Mixed microalgae culture for ammonium removal in the absence of phosphorus: Effect of phosphorus supplementation and process modeling

Microalgal growth and ammonium removal in a P-free medium have been studied in two batch photobioreactors seeded with a mixed microalgal culture and operated for 46 days. A significant amount of ammonium (106 mg NH₄-Nl⁻¹) was removed in a P-free medium, showing that microalgal growth and phosphorus uptake are independent processes. The ammonium removal rate decreased during the experiment, partly due to a decrease in the cellular phosphorus content. After a single phosphate addition in the medium of one of the reactors, intracellular phosphorus content of the corresponding microalgal culture rapidly increased, and so did the ammonium removal rate. These results show how the amount of phosphorus internally stored affects the ammonium removal rate. A mathematical model was proposed to reproduce these observations. The kinetic expression for microalgae growth includes a Monod term and a Hill's function to represent the effect of ammonium and stored polyphosphate concentrations, respectively. The proposed model accurately reproduced the experimental data (r = 0.952, P-value <0.01).     

Sulfide induces phosphate release from polyphosphate in cultures of a marine Beggiatoa strain

Sulfur bacteria such as Beggiatoa or Thiomargarita have a particularly high capacity for storage because of their large size. In addition to sulfur and nitrate, these bacteria also store phosphorus in the form of polyphosphate. Thiomargarita namibiensis has been shown to release phosphate from internally stored polyphosphate in pulses creating steep peaks of phosphate in the sediment and thereby inducing the precipitation of phosphorus-rich minerals. Large sulfur bacteria populate sediments at the sites of recent phosphorite formation and are found as fossils in ancient phosphorite deposits. Therefore, it can be assumed that this physiology contributes to the removal of bioavailable phosphorus from the marine system and thus is important for the global phosphorus cycle. We investigated under defined laboratory conditions which parameters stimulate the decomposition of polyphosphate and the release of phosphate in a marine Beggiatoa strain. Initially, we tested phosphate release in response to anoxia and high concentrations of acetate, because acetate is described as the relevant stimulus for phosphate release in activated sludge. To our surprise, the Beggiatoa strain did not release phosphate in response to this treatment. Instead, we could clearly show that increasing sulfide concentrations and anoxia resulted in a decomposition of polyphosphate. This physiological reaction is a yet unknown mode of bacterial polyphosphate usage and provides a new explanation for high phosphate concentrations in sulfidic marine sediments.     

P-31 in-vivo NMR investigation on the function of polyphosphates as phosphate-and energysource during the regreening of the green alga Chlorella fusca

The green alga Chlorella fusca accumulates polyphosphates under conditions of nitrogen starvation while deassembling the photosynthetic apparatus. The polyphosphate content of cells regreening after resupply with nitrate under different culture conditions was investigated by P-31 in-vivo NMR spectroscopy. Neither phosphate deficiency nor anaerobiosis during the first hours of regreening inhibited the recovery of the cells. Polyphosphates were degraded during regeening. Differences in the amount of polyphosphates of phosphate supplied and deficient cells occurred only after more then 8 h. After 16 h phosphate deficient cells had still 75% of the polyphosphate content of phosphate suppled cells. In cells kept under anaerobic conditions polyphosphate degradation was much higher than in oxygen supplied cells. After 8 h they contained less than 50% of the polyphosphate content of oxygen supplied cells. These data suggest that polyphosphates serve as obligatory phosphate source during regreening and may be used as an energy source.Non standard abbreviations EDTA Ethylene diamine tetraacetic acid - FID Free induction decay - MOPSO 3-(N-morpholine)-2-hydroxy-propanesulfonic acid - NMR Nuclear magnetic resonance - PP Polyphosphates - PP₄ central phosphate groups of polyphosphates     

Regulation of inorganic polyphosphate is required for proper vacuolar proteolysis in fission yeast

Regulation of cellular proliferation and quiescence is a central issue in biology that has been studied using model unicellular eukaryotes, such as the fission yeast Schizosaccharomyces pombe. We previously reported that the ubiquitin/proteasome pathway and autophagy are essential to maintain quiescence induced by nitrogen deprivation in S. pombe; however, specific ubiquitin ligases that maintain quiescence are not fully understood. Here we investigated the SPX-RING-type ubiquitin ligase Pqr1, identified as required for quiescence in a genetic screen. Pqr1 is found to be crucial for vacuolar proteolysis, the final step of autophagy, through proper regulation of phosphate and its polymer polyphosphate. Pqr1 restricts phosphate uptake into the cell through ubiquitination and subsequent degradation of phosphate transporters on plasma membranes. We hypothesized that Pqr1 may act as the central regulator for phosphate control in S. pombe, through the function of the SPX domain involved in phosphate sensing. Deletion of pqr1⁺ resulted in hyperaccumulation of intracellular phosphate and polyphosphate and in improper autophagy-dependent proteolysis under conditions of nitrogen starvation. Polyphosphate hyperaccumulation in pqr1⁺-deficient cells was mediated by the polyphosphate synthase VTC complex in vacuoles. Simultaneous deletion of VTC complex subunits rescued Pqr1 mutant phenotypes, including defects in proteolysis and loss of viability during quiescence. We conclude that excess polyphosphate may interfere with proteolysis in vacuoles by mechanisms that as yet remain unknown. The present results demonstrate a connection between polyphosphate metabolism and vacuolar functions for proper autophagy-dependent proteolysis, and we propose that polyphosphate homeostasis contributes to maintenance of cellular viability during quiescence.     

Stoichiometric model of the aerobic metabolism of the biological phosphorus removal process

In the aerobic phase of the biological phosphorus removal process, poly-beta-hydroxybutyrate, produced during anaerobic conditions, is used for cell growth, phosphate uptake, and glycogen formation. A metabolic model of this process has been developed. The yields for growth, polyphosphate and glycogen formation are quantified using the coupling of all these conversions to the oxygen consumption. The uptake of phosphate and storage as polyphosphate is shown to have a direct effect on the observed oxygen consumption in the aerobic phase. The overall energy requirements for the P-metabolism are substantial: 25% of the acetate consumed during anaerobic conditions and 60% of the oxygen consumptions is used for the synthesis of polyphosphate and glycogen. (c) 1994 John Wiley & Sons, Inc.     

Observation of polyphosphate bodies and DNA during the cell division cycle of Synechococcus elongatus PCC 7942

Although most cyanobacterial cells contain prominent polyphosphate bodies in the central cytoplasmic area enclosed by the peripheral thylakoid membranes, their roles are not fully understood. Storing phosphate for nucleotide production might be one of their important roles in survival of the cells. As a step towards identifying a possible contribution of the polyphosphate bodies to DNA synthesis, the relationship between polyphosphate bodies and DNA throughout cell division cycle of Synechococcus elongatus PCC 7942 cells cultured under light/dark cycles was investigated with light and electron microscopy. During the dark period, the average size of polyphosphate bodies increased gradually without significant change in their number and distribution. However, during the light period, the number of polyphosphate bodies increased, while the size of each polyphosphate body decreased and cells elongated until the end of the light period, when most cells divided. The ratio of the content of polyphosphate bodies to cell length increased gradually during the dark period and decreased during the light period. Hoechst 33342‐stained DNA appeared diffuse during the dark period, but in the light period it became condensed and eventually formed a wavy, rope‐like structure prior to cell division. Close association between fibres containing DNA and polyphosphate bodies was demonstrated by TEM using DNA‐specific staining and BrdU labelling. These regular coordinated changes of polyphosphate bodies and DNA shape during the cell division cycle, together with their intimate interaction, imply a role of polyphosphate bodies in supplying material for DNA.     

Incorporation of phosphorus into phased cells ofCandida utilis using32P and33P

Changes in phosphorus metabolism were studied by examining the incorporation of³²P and³³P into cells ofCandida utilis growing in phased culture during a 6 h cell cycle and a post-cycle period of 6 h. Three different chemically defined media were used; these were phosphorus, nitrogen and carbon limited. The patterns of incorporation of phosphorus into RNA, DNA, lipid and cold water extractable phosphate fractions showed a non-uniform behaviour during both cell cycle and post-cycle periods. The patterns were different in all three types of media. The results showed that a cell can grow and develop at a fixed growth rate in different ways: so that the pattern of behaviour during a cell cycle is not stereotyped for a given doubling time, but largely depends upon the nutrient environment in which the cell exists.     

Effect of phosphorus deficiency on levels of phosphorus compounds in spirodela

When Spirodela plants are transferred to a phosphate-deficient medium, growth slows down immediately, and ceases after 14 days. During this time, inorganic phosphate content falls from 30 to 0.7 μmoles/g fresh weight of tissue, phosphate ester content from 3.5 to 0.6 μmoles/g, phospholipid content from 3.5 to 1.2 μmoles/g, and residual phosphate (mainly RNA) content from 7.5 to 2.0 μmoles/g. Relative proportions of the various phosphate esters, and relative proportions of the various phospholipids, are not markedly affected by phosphate deficiency. Turnover rates of phosphate esters are somewhat higher in phosphate-deficient tissue. In control tissue, inorganic phosphate is present in 2 pools; a metabolic (12%) and a non-metabolic pool (88%). In phosphate-deficient tissues, most of the inorganic phosphate (>90%) is in the metabolic pool. Non-metabolic phosphate is presumably stored in the vacuole, and is not readily accessible to the tissue, so that growth normally occurs at the expense of external phosphate. During deficiency, growth is limited by the rate at which phosphate can be transported through the tonoplast and tissue to the growing point. Growth ceases when the supply of non-metabolic phosphate is exhausted. Metabolic phosphate is presumably located in the cytoplasm: it can not be used for growth. Nor can the plant respond to deficiency by making some phosphorus compounds at the expense of others. In this respect, phosphorus deficiency and nitrogen deficiency are dissimilar.     

Regulation of polyphosphate metabolism in Acinetobacter strain 210A grown in carbon- and phosphate-limited continuous cultures

The response of Acinetobacter strain 210A to low phosphate concentrations was investigated in P- or C-limited chemostat cultures. The organism accumulated poly--hydroxybutyric acid under P-deprivation, at phosphate concentrations ranging from 0.1 to 0.7 mM. The amount of biomass was proportional to the phosphate concentration in the medium and no polyphosphate was formed. When shifting a culture from P- to C-limitation phosphate was accumulated as polyphosphate. No poly--hydroxybutyrate could be detected in these cells. The amount of polyphosphate in the cell showed a hysteresis. When cultures were shifted from low to high phosphate concentrations, polyphosphate reached a maximum of about 60 mg P per gram of dry weight at about 3 times excess phosphate (ca. 2.5 mM P_i). It decreased to 45 mg P per gram dry weight at approximately 5 times the phosphate needed for growth (ca. 3.5 mM P_i). In the reverse case (high to low) polyphosphate did never exceed 45 mg P per gram dry weight. The specific activities of alkaline phosphatase and the phosphate uptake system were induced at residual P_i concentrations below the detection limit (<10 M). The specific uptake rate followed also a hysteresis. The specific activities of polyphosphatase and polyphosphate: AMP phosphotransferase increased when polyphosphate formation was possible.Abbreviations HPP High polymeric polyphosphates - PHB Poly--hydroxybutyric acid - PP_n Polyphosphate - PQQ Pyrrolo-quinoline quinone - U 1 mol product formed · min^-1     

<Superscript>31</Superscript>P NMR study of polyphosphate levels during different growth phases of <Emphasis Type="Italic">Phycomyces blakesleeanus</Emphasis>

The changes in relative polyphosphate content, estimated as the intensity ratio of core polyphosphate signal and intracellular inorganic phosphate signal from ³¹P NMR spectra, during the growth of Phycomyces blakesleeanus are reported. The ratio increases from 16 h to 28 h of growth, the minimum occurs at 32 h, followed by sharp increase up to 36 h, and a steady decrease afterwards. The changes in the biomass during mycelium growth showed steady increases, with a stagnation period between 32 h and 36 h during which a pronounced increase in the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal occurred. The reduction of growth temperature from 22°C to 18°C significantly decreased the rate and intensity of growth, but the pattern of polyphosphate changes remained unchanged. The changes of the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal are linked to characteristic stages of sporangiophore development. Analysis of core polyphosphates, intracellular inorganic phosphate and β-ATP signal intensities suggest the role of polyphosphates as an energy and/or a phosphate reserves during Phycomyces development.     

Intracellular Accumulation of Polyphosphate by the Yeast Candida humicola G-1 in Response to Acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.     

Radiolabelled proteomics to determine differential functioning of Accumulibacter during the anaerobic and aerobic phases of a bioreactor operating for enhanced biological phosphorus removal

Proteins synthesized by the mixed microbial community of two sequencing batch reactors run for enhanced biological phosphorus removal (EBPR) during aerobic and anaerobic reactor phases were compared, using mass spectrometry‐based proteomics and radiolabelling. Both sludges were dominated by polyphosphate‐accumulating organisms belonging to Candidatis Accumulibacter and the majority of proteins identified matched closest to these bacteria. Enzymes from the Embden–Meyerhof–Parnas pathway were identified, suggesting this is the major glycolytic pathway for these Accumulibacter populations. Enhanced aerobic synthesis of glyoxylate cycle enzymes suggests this cycle is important during the aerobic phase of EBPR. In one sludge, several TCA cycle enzymes showed enhanced aerobic synthesis, suggesting this cycle is unimportant anaerobically. The second sludge showed enhanced synthesis of TCA cycle enzymes under anaerobic conditions, suggesting full or partial TCA cycle operation anaerobically. A phylogenetic analysis of Accumulibacter polyphosphate kinase genes from each sludge demonstrated different Accumulibacter populations dominated the two sludges. Thus, TCA cycle activity differences may be due to Accumulibacter strain differences. The major fatty acids present in Accumulibacter‐dominated sludge include palmitic, hexadecenoic and cis‐vaccenic acid and fatty acid content increased by approximately 20% during the anaerobic phase. We hypothesize that this is associated with increased anaerobic phospholipid membrane biosynthesis, to accommodate intracellular polyhydroxyalkanoate granules.     

Regulation of Phosphate Accumulation in the Unicellular Cyanobacterium Synechococcus

The phosphorus contents of acid-soluble pools, lipid, ribonucleic acid, and acid-insoluble polyphosphate were lowered in Synechococcus in proportion to the reduction in growth rate in phosphate-limited but not in nitrate-limited continuous culture. Phosphorus in these cell fractions was lost proportionately during progressive phosphate starvation of batch cultures. Acid-insoluble polyphosphate was always present in all cultural conditions to about 10% of total cell phosphorus and did not turn over during balanced exponential growth. Extensive polyphosphate formation occurred transiently when phosphate was given to cells which had been phosphate limited. This material was broken down after 8 h even in the presence of excess external orthophosphate, and its phosphorus was transferred into other cell fractions, notably ribonucleic acid. Phosphate uptake kinetics indicated an invariant apparent K(m) of about 0.5 muM, but V(max) was 40 to 50 times greater in cells from phosphate-limited cultures than in cells from nitrate-limited or balanced batch cultures. Over 90% of the phosphate taken up within the first 30 s at 15 degrees C was recovered as orthophosphate. The uptake process is highly specific, since neither phosphate entry nor growth was affected by a 100-fold excess of arsenate. The activity of polyphosphate synthetase in cell extracts increased at least 20-fold during phosphate starvation or in phosphate-restricted growth, but polyphosphatase activity was little changed by different growth conditions. The findings suggest that derepression of the phosphate transport and polyphosphate-synthesizing systems as well as alkaline phosphatase occurs in phosphate shortage, but that the breakdown of polyphosphate in this organism is regulated by modulation of existing enzyme activity.     

Investigations on phosphate uptake and polyphosphate metabolism by mycorrhized and nonmycorrhized roots of beech and pine as investigated by in vivo 31P-NMR

Comparative in vivo ³¹P-NMR studies of mycorrhized and nonmycorrhized roots of Fagus sylvatica and Pinus sylvestris and of the fungus Suillus bovinus in pure culture have produced interesting new data. With respect to intracellular compartments and pH, ³¹P-NMR spectroscopy showed that the spectrum of the mycorrhiza results from simple superimposition of the spectra of its symbionts. A special method of cyclic phosphate supply followed by block averaging of the NMR spectra was used to determine the kinetic behaviour of phosphate uptake and storage and its incorporation into polyphosphate at a constant external pH of 5.5. Mycorrhized roots and pure fungus showed transformation of accumulated inorganic phosphate into mobile polyphosphate with a medium chain length. Transformation of mobile into immobile polyphosphate either with a long chain length or in a granular state was also observed. Thus, two different types of fungal polyphosphate could be verified. Deficiency of external phosphate initiated the mobilization of internal phosphate, transforming stored polyphosphate into phosphate. It could be shown that a high fungal mass renders mycorrhizal phosphate metabolism less sensitive to external variation in nutrient concentration. The central role of the fungus in regulating mycorrhizal phosphate metabolism is discussed.     

Uranyl Precipitation by Pseudomonas aeruginosa via Controlled Polyphosphate Metabolism

The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions.     

External Iron Regulates Polyphosphate Content in the Acidophilic, Thermophilic Alga Cyanidium caldarium

Transmission electron microscopy revealed the presence of electron-dense bodies (EDB) in the cytosol of the acidophilic, thermophilic red alga Cyanidium caldarium. These bodies contain almost exclusively Fe, P, and O and can play a role in Fe storage. ³¹P-nuclear magnetic resonance analysis identified a sharp signal at −23.3 ppm, which was attributed to the phosphate groups of the inner portions of polyphosphate chains. From this evidence, as well as that of a previous ESR study (Nagasaka et al., BioMetals 16:465–470, 2003), it can be concluded that polyphosphates are the major anionic constituents of the EDB. Omission of Fe from the culture medium resulted in substantially decreased polyphosphate levels, demonstrating the control of cellular polyphosphate content by the Fe status of the culture medium.     

Engineering polyphosphate metabolism in Escherichia coli: implications for bioremediation of inorganic contaminants

Polyphosphate metabolism plays an important role in the bioremediation of phosphate contamination in municipal wastewater, and may play a key role in heavy metal tolerance and bioremediation. However, little is known about the regulation of polyphosphate metabolism in microorganisms and its role in heavy metal toxicity. We have manipulated polyphosphate metabolism in Escherichia coli by overexpressing the genes for polyphosphate kinase (ppk) and for polyphosphatase (ppx) under control of their native promoters and inducible promoters. Overexpression of ppk results in high levels of intracellular polyphosphate, improved phosphate uptake, but no increase in tolerance to heavy metals. Overexpression of both ppk and ppx results in lower levels of intracellular polyphosphate, secretion of phosphate from the cell, and increased tolerance to heavy metals. Metabolic flux analysis indicates that the cell responds to increased flux through the PPK-PPX pathway by altering flux through the TCA cycle.     

Establishment of a shortened annual cycle system; a tool for the analysis of annual re-translocation of phosphorus in the deciduous woody plant (Populus alba L.)

The supply of phosphorus, the essential element for plant growth and development, is often limited in natural environments. Plants employ multiple physiological strategies to minimize the impact of phosphate deficiency. In deciduous trees, phosphorus is remobilized from senescing leaves in autumn and stored in other tissues for reuse in the following spring. We previously monitored the annual changes in leaf phosphate content of white poplar (Populus alba) growing under natural conditions and found that about 75 % of inorganic and 60 % of organic leaf phosphates observed in May were remobilized by November. In order to analyze this process (such annual events), we have established a model system, in which an annual cycle of phosphate re-translocation in trees can be simulated under laboratory conditions by controlling temperature and photoperiod (=‘shortened annual cycle’). This system evidently allowed us to monitor the annual changes in leaf color, phosphate remobilization from senescent leaves, and bud break in the next spring within five months. This will greatly facilitate the analysis of cellular and molecular mechanisms of annual phosphate re-translocation in deciduous trees.     

Polyphosphate Stores Enhance the Ability of Vibrio cholerae To Overcome Environmental Stresses in a Low-Phosphate Environment

Vibrio cholerae, the causative agent of Asiatic cholera, has been reported to make large quantities of polyphosphate. Inorganic polyphosphate is a ubiquitous molecule with a variety of functions in prokaryotic and eukaryotic cells. We constructed a V. cholerae mutant with a deletion in the polyphosphate kinase (ppk) gene. The mutant was defective in polyphosphate biosynthesis. Deletion of ppk had no significant effect on production of cholera toxin, hemagglutinin/protease, motility, biofilm formation, and colonization of the suckling mouse intestine. The wild type and mutant had similar growth rates in rich and minimal medium and exhibited similar phosphate uptake and alkaline phosphatase induction. In contrast to ppk mutants from other gram-negative bacteria, the V. cholerae mutant survived prolonged starvation in LB medium and artificial seawater basal salts. The ppk mutant was significantly more sensitive to low pH, high salinity, and oxidative stress when it was cultured in low-phosphate minimal medium. The ppk mutant failed to induce catalase when it was downshifted to phosphorus-limiting conditions. Furthermore, the increased sensitivity of the ppk mutant to environmental stressors in phosphate-limited medium correlated with a diminished capacity to synthesize ATP from intracellular reservoirs. We concluded that polyphosphate protects V. cholerae from environmental stresses under phosphate limitation conditions. It has been proposed that toxigenic V. cholerae can survive in estuaries and brackish waters in which phosphorus and/or nitrogen can be a limiting nutrient. Thus, synthesis of large polyphosphate stores could enhance the ability of V. cholerae to survive in the aquatic environment.