions. Inorganic phosphate (Pi) usually negatively regulates biologically-active molecule production in Actinomycetes, while it has been reported to have a positive control on lantibiotic production in Firmicutes strains. So far, no information is available concerning the Pi effect on lantibiotic biosynthesis in Actinomycetes.ResultsAfter having developed a suitable defined medium, Pi-limiting conditions were established and confirmed by quantitative analysis of polyphosphate accumulation and of expression of selected Pho regulon genes, involved in the Pi-limitation stress response. Then, the effect of Pi on Microbispora growth and NAI-107 biosynthesis was investigated in a defined medium containing increasing Pi amounts. Altogether, our analyses revealed that phosphate is necessary for growth and positively influences both growth and NAI-107 production up to a concentration of 5 mM. Higher Pi concentrations were not found to further stimulate Microbispora growth and NAI-107 production.ConclusionThese results, on one hand, enlarge the knowledge on Microbispora physiology, and, on the other one, could be helpful to develop a robust and economically feasible production process of NAI-107 as a drug for human use.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0133-0) contains supplementary material, which is available to authorized users.

Polyphosphate accumulation is driven by transcriptome alterations that lead to near-synchronous and near-equivalent uptake of inorganic cations in an arbuscular mycorrhizal fungus.

Arbuscular mycorrhizal (AM) fungi accumulate a massive amount of phosphate as polyphosphate to deliver to the host, but the underlying physiological and molecular mechanisms have yet to be elucidated. In the present study, the dynamics of cationic components during polyphosphate accumulation were investigated in conjunction with transcriptome analysis. Rhizophagus sp. HR1 was grown with Lotus japonicus under phosphorus-deficient conditions, and extraradical mycelia were harvested after phosphate application at prescribed intervals. Levels of polyphosphate, inorganic cations and amino acids were measured, and RNA-Seq was performed on the Illumina platform. Phosphate application triggered not only polyphosphate accumulation but also near-synchronous and near-equivalent uptake of Na(+) , K(+) , Ca(2+) and Mg(2+) , whereas no distinct changes in the levels of amino acids were observed. During polyphosphate accumulation, the genes responsible for mineral uptake, phosphate and nitrogen metabolism and the maintenance of cellular homeostasis were up-regulated. The results suggest that inorganic cations play a major role in neutralizing the negative charge of polyphosphate, and these processes are achieved by the orchestrated regulation of gene expression. Our findings provide, for the first time, a global picture of the cellular response to increased phosphate availability, which is the initial process of nutrient delivery in the associations.

Metabolic characteristics of a glycogen-accumulating organism in Defluviicoccus cluster II revealed by comparative genomics.

Glycogen-accumulating organisms (GAOs) may compete with phosphate-accumulating organisms (PAOs) for short-chain fatty acids (VFAs) in anaerobic polyhydroxyalkanoates (PHA) synthesis, but no consequently aerobic polyphosphate accumulation in enhanced biological phosphorus removal (EBPR) process, thus deteriorating the EBPR process. They are detected frequently in the deteriorated EBPR process, but their metabolisms are still far from our comprehensions for there is seldom pure culture. In this study, a nearly complete draft genome of a GAOs in Defluviicoccus cluster II, GAO-HK, is recruited from the metagenome of activated sludge in a full-scale industrial anoxic/aerobic wastewater plant. Comparative genomics reveal similar metabolisms of PHA and glycogen in GAOs of GAO-HK, Defluviicoccus tetraformis TFO71 (TFO71) and Competibacter phosphatis clade IIA (CPIIA), and PAOs of Accumulibacter clade IIA UW-1 (UW-1) and Tetrasphaera elongata Lp2 (Lp2). Although there are similar gene cassettes related with polyphosphate metabolism in these GAOs and PAOs, especially for Defluviicoccus-relative bacteria and UW-1, ppk1 in GAOs are diverse from those in the identified PAOs, implying the difference of polyphosphate metabolism in GAOs and PAOs. Additionally, genes related to the dissimilatory denitrification are absent in TFO71 and GAO-HK, implying that additional nitrate or nitrite may favor PAOs over Defluviicoccus-relative GAOs. Therefore, PAOs suffering from competition of Defluviicoccus-relative GAOs might be rescued with the additional nitrate/nitrite, which is important to improve the stability of EBPR processes.

Luxury uptake of phosphorus by microalgae in waste stabilisation ponds: current understanding and future direction

There is a need to improve phosphorus removal for the tens of thousands of small communities around the world that currently rely on waste stabilisation ponds for their wastewater treatment. We now know that microalgae in pond systems are capable of accumulating phosphorus within their cells as polyphosphate in a process known as luxury uptake, but this knowledge has not yet been applied to engineer a new process to improve phosphorus removal. This paper summarises the current understanding of this mechanism, discusses the key factors influencing polyphosphate accumulation and provides future direction for research in this field. There have only been a limited number of studies that have focussed on luxury uptake of polyphosphate by microalgae in high phosphorus concentration environments such as those found in waste stabilisation pond systems. However, from this review it has been shown that the environmental factors which influence polyphosphate storage are the phosphate concentration, light intensity and temperature. Currently we are limited to a black box understanding of the bulk population and as a result future research needs to focus on a systematic evaluation of different microalgal genera under a wide range of environmental, biological and process variables in order to reveal the conditions needed to reliably trigger this phenomenon. This will then provide the basis for developing a new algal biotechnology optimised for luxury uptake of polyphosphate. While there are still several key questions that need to be answered there is potential for this to lead to a process which could be as significant to pond systems as the development of enhanced biological phosphorus removal was for the activated sludge process.

Monitoring intracellular polyphosphate accumulation in enhanced biological phosphorus removal systems by quantitative image analysis.

A rapid methodology for intracellular storage polyphosphate (poly-P) identification and monitoring in enhanced biological phosphorus removal (EBPR) systems is proposed based on quantitative image analysis (QIA). In EBPR systems, 4',6-diamidino-2-phenylindole (DAPI) is usually combined with fluorescence in situ hybridization to evaluate the microbial community. The proposed monitoring technique is based on a QIA procedure specifically developed for determining poly-P inclusions within a biomass suspension using solely DAPI by epifluorescence microscopy. Due to contradictory literature regarding DAPI concentrations used for poly-P detection, the present work assessed the optimal DAPI concentration for samples acquired at the end of the EBPR aerobic stage when the accumulation occurred. Digital images were then acquired and processed by means of image processing and analysis. A correlation was found between average poly-P intensity values and the analytical determination. The proposed methodology can be seen as a promising alternative procedure for quantifying intracellular poly-P accumulation in a faster and less labour-intensive way.

Cytoplasmic inorganic polyphosphate participates in the heavy metal tolerance of Cryptococcus humicola

The basidiomycetous yeast Cryptococcus humicola was shown to be tolerant to manganese, cobalt, nickel, zinc, lanthanum, and cadmium cations at a concentration of 2.5 mmol/L, which is toxic for many yeasts. The basidiomycetous yeast Cryptococcus terreus was sensitive to all these ions and did not grow at the above concentration. In the presence of heavy metal cations, С. humicola, as opposed to C. terreus, was characterized by the higher content of acid-soluble inorganic polyphosphates. In vivo 4',6'-diamino-2-phenylindole dihydrochloride staining revealed polyphosphate accumulation in the cell wall and cytoplasmic inclusions of С. humicola in the presence of heavy metals. In C. terreus, polyphosphates in the presence of heavy metals accumulate mainly in vacuoles, which results in morphological changes in these organelles and, probably, disturbance of their function. The role of polyphosphate accumulation and cellular localization as factors of heavy metal tolerance of Cryptococcus humicola is discussed.

Accumulation of Polyphosphate in Lactobacillus spp. and Its Involvement in Stress Resistance

Polyphosphate (poly-P) is a polymer of phosphate residues synthesized and in some cases accumulated by microorganisms, where it plays crucial physiological roles such as the participation in the response to nutritional stringencies and environmental stresses. Poly-P metabolism has received little attention in Lactobacillus, a genus of lactic acid bacteria of relevance for food production and health of humans and animals. We show that among 34 strains of Lactobacillus, 18 of them accumulated intracellular poly-P granules, as revealed by specific staining and electron microscopy. Poly-P accumulation was generally dependent on the presence of elevated phosphate concentrations in the culture medium, and it correlated with the presence of polyphosphate kinase (ppk) genes in the genomes. The ppk gene from Lactobacillus displayed a genetic arrangement in which it was flanked by two genes encoding exopolyphosphatases of the Ppx-GppA family. The ppk functionality was corroborated by its disruption (LCABL_27820 gene) in Lactobacillus casei BL23 strain. The constructed ppk mutant showed a lack of intracellular poly-P granules and a drastic reduction in poly-P synthesis. Resistance to several stresses was tested in the ppk-disrupted strain, showing that it presented a diminished growth under high-salt or low-pH conditions and an increased sensitivity to oxidative stress. These results show that poly-P accumulation is a characteristic of some strains of lactobacilli and may thus play important roles in the physiology of these microorganisms.

Phosphate application enhances the resistance of arbuscular mycorrhizae in clover plants to cadmium via polyphosphate accumulation in fungal hyphae

Both phosphorus (P) application and arbuscular mycorrhizal (AM) fungal inoculation can enhance plant resistance to Cd toxicity, however, their interaction and the underlying mechanism are still unclear. The aims of this study were to investigate the effect of P application on AM resistance to Cd and to reveal the corresponding mechanism. We designed a compartmented system to separate extraradical hyphae (EH) of Rhizophagus irregularis from the roots of clover (Trifolium ripense L.). EH was applied with different levels of P and cadmium (Cd). The content and uptake of P and Cd were monitored. Polyphosphate (polyP) accumulation in EH was quantified. The localization of polyP and Cd was visualized using fluorescent staining. Results indicated that Cd greatly decreased EH biomass and polyP accumulation while P increased polyP accumulation. PolyP accumulation in EH enhanced AM resistance to Cd, and also reduced Cd uptake by plants. Fluorescent images indicated the overlapping of Cd signals and polyP signals. These data suggest that P application can enhance the resistance of AM to Cd via polyP chelation, and this represents another mechanism underlying the detoxification of Cd by AM hyphae other than the absorption by hyphae.

Stable polyphosphate accumulation by a pseudo-revertant of an Escherichia coli phoU mutant

phoU mutants of bacteria are potentially useful for the removal of inorganic phosphate (Pi) from sewage because they can accumulate a large amounts of polyphosphate (polyP). However, the growth of phoU mutants is severely defective and is easily outgrown by revertant(s) that have lost the ability to accumulate polyP during growth in a nutrient-rich medium. We found that a pseudo-revertant, designated LAP[+], that appeared in a culture of an Escherichia coli phoU mutant that could accumulate polyP even after ten serial passages. Reduction in the expression of the Pi-specific transporter Pst in LAP[+] may contribute to relieving stresses such as excess Pi incorporation that could stimulate reversions. The discovery of a LAP[+] provides a clue to generate phoU mutants that accumulate polyP in a stable manner.

Long-term performance of enhanced biological phosphorus removal with increasing concentrations of silver nanoparticles and ions

The wide use of silver nanoparticles (AgNPs) and silver ions (Ag+) as antimicrobial agents have caused concerns about their potential effects on the environment, such as wastewater treatment plants. This paper reports the effects of increasing the influent AgNPs and Ag+ from 1 to 5 mg L−1 on the performance of enhanced biological phosphorus removal (EBPR). The increase of AgNPs showed no significant influence on EBPR in both short-term and long-term experiments. Phosphorus removal, however, was decreased by a sudden increase in Ag+ concentration and it was gradually recovered after long-term culture. The mechanism studies showed that the transformation of polyhydroxyalkanoates and glycogen and the activity of exopolyphosphatase declined when Ag+ was increased suddenly, but they were recovered after long-term culture. The denatured gradient gel electrophoresis and fluorescence in situ hybridization analyses indicated that the bacterial community structure and the abundance of polyphosphate accumulation organisms were not changed by AgNPs or Ag+. Further investigations revealed that the long-term exposure of Ag+ increased the amount of sludge extracellular polymeric substances, which accounted for the mitigation of Ag+ toxicity to the EBPR key enzyme and microbes.

A metabolic model for members of the genus Tetrasphaera involved in enhanced biological phosphorus removal

Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to 'Candidatus Accumulibacter phosphatis' is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by 'Candidatus Accumulibacter phosphatis', and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation.

Inorganic polyphosphate accumulation by Cunninghamella elegans (UCP 542) and its influence in the decolorization of textile azo dye Orange II

The inappropriate disposal of dyes in wastewater constitutes an environmental problem and can cause damage to the ecosystem. Alternative treatments have been reported that employ fungi that are particularly effective in the decolorization of textile effluents. In these methods, the biomass can be used for industrial removal of dyes by biosorption. The current study has investigated the accumulation of polyphosphate (poly P) in the fungus Cunninghamella elegans UCP 542 and evaluated the capacity of the inactivated biomass, obtained from 144 h of incubation, to decolorize the dye Orange II. The results suggest the transport of the inorganic phosphorus, as demonstrated through the cellular bioaccumulation. The biochemical and physiological aspects of bioaccumulation in the poly P form were related to the process of removal of the color of Orange II, with the reduction of 60 % of the dye solution.

Accumulation of phosphate and polyphosphate by Cryptococcus humicola and Saccharomyces cerevisiae in the absence of nitrogen

The search for new phosphate-accumulating microorganisms is of interest in connection with the problem of excess phosphate in environment. The ability of some yeast species belonging to ascomycetes and basidiomycetes for phosphate (P (i) ) accumulation in nitrogen-deficient medium was studied. The ascomycetous Saccharomyces cerevisiae and Kuraishia capsulata and basidiomycetous Cryptococcus humicola, Cryptococcus curvatus, and Pseudozyma fusiformata were the best in P (i) removal. The cells of Cryptococcus humicola and S.cerevisiae took up 40% P (i) from the media containing P (i) and glucose (5 and 30mM, respectively), and up to 80% upon addition of 5mM MgSO(4) (.) The cells accumulated P (i) mostly in the form of polyphosphate (PolyP). In the presence of Mg(2+) , the content of PolyP with longer average chain length increased in both yeasts; they both had numerous inclusions fluorescing in the yellow region of the spectrum, typical of DAPI-PolyP complexes. Among the yeast species tested, Cryptococcus humicola is a new promising model organisms to study phosphorus removal from the media and biomineralization in microbial cells.

Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription

In hematology there has recently been increasing interest in inorganic polyphosphate. This polymer accumulates in platelet granules and its functions include modulating various stages of blood coagulation, inducing angiogenesis, and provoking apoptosis of plasma cells. In this study we evaluated the characteristics of intracellular polyphosphate in myeloma cell lines, in primary myeloma cells from patients, and in other human B-cell populations from healthy donors. We have developed a novel flow cytometric method for detecting levels of polyphosphate in cell populations. We also used confocal microscopy and enzymatic analysis to study polyphosphate localization and characteristics. We found that myeloma plasma cells contain higher levels of intracellular polyphosphate than normal plasma cells and other B-cell populations. Localization experiments indicated that high levels of polyphosphate accumulate in the nucleolus of myeloma cells. As the principal function of the nucleolus involves transcription of ribosomal DNA genes, we found changes in the cellular distribution of polyphosphate after the inhibition of nucleolar transcription. In addition, we found that RNA polymerase I activity, responsible for transcription in the nucleolus, is also modulated by polyphosphate, in a dose-dependent manner. Our results show an unusually high accumulation of polyphosphate in the nucleoli of myeloma cells and a functional relationship of this polymer with nucleolar transcription.

Transcriptome profiling of a curdlan-producing Agrobacterium reveals conserved regulatory mechanisms of exopolysaccharide biosynthesis

BackgroundThe ability to synthesize exopolysaccharides (EPS) is widespread among microorganisms, and microbial EPS play important roles in biofilm formation, pathogen persistence, and applications in the food and medical industries. Although it is well established that EPS synthesis is invariably in response to environmental cues, it remains largely unknown how various environmental signals trigger activation of the biochemical synthesis machinery.ResultsWe report here the transcriptome profiling of Agrobacterium sp. ATCC 31749, a microorganism that produces large amounts of a glucose polymer known as curdlan under nitrogen starvation. Transcriptome analysis revealed a nearly 100-fold upregulation of the curdlan synthesis operon upon transition to nitrogen starvation, thus establishing the prominent role that transcriptional regulation plays in the EPS synthesis. In addition to known mechanisms of EPS regulation such as activation by c-di-GMP, we identify novel mechanisms of regulation in ATCC 31749, including RpoN-independent NtrC regulation and intracellular pH regulation by acidocalcisomes. Furthermore, we show evidence that curdlan synthesis is also regulated by conserved cell stress responses, including polyphosphate accumulation and the stringent response. In fact, the stringent response signal, pppGpp, appears to be indispensible for transcriptional activation of curdlan biosynthesis.ConclusionsThis study identifies several mechanisms regulating the synthesis of curdlan, an EPS with numerous applications. These mechanisms are potential metabolic engineering targets for improving the industrial production of curdlan from Agrobacterium sp. ATCC 31749. Furthermore, many of the genes identified in this study are highly conserved across microbial genomes, and we propose that the molecular elements identified in this study may serve as universal regulators of microbial EPS synthesis.

The incorporation of a zone of calcified cartilage improves the interfacial shear strength between in vitro-formed cartilage and the underlying substrate

A major challenge for cartilage tissue engineering remains the proper integration of constructs with surrounding tissues in the joint. Biphasic osteochondral constructs that can be anchored in a joint through bone ingrowth partially address this requirement. In this study, a methodology was devised to generate a cell-mediated zone of calcified cartilage (ZCC) between the in vitro-formed cartilage and a porous calcium polyphosphate (CPP) bone substitute in an attempt to improve the mechanical integrity of that interface. To do so, a calcium phosphate (CaP) film was deposited on CPP by a sol–gel process to prevent the accumulation of polyphosphates and associated inhibition of mineralization as the substrate degrades. Cartilage formed in vitro on the top surface of CaP-coated CPP by deep-zone chondrocytes was histologically and biochemically comparable to that formed on uncoated CPP. Furthermore, the mineral in the ZCC was similar in crystal structure, morphology and length to that formed on uncoated CPP and native articular cartilage. The generation of a ZCC at the cartilage–CPP interface led to a 3.3-fold increase in the interfacial shear strength of biphasic constructs. Improved interfacial strength of these constructs may be critical to their clinical success for the repair of large cartilage defects.

A metabolic network of a phosphate-accumulating organism provides new insights into enhanced biological phosphorous removal

Here we present a metabolic network representing the central carbon metabolism as well as the synthesis of polyhydroxyalcanohates and the polyphosphate accumulation mechanisms of the bacterium Candidatus Accumulibacter phosphatis, which was previously identified from metagenomic studies in enhanced biological phosphorous removal sludges. The reconstructed metabolic network, together with flux balance analysis can be used to provide new insights into controversial aspects of the metabolism of phosphate-accumulating organisms and is also a tool that can be used in to help enhanced biological phosphorous removal (EBPR) process design and operation.

Accumulation of inorganic polyphosphates in Saccharomyces cerevisiae under nitrogen deprivation: Stimulation by magnesium ions and peculiarities of localization

The yeast Saccharomyces cerevisiae was shown to have a high potential as a phosphate-accumulating organism under growth suppression by nitrogen limitation. The cells took up over 40% of phosphate from the medium containing 30 mM glucose and 5 mM potassium phosphate and over 80% of phosphate on addition of 5 mM magnesium sulfate. The major part of accumulated Pi was reserved as polyphosphates. The content of polyphosphates was ∼57 and ∼75% of the phosphate accumulated by the cells in the absence and presence of magnesium ions, respectively. The content of long-chain polyphosphates increased in the presence of magnesium ions, 5-fold for polymers with the average length of ∼45 phosphate residues, 3.7-fold for polymers with the average chain length of ∼75 residues, and more than 10-fold for polymers with the average chain length of ∼200 residues. On the contrary, the content of polyphosphates with the average chain length of ∼15 phosphate residues decreased threefold. According to the data of electron and confocal microscopy and X-ray microanalysis, the accumulated polyphosphates were localized in the cytoplasm and vacuoles. The cytoplasm of the cells accumulating polyphosphates in the presence of magnesium ions had numerous small phosphorus-containing inclusions; some of them were associated with large electron-transparent inclusions and the cytoplasmic membrane.

Structural, mass and elemental analyses of storage granules in methanogenic archaeal cells

Storage granules are an important component of metabolism in many organisms spanning the bacterial, eukaryal and archaeal domains, but systematic analysis of their organization inside cells is lacking. In this study, we identify and characterize granule-like inclusion bodies in a methanogenic archaeon, Methanospirillum hungatei, an anaerobic microorganism that plays an important role in nutrient recycling in the ecosystem. Using cryo electron microscopy, we show that granules in mature M. hungatei are amorphous in structure with a uniform size. Energy dispersive X-ray spectroscopy analysis establishes that each granule is a polyphosphate body (PPB) that consists of high concentrations of phosphorous and oxygen, and increased levels of iron and magnesium. By scanning transmission electron tomography, we further estimate that the mass density within a PPB is a little less than metal titanium at room temperature and is about four times higher than that of the surrounding cytoplasm. Finally, three-dimensional cryo electron tomography reveals that PPBs are positioned off-centre in their radial locations relative to the cylindrical axis of the cell, and almost uniformly placed near cell ends. This positioning ability points to a genetic program that spatially and temporally directs the accumulation of polyphosphate into a storage granule, perhaps for energy-consuming activities, such as cell maintenance, division or motility.