Characterisation of Phosphate Accumulating Organisms and Techniques for Polyphosphate Detection: A Review
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paper_title: Phosphorus management in Europe in a changing world

paper_content:

Food production in Europe is dependent on imported phosphorus (P) fertilizers, but P use is inefficient and losses to the environment high. Here, we discuss possible solutions by changes in P management. We argue that not only the use of P fertilizers and P additives in feed could be reduced by fine-tuning fertilization and feeding to actual nutrient requirements, but also P from waste has to be completely recovered and recycled in order to close the P balance of Europe regionally and become less dependent on the availability of P-rock reserves. Finally, climate-smart P management measures are needed, to reduce the expected deterioration of surface water quality resulting from climate-change-induced P loss.
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paper_title: The Relevance of Phosphorus and Iron Chemistry to the Recovery of Phosphorus from Wastewater: A Review

paper_content:

The addition of iron is a convenient way for removing phosphorus from wastewater, but this is often considered to limit phosphorus recovery. Struvite precipitation is currently used to recover phosphorus, and this approach has attracted much interest. However, it requires the use of enhanced biological phosphorus removal (EBPR). EBPR is not yet widely applied and the recovery potential is low. Other phosphorus recovery methods, including sludge application to agricultural land or recovering phosphorus from sludge ash, also have limitations. Energy-producing wastewater treatment plants increasingly rely on phosphorus removal using iron, but the problem (as in current processes) is the subsequent recovery of phosphorus from the iron. In contrast, phosphorus is efficiently mobilized from iron by natural processes in sediments and soils. Iron-phosphorus chemistry is diverse, and many parameters influence the binding and release of phosphorus, including redox conditions, pH, presence of organic substances, and particle morphology. We suggest that the current poor understanding of iron and phosphorus chemistry in wastewater systems is preventing processes being developed to recover phosphorus from iron-phosphorus rich wastes like municipal wastewater sludge. Parameters that affect phosphorus recovery are reviewed here, and methods are suggested for manipulating iron-phosphorus chemistry in wastewater treatment processes to allow phosphorus to be recovered.
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paper_title: Optimizing the production of Polyphosphate from Acinetobacter towneri

paper_content:

Inorganic polyphosphates (PolyP) are linear polymers of few to several hundred orthophosphate residues, linked by energy-rich phosphoanhydride bonds. Four isolates had been screened from soil sample. By MALDI-TOF analysis, they were identified as Bacillius cereus, Acinetobacter towneri, B. megaterium and B. cereus. The production of PolyP in four isolates was studied in phosphate uptake medium and sulfur deficient medium at pH 7. These organisms had shown significant production of PolyP after 22h of incubation. PolyP was extracted from the cells using alkaline lysis method. Among those isolates, Acinetobacter towneri was found to have high (24.57% w/w as P) accumulation of PolyP in sulfur deficient medium. The media optimization for sulfur deficiency was carried out using Response surface methodology (RSM). It was proven that increase in phosphate level in the presence of glucose, under sulfur limiting condition, enhanced the phosphate accumulation by Acinetobacter towneri and these condition can be simulated for the effective removal of phosphate from wastewater sources.
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paper_title: Phosphorus recovery from wastewater through microbial processes

paper_content:

Waste streams offer a compelling opportunity to recover phosphorus (P). 15–20% of world demand for phosphate rock could theoretically be satisfied by recovering phosphorus from domestic waste streams alone. For very dilute streams ( −1 ), including domestic wastewater, it is necessary to concentrate phosphorus in order to make recovery and reuse feasible. This review discusses enhanced biological phosphorus removal (EBPR) as a key technology to achieve this. EBPR relies on polyphosphate accumulating organisms (PAOs) to take up phosphorus from waste streams, so concentrating phosphorus in biomass. The P-rich biosolids can be either directly applied to land, or solubilized and phosphorus recovered as a mineral product. Direct application is effective, but the product is bulky and carries contaminant risks that need to be managed. Phosphorus release can be achieved using either thermochemical or biochemical methods, while recovery is generally by precipitation as struvite. We conclude that while EBPR technology is mature, the subsequent phosphorus release and recovery technologies need additional development.
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paper_title: A method for screening remove polyphosphate-accumulating mutants which remove phosphate efficiently from synthetic wastewater

paper_content:

The biological process for phosphorus removal from wastewater is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyp). We previously showed that a phoU mutation leads to polyp accumulation in Escherichia coli. The phoU mutant could be easily screened on agar plates containing 5-bromo-4-chloro-3-indolyl-phosphate (X-Pi) after N-methyl-N′-vitro-N-nitrosoguanidine (NTG) mutagenesis. Here, we demonstrate that this method is also useful for screening polyp-accumulating mutants of bacterial strains isolated from soil and activated sludge samples.
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paper_title: Advances in enhanced biological phosphorus removal: from micro to macro scale

paper_content:

The enhanced biological phosphorus removal (EBPR) process has been implemented in many wastewater treatment plants worldwide. While the EBPR process is indeed capable of efficient phosphorus (P) removal performance, disturbances and prolonged periods of insufficient P removal have been observed at full-scale plants on numerous occasions under conditions that are seemingly favourable for EBPR. Recent studies in this field have utilised a wide range of approaches to address this problem, from studying the microorganisms that are primarily responsible for or detrimental to this process, to determining their biochemical pathways and developing mathematical models that facilitate better prediction of process performance. The overall goal of each of these studies is to obtain a more detailed insight into how the EBPR process works, where the best way of achieving this objective is through linking together the information obtained using these different approaches. This review paper critically assesses the recent advances that have been achieved in this field, particularly relating to the areas of EBPR microbiology, biochemistry, process operation and process modelling. Potential areas for future research are also proposed. Although previous research in this field has undoubtedly improved our level of understanding, it is clear that much remains to be learned about the process, as many unanswered questions still remain. One of the challenges appears to be the integration of the existing and growing scientific knowledge base with the observations and applications in practice, which this paper hopes to partially achieve.
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paper_title: Inorganic polyphosphate: essential for growth and survival.

paper_content:

Inorganic polyphosphate (Poly P) is a polymer of tens to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds as in ATP. Found in abundance in all cells in nature, it is unique in its likely role in the origin and survival of species. Here, we present extensive evidence that the remarkable properties of Poly P as a polyanion have made it suited for a crucial role in the emergence of cells on earth. Beyond that, Poly P has proved in a variety of ways to be essential for growth of cells, their responses to stresses and stringencies, and the virulence of pathogens. In this review, we pay particular attention to the enzyme, polyphosphate kinase 1 (Poly P kinase 1 or PPK1), responsible for Poly P synthesis and highly conserved in many bacterial species, including 20 or more of the major pathogens. Mutants lacking PPK1 are defective in motility, quorum sensing, biofilm formation, and virulence. Structural studies are cited that reveal the conserved ATP-binding site of PPK1 at atomic resolution and reveal that the site can be blocked with minute concentrations of designed inhibitors. Another widely conserved enzyme is PPK2, which has distinctive kinetic properties and is also implicated in the virulence of some pathogens. Thus, these enzymes, absent in yeast and animals, are novel attractive targets for treatment of many microbial diseases. Still another enzyme featured in this review is one discovered in Dictyostelium discoideum that becomes an actin-like fiber concurrent with the synthesis, step by step, of a Poly P chain made from ATP. The Poly P-actin fiber complex, localized in the cell, lengthens and recedes in response to metabolic signals. Homologs of DdPPK2 are found in pathogenic protozoa and in the alga Chlamydomonas. Beyond the immediate relevance of Poly P as a target for anti-infective drugs, a large variety of cellular operations that rely on Poly P will be considered.
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paper_title: Isolation and Phylogenetic Analysis of Polyphosphate Accumulating Organisms in Water and Sludge of Intensive Catfish Ponds in the Mekong Delta, Vietnam

paper_content:

Polyphosphate accumulating organisms were isolated from water and sludge samples of intensive catfish ponds in the Mekong Delta, Vietnam. The Results of estimation of intracellular polyphosphate concentration conducted on each of monocultures indicated that the content of intracellular polyphosphate varied from 2 mg/l to 148.1 mg/l after 6 days of incubation in the medium. Of 191 isolates, twenty-one have uptake and store intracellular phosphate from 19.6 to 148.1 mg/l. They have shaped like a rods and short rods or cocci, a few of them were slightly curved or straight or curved rods. The majority of them are gram-positive (76.2%) and the remains are gram-negative. The partial 16S rRNA genes of these isolates were sequenced and compared with bacterial 16S rRNA genes in Genbank using BlastN Program. Phylogenetic tree was constructed on the basic 16S rRNA gene sequences demonstrating the population of high phosphate accumulating bacteria obtained from samples of catfish ponds were affiliated with four major bacterial lineages. Twenty-one bacteria isolates from samples of catfish ponds included in four classes: Bacilli, Actinobacteria, Beta-proteobacteria, Gamma-proteobacteria. The majority of the strains showed excess phosphate accumulation. Strains related to Bacillus sp. were dominant bacteria group constituted up to 52.4% of all identified isolates, but high phosphate accumulating bacteria are Burkholderia vietnamiensis TVT003L within class Beta-proteobacteria, Acinetobacter radioresistens TGT013L within Gamma-proteobacteria and Arthrobacter protophomiae VLT002L within class Actinobacteria. Methyl blue Loeffler’s staning and electron microscopy examination confirmed that the bacteria had stored polyphosphate granules intracellularly.
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paper_title: Methods for Detection and Quantification of Polyphosphate and Polyphosphate Accumulating Microorganisms in Aquatic Sediments

paper_content:

It has been speculated that the microbial P pool is highly variable in the uppermost layer of various aquatic sediments, especially when an excessive P accumulation in form of polyphosphate (Poly-P) occurs. Poly-P storage is a universal feature of many different organisms and has been technically optimised in wastewater treatment plants (WWTP) with enhanced biological phosphorus removal (EBPR). In the recent past, new insights into mechanisms of P elimination in WWTP almost exclusively depended on the development and application of novel methods like 31P-NMR spectroscopy and molecular methods for identifying Poly-P accumulating microorganisms (PAO). The aim of the present review is to compile current methods potentially available for detection and quantification of Poly-P in sediments and to complement it with yet unpublished results to validate their application in natural sediments. The most powerful tool for reliable Poly-P quantification in sediments is the liquid 31P-NMR technique which has been successfully used for Poly-P measurements in a variety of aquatic sediments. But the microorganisms as well as mechanisms involved in Poly-P storage and cycling are largely unknown. Therefore, we also intend to stimulate future studies focusing on these encouraging topics in sediment research via the implementation of novel methods. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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paper_title: Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms.

paper_content:

Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAO's metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.
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paper_title: Advances in techniques for phosphorus analysis in biological sources.

paper_content:

In general, conventional P analysis methods suffer from not only the fastidious extraction and pre-treatment procedures required but also the generally low specificity and poor resolution regarding the P composition and its temporal and spatial dynamics. More powerful yet feasible P analysis tools are in demand to help elucidating the biochemistry nature, roles and dynamics of various phosphorus-containing molecules in vitro and in vivo. Recent advances in analytical chemistry, especially in molecular and atomic spectrometry such as NMR, Raman and X-ray techniques, have enabled unique capability of P analysis relevant to submicron scale biochemical processes in individual cell and in natural samples without introducing too complex and invasive pretreatment steps. Great potential still remains to be explored in wider and more combined and integrated requests of these techniques to allow for new possibilities and more powerful P analysis in biological systems. This review provides a comprehensive summary of the available methods and recent developments in analytical techniques and their applications for characterization and quantification of various forms of phosphorus, particularly polyphosphate, in different biological sources.
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paper_title: Identification of functionally relevant populations in enhanced biological phosphorus removal processes based on intracellular polymers profiles and insights into the metabolic diversity and heterogeneity.

paper_content:

This study proposed and demonstrated the application of a new Raman microscopy-based method for metabolic state-based identification and quantification of functionally relevant populations, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), in enhanced biological phosphorus removal (EBPR) system via simultaneous detection of multiple intracellular polymers including polyphosphate (polyP), glycogen, and polyhydroxybutyrate (PHB). The unique Raman spectrum of different combinations of intracellular polymers within a cell at a given stage of the EBPR cycle allowed for its identification as PAO, GAO, or neither. The abundance of total PAOs and GAOs determined by Raman method were consistent with those obtained with polyP staining and fluorescence in situ hybridization (FISH). Different combinations and quantities of intracellular polymer inclusions observed in single cells revealed the distribution of different sub-PAOs groups among the total PAO populations, which exhibit phenotypic and metabolic heterogeneity and diversity. These results also provided evidence for the hypothesis that different PAOs may employ different extents of combination of glycolysis and TCA cycle pathways for anaerobic reducing power and energy generation and it is possible that some PAOs may rely on TCA cycle solely without glycolysis. Sum of cellular level quantification of the internal polymers associated with different population groups showed differentiated and distributed trends of glycogen and PHB level between PAOs and GAOs, which could not be elucidated before with conventional bulk measurements of EBPR mixed cultures.
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paper_title: Dominant and novel clades of Candidatus Accumulibacter phosphatis in 18 globally distributed full-scale wastewater treatment plants

paper_content:

Here we employed quantitative real-time PCR (qPCR) assays for polyphosphate kinase 1 (ppk1) and 16S rRNA genes to assess relative abundances of dominant clades of Candidatus Accumulibacter phosphatis (referred to Accumulibacter) in 18 globally distributed full-scale wastewater treatment plants (WWTPs) from six countries. Accumulibacter were not only detected in the 6 WWTPs performing biological phosphorus removal, but also inhabited in the other 11 WWTPs employing conventional activated sludge (AS) with abundances ranging from 0.02% to 7.0%. Among the AS samples, clades IIC and IID were found to be dominant among the five Accumulibacter clades. The relative abundance of each clade in the Accumulibacter lineage significantly correlated (p < 0.05) with the influent total phosphorus and chemical oxygen demand instead of geographical factors (e.g. latitude), which showed that the local wastewater characteristics and WWTPs configurations could be more significant to determine the proliferation of Accumulibacter clades in full-scale WWTPs rather than the geographical location. Moreover, two novel Accumulibacter clades (IIH and II-I) which had not been previously detected were discovered in two enhanced biological phosphorus removal (EBPR) WWTPs. The results deepened our understanding of the Accumulibacter diversity in environmental samples.
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paper_title: Phosphorus accumulation by bacteria isolated from a continuous-flow two-sludge system.

paper_content:

In this article, polyphosphate-accumulating organisms (PAOs) from a lab-scale continuous-flow two-sludge system was isolated and identified, the different phosphorus accumulation characteristics of the isolates under anoxic and aerobic conditions were investigated. Two kinds of PAOs were both found in the anoxic zones of the two-sludge system, one of them utilized only oxygen as electron acceptor, and the other one utilized either nitrate or oxygen as electron acceptor. Of the total eight isolates, five isolates were capable of utilizing both nitrate and oxygen as electron acceptors to uptake phosphorus to some extent. And three of the five isolates showed good phosphorus accumulative capacities both under anoxic or aerobic conditions, two identified as Alcaligenes and one identified as Pseudomonas. Streptococcus was observed weak anoxic phosphorus accumulation because of its weak denitrification capacity, but it showed good phosphorus accumulation capacity under aerobic conditions. One isolates identified as Enterobacteriaceae was proved to be a special species of PAOs, which could only uptake small amounts of phosphorus under anoxic conditions, although its denitrification capacity and aerobic phosphorus accumulation capacity were excellent.
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paper_title: Extraction and detection methods for polyphosphate storage in autotrophic planktonic organisms

paper_content:

Different extraction procedures were employed to characterise the polyphosphate granules in autotrophic planktonic organisms, the green microalgae Chlorella vulgaris and the cyanobacterium Synechocystis sp. strain PPC 6803. The effectiveness of these methods was assessed using epifluorescence microscopic analysis of DAPI stained specimens as well as by electron spectroscopic imaging. The results clearly indicate that NaOH and hot water treatment followed by filtration of the extracts are suitable to obtain a cell free suspension of intact polyphosphate granules without hydrolysing the polymers. The methods described are useful to gain physiological information on the phosphorus status of autotrophic planktonic organisms.
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paper_title: Inorganic polyphosphate in industry, agriculture and medicine: Modern state and outlook

paper_content:

Abstract Inorganic polyphosphates (PolyP) are linear polymers containing a few to several hundred orthophosphate residues linked by energy-rich phosphoanhydride bonds. PolyPs are widely used as reagents in water treatment, fertilizers, flame retardants and food additives due to its unique properties, inexpensiveness, nontoxicity and biodegradability. The practice of enhanced biological phosphorus removal (EBPR), based on PolyP accumulation by sludge bacteria, is an accepted and low-cost strategy for controlling eutrophication. PolyPs are present in the cells of all living organisms, from bacteria to mammals. They perform numerous functions in the cells: phosphate and energy storage, sequestration and storage of cations, formation of membrane channels, cell envelope formation and function, gene activity control, regulation of enzyme activities, stress response and stationary phase adaptation. PolyPs participate in bone tissue development and in the blood coagulation cascade and are promising candidates in therapy for bone and blood diseases. They may also have application in creating novel bone substitute materials, serving as carriers for prolonged action drugs, and acting as a phosphodonor in enzymatic synthesis of biologically active compounds. The importance of polyphosphate kinases in the virulence of pathogens forms a basis for the development of new antibiotics. Further study of PolyP biochemistry and cell biology can be applied to medicine, environmental protection and agriculture.
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paper_title: A high throughput method and culture medium for rapid screening of phosphate accumulating microorganisms

paper_content:

Abstract A novel PA Medium (PAM) for efficient screening of phosphate-accumulating organisms (PAOs) was developed taking Serratia marcescens NBRI1213 as model organism. The defined National Botanical Research Institute’s growth medium (NBRI) supplemented with 0.1% maltose, designed for quantitative estimation of phosphate accumulation was designated as PAM. Our work suggested usage of PAM for efficient qualitative screening and as a microbiological medium for preferential selection of PAOs on Petri-plates. For qualitative screening of PAOs, Toluidine blue-O dye (TBO) was supplemented in PAM, designated as PAM-TBO. Qualitative analysis of phosphate accumulated by various groups correlated well with grouping based upon quantitative analysis of PAOs, effect of carbon, nitrogen, salts, and phosphate accumulation-defective transposon mutants. For significantly increasing sample throughput, efficiency of screening PAOs was further enhanced by adaptation of PAM-TBO assay to microtiter plate based method. It is envisaged that usage of this medium will be salutary for quick screening of PAOs from environment.
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paper_title: Differentiation of polyphosphate metabolism between the extra- and intraradical hyphae of arbuscular mycorrhizal fungi

paper_content:

Summary ::: ::: • Regulation of polyphosphate metabolism is reported in arbuscular mycorrhizal symbiosis. ::: ::: ::: • Marigold (Tagetes patula) plants inoculated with Glomus coronatum or Glomus etunicatum were grown in mesh bags. Exopolyphosphatase activity in extra- and intraradical hyphae was measured and characterized. The hyphae were stained with Neutral red to show acidic vacuoles in which polyphosphate synthesis might occur. ::: ::: ::: • Exopolyphosphate activity was differently expressed between the extra- and intraradical hyphae, as indicated by different pH optima; high activity was observed at pH 5.0 in the intraradical hyphae of both fungal species. Km values were lower at neutral pH with long-chain polyphosphate, whereas acidic activity showed lower Km with short-chain polyphosphate. Both extra- and intradical hyphae had acidic vacuoles. Polyphosphate occurred in the hyphae of the high-P, but not the low-P treatment. By contrast, exopolyphosphatase activity and vacuolar acidity were relatively constant irrespective of polyphosphate status. ::: ::: ::: • The fungi have at least two different exopolyphosphatase-type enzymes which are differently expressed between extra- and intraradical hyphae; polyphosphate accumulation might be a dynamic balance between synthesis and hydrolysis.
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paper_title: Methods for Detection and Quantification of Polyphosphate and Polyphosphate Accumulating Microorganisms in Aquatic Sediments

paper_content:

It has been speculated that the microbial P pool is highly variable in the uppermost layer of various aquatic sediments, especially when an excessive P accumulation in form of polyphosphate (Poly-P) occurs. Poly-P storage is a universal feature of many different organisms and has been technically optimised in wastewater treatment plants (WWTP) with enhanced biological phosphorus removal (EBPR). In the recent past, new insights into mechanisms of P elimination in WWTP almost exclusively depended on the development and application of novel methods like 31P-NMR spectroscopy and molecular methods for identifying Poly-P accumulating microorganisms (PAO). The aim of the present review is to compile current methods potentially available for detection and quantification of Poly-P in sediments and to complement it with yet unpublished results to validate their application in natural sediments. The most powerful tool for reliable Poly-P quantification in sediments is the liquid 31P-NMR technique which has been successfully used for Poly-P measurements in a variety of aquatic sediments. But the microorganisms as well as mechanisms involved in Poly-P storage and cycling are largely unknown. Therefore, we also intend to stimulate future studies focusing on these encouraging topics in sediment research via the implementation of novel methods. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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paper_title: Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms.

paper_content:

Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAO's metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.
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paper_title: Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4',6'-diamidino-2-phenylindole) and tetracycline labeling.

paper_content:

Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a "Candidatus Accumulibacter"-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.
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paper_title: A novel method for determination of inorganic polyphosphates using the fluorescent dye fura-2.

paper_content:

Abstract A method for determining inorganic polyphosphate, which is based on the Mn 2+ -induced quenching of the fluorescence of the calcium indicator fura-2, is described. The effect of Mn 2+ ions on fura-2 fluorescence is gradually abolished in the presence of increasing concentrations of polyphosphate; this allows the quantification both of synthetic polyphosphates and of the naturally occurring polymer isolated from tissues or cells. The described method has some advantages compared to conventional procedures for detection of polyphosphates based on the metachromatic effect on toluidine blue. It can be applied for the determination of pyrophosphate, tripolyphosphate and other short-chain polyphosphates not detectable by toluidine blue and it can be used for measurement both of pyrophosphatase and exopolyphosphatase activity.
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paper_title: Biodiversity of Polyphosphate Accumulating Bacteria in Eight WWTPs with Different Modes of Operation

paper_content:

AbstractEnhanced biological phosphorous removal (EBPR) from wastewater has been successfully used for more than three decades and is considered to be an environmentally friendly wastewater-treatment process. Biologically, this process is realized by incorporation of phosphate as polyphosphate (polyP) granules in activated sludge bacteria. Important groups of bacteria responsible for P removal have been identified, but the full microbial diversity involved in this process is still unknown. This paper reports on the microbial composition of activated sludge communities in eight wastewater-treatment plants (WWTPs) with different sizes and modes of operation. The polyphosphate accumulating organisms (PAOs) within this complex biocenosis were identified by fluorescent dye staining and classified by in situ hybridization techniques. Of the bacteria in the aerobic basin, 5–13% contained polyP granules. In addition, flow cytometry was used to quantify PAOs after tetracycline staining and to separate these cells. ...
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paper_title: Fixation procedures for flow cytometric analysis of environmental bacteria

paper_content:

Abstract Analysis of environmental bacteria on the single cell level often requires fixation to store the cells and to keep them in a state as near life-like as possible. Fixation procedures should furthermore counteract the increase of autofluorescence, cell clogging, and distortion of surface characteristics. Additionally, they should meet the specific fixation demands of both aerobically and anaerobically grown bacteria. A fixation method was developed based on metal solutions in combination with sodium azide. The fixation efficiencies of aluminium, barium, bismuth, cobalt, molybdenum, nickel, and tungsten salts were evaluated by flow cytometric measurement of the DNA contents as a bacterial population/community stability marker. Statistical equivalence testing was involved to permit highly reliable flow cytometric pattern evaluation. Investigations were carried out with pure cultures representing environmentally important metabolic and respiratory pathways as controls and with activated sludge as an example for highly diverse bacterial communities. A mixture of 5 mM barium chloride and nickel chloride, each and 10% sodium azide was found to be a suitable fixative for all tested bacteria. The described method provided good sample stability for at least 9 days.
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paper_title: Diversity of nitrite reductase genes in "Candidatus Accumulibacter phosphatis"-dominated cultures enriched by flow-cytometric sorting.

paper_content:

"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.
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paper_title: Correlation of Community Dynamics and Process Parameters As a Tool for the Prediction of the Stability of Wastewater Treatment

paper_content:

Wastewater treatment often suffers from instabilities and the failure of specific functions such as biological phosphorus removal by polyphosphate accumulating organisms. Since most of the microorganisms involved in water clarification are unknown it is challenging to operate the process accounting for the permanent varying abiotic parameters and the complex composition and unrevealed metabolic capacity of a wastewater microbial community. Fulfilling the demands for water quality irrespective of substrate inflow conditions may emit severe problems if the limited management resources of municipal wastewater treatment plants are regarded. We used flow cytometric analyses of cellular DNA and polyphosphate to create patterns mirroring dynamics in community structure. These patterns were resolved in up to 15 subclusters, the presence and abundances of which correlated with abiotic data. The study used biostatistics to determine the kind and strength of the correlation. Samples investigated were obtained from a...
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paper_title: Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4',6'-diamidino-2-phenylindole) and tetracycline labeling.

paper_content:

Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a "Candidatus Accumulibacter"-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.
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paper_title: Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription

paper_content:

Background. In hematology there has recently been increasing interest in inorganic polyphosphate. This polymer accumulates in platelet granules and its functions include modulating various stages in blood coagulation, inducing angiogenesis, and provoking apoptosis of plasma cells. In this work, we evaluate 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. ::: ::: Design and Methods. We have developed a novel method for detecting levels of polyphosphate in cell populations using flow cytometry. We also have studied polyphosphate localization and characteristics, using confocal microscopy and enzymatic analysis experiments. ::: ::: Results. We have found that myeloma plasma cells present higher levels of intracellular polyphosphate than normal plasma cells and other B-cell populations. Localization experiments indicated that polyphosphate accumulates at high levels in the nucleolus of myeloma cells. As the principal function of the nucleolus involves the transcription of ribosomal DNA genes, we have found changes in the cellular distribution of polyphosphate after the inhibition of the nucleolar transcription. In addition, we have found that RNA polymerase I activity, responsible for transcription in the nucleolus, is also modulated by polyphosphate, in a dose-dependent manner. ::: ::: Conclusions. Our results show an unusual high accumulation of polyphosphate in the nucleoli of myeloma cells and a functional relationship of this polymer with the nucleolar transcription.
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paper_title: In situ identification of polyphosphate- and polyhydroxyalkanoate-accumulating traits for microbial populations in a biological phosphorus removal process

paper_content:

Polyphosphate- and polyhydroxyalkanoate (PHA)-accumulating traits of predominant microorganisms in an efficient enhanced biological phosphorus removal (EBPR) process were investigated systematically using a suite of non-culture-dependent methods. Results of 16S rDNA clone library and fluorescence in situ hybridization (FISH) with rRNA-targeted, group-specific oligonucleotide probes indicated that the microbial community consisted mostly of the α- (9.5% of total cells), β- (41.3%) and γ- (6.8%) subclasses of the class Proteobacteria, Flexibacter–Cytophaga (4.5%) and the Gram-positive high G+C (HGC) group (17.9%). With individual phylogenetic groups or subgroups, members of Candidatus Accumulibacter phosphatis in the β-2 subclass, a novel HGC group closely related to Tetrasphaera spp., and a novel γ-proteobacterial group were the predominant populations. Furthermore, electron microscopy with energy-dispersive X-ray analysis was used to validate the staining specificity of 4,6-diamino-2-phenylindole (DAPI) for intracellular polyphosphate and revealed the composition of polyphosphate granules accumulated in predominant bacteria as mostly P, Ca and Na. As a result, DAPI and PHA staining procedures could be combined with FISH to identify directly the polyphosphate- and PHA-accumulating traits of different phylogenetic groups. Members of Accumulibacter phosphatis and the novel gamma-proteobacterial group were observed to accumulate both polyphosphate and PHA. In addition, one novel rod-shaped group, closely related to coccus-shaped Tetrasphaera, and one filamentous group resembling Candidatus Nostocoidia limicola in the HGC group were found to accumulate polyphosphate but not PHA. No cellular inclusions were detected in most members of the α-Proteobacteria and the Cytophaga–Flavobacterium group. The diversified functional traits observed suggested that different substrate metabolisms were used by predominant phylogenetic groups in EBPR processes.
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paper_title: Characteristics of Denitrifying Phosphate Accumulating Organisms in an Anaerobic-Intermittently Aerobic Process

paper_content:

The anaerobic-intermittently aerobic (AIA) process was operated for the enhanced biological phosphorus and nitrogen removal over 2 years. A bench-scale AIA reactor operated in a continuous-flow anaerobic and alternating anoxic–aerobic mode was demonstrated to accomplish nitrification, denitrification, and phosphorus removal. Under the anaerobic zone, carbon source was taken up, polyhydroxyalkanoates (PHAs) were formed, and accomplished phosphorus release. The simultaneous phosphate uptake and denitrification by the denitrifying phosphate accumulating organisms (DePAOs) was observed even though the PHAs in cells were oxidized in the aerobic phase before the anoxic phase. Ammonium was oxidized to nitrate under the aerobic phase, and nitrate was reduced to nitrogen gas under the anoxic phase. As the nitrate concentration increased, phosphate uptake rate and denitrification rate decreased, whereas the release of phosphate was accelerated with the addition of the external carbon source. The secondary phosphate...
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paper_title: Anaerobic glyoxylate cycle activity during simultaneous utilization of glycogen and acetate in uncultured Accumulibacter enriched in enhanced biological phosphorus removal communities

paper_content:

Enhanced biological phosphorus removal (EBPR) communities protect waterways from nutrient pollution and enrich microorganisms capable of assimilating acetate as polyhydroxyalkanoate (PHA) under anaerobic conditions. Accumulibacter, an important uncultured polyphosphate-accumulating organism (PAO) enriched in EBPR, was investigated to determine the central metabolic pathways responsible for producing PHA. Acetate uptake and assimilation to PHA in Accumulibacter was confirmed using fluorescence in situ hybridization (FISH)-microautoradiography and post-FISH chemical staining. Assays performed with enrichments of Accumulibacter using an inhibitor of glyceraldehyde-3-phosphate dehydrogenase inferred anaerobic glycolysis activity. Significant decrease in anaerobic acetate uptake and PHA production rates were observed using inhibitors targeting enzymes within the glyoxylate cycle. Bioinformatic analysis confirmed the presence of genes unique to the glyoxylate cycle (isocitrate lyase and malate synthase) and gene expression analysis of isocitrate lyase demonstrated that the glyoxylate cycle is likely involved in PHA production. Reduced anaerobic acetate uptake and PHA production was observed after inhibition of succinate dehydrogenase and upregulation of a succinate dehydrogenase gene suggested anaerobic activity. Cytochrome b/b6 activity inferred that succinate dehydrogenase activity in the absence of external electron acceptors may be facilitated by a novel cytochrome b/b6 fusion protein complex that pushes electrons uphill to more electronegative electron carriers. Identification of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase genes in Accumulibacter demonstrated the potential for interconversion of C3 intermediates of glycolysis and C4 intermediates of the glyoxylate cycle. Our findings along with previous hypotheses from analysis of microbiome data and metabolic models for PAOs were used to develop a model for anaerobic carbon metabolism in Accumulibacter.
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paper_title: Biological phosphorus removal from real wastewater in a sequencing batch reactor operated as aerobic/extended-idle regime

paper_content:

Abstract Recently, it has been reported that biological phosphorus removal (BPR) could be achieved in a sequencing batch reactor (SBR) with aerobic/extended-idle (A/EI) regime using synthetic medium. This paper first examined the feasibility and stability of the A/EI regime treating real domestic wastewater. The results showed that the A/EI-SBR removed 1.32 ± 0.03–3.55 ± 0.04 mg of phosphorus per g of volatile suspended solids during the steady-state operation, suggesting that BPR from domestic wastewater could be well realised in the A/EI regime. Then, another SBR operated as the conventional anaerobic/oxic (A/O) regime was conducted to compare the soluble orthophosphate (SOP) removal with the A/EI regime. The results clearly showed that the A/EI regime achieved higher SOP removal than the A/O regime. Finally, the mechanism for the A/EI-SBR driving superior SOP removal was investigated. It was found that the sludge cultured by the A/EI regime had more polyphosphate accumulating organisms and less glycogen accumulating organisms than that by the A/O regime. Further investigations showed that the A/EI-SBR had a lower glycogen transformation and a higher PHB/PHV ratio, which correlated well with the superior phosphorus removal.
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paper_title: Simultaneous COD, nitrogen, and phosphate removal by aerobic granular sludge

paper_content:

Aerobic granular sludge technology offers a possibility to design compact wastewater treatment plants based on simultaneous chemical oxygen demand (COD), nitrogen and phosphate removal in one sequencing batch reactor. In earlier studies, it was shown that aerobic granules, cultivated with an aerobic pulse-feeding pattern, were not stable at low dissolved oxygen concentrations. Selection for slow-growing organisms such as phosphate-accumulating organisms (PAO) was shown to be a measure for improved granule stability, particularly at low oxygen concentrations. Moreover, this allows long feeding periods needed for economically feasible full-scale applications. Simultaneous nutrient removal was possible, because of heterotrophic growth inside the granules (denitrifying PAO). At low oxygen saturation (20%) high removal efficiencies were obtained; 100% COD removal, 94% phosphate (P-) removal and 94% total nitrogen (N-) removal (with 100% ammonium removal). Experimental results strongly suggest that P-removal occurs partly by (biologically induced) precipitation. Monitoring the laboratory scale reactors for a long period showed that N-removal efficiency highly depends on the diameter of the granules. © 2005 Wiley Periodicals, Inc.
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paper_title: Operation and control of SBR processes for enhanced biological nutrient removal from wastewater

paper_content:

In the last decades, the awareness of environmental issues has increased in society considerably. There is an increasing need to improve the effluent quality of domestic wastewater treatment processes. This thesis describes the application of the Sequencing Batch Reactor (SBR) technology for Biological Nutrient Removal (BNR) from the wastewater. In particular, the work presented evolves from the nitrogen removal to the biological nutrient removal (i.e. nitrogen plus phosphorous removal) with special attention to the operational strategy design, the identification of possible reactor cycle controls or the influent composition related to the process efficiency. In such sense, also the use of ethanol as an external carbon (when low influent Carbon:Phosphorus (C:P) or Carbon:Nitrogen (C:N) ratios are presented) are studied as an alternative to maintain the BNR efficiency.
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paper_title: Ecophysiology of polyphosphate-accumulating organisms and glycogen-accumulating organisms in a continuously aerated enhanced biological phosphorus removal process

paper_content:

Aims: To investigate the ecophysiology of populations of polyphosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) in communities of a novel acetate fed process removing phosphate from wastewater. Attempts were made to see if acetate could be replaced by an alternative carbon source which did not support the growth of the GAO. ::: ::: ::: ::: Methods and Results: A continuously aerated sequencing batch reactor was operated with different acetate feed levels. Fluorescence in situ hybridization (FISH) showed that Defluviicoccus GAO numbers increased at lower acetate feed levels. With FISH/microautoradiography (MAR) both detected morphotypes of Defluviicoccus assimilated a wider range of substrates aerobically than Accumulibacter PAO. Their uptake profile differed from that reported for the same phylotype in full scale anaerobic : aerobic EBPR plants. ::: ::: ::: ::: Conclusions: This suggests that replacing acetate with another substrate is unlikely to provide Accumulibacter with a selective advantage in this process. Why Defluviicoccus appeared to out-compete Accumulibacter at lower acetate concentrations was not clear. Data suggest physiological and morphological diversity may exist within a single Defluviicoccus phylotype. ::: ::: ::: ::: Significance and Impact of the Study: This study implies that the current FISH probes for Defluviicoccus GAO may not reveal the full extent of their biodiversity, and that more information is required before strategies for their control can be devised.
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paper_title: Optimizing the production of Polyphosphate from Acinetobacter towneri

paper_content:

Inorganic polyphosphates (PolyP) are linear polymers of few to several hundred orthophosphate residues, linked by energy-rich phosphoanhydride bonds. Four isolates had been screened from soil sample. By MALDI-TOF analysis, they were identified as Bacillius cereus, Acinetobacter towneri, B. megaterium and B. cereus. The production of PolyP in four isolates was studied in phosphate uptake medium and sulfur deficient medium at pH 7. These organisms had shown significant production of PolyP after 22h of incubation. PolyP was extracted from the cells using alkaline lysis method. Among those isolates, Acinetobacter towneri was found to have high (24.57% w/w as P) accumulation of PolyP in sulfur deficient medium. The media optimization for sulfur deficiency was carried out using Response surface methodology (RSM). It was proven that increase in phosphate level in the presence of glucose, under sulfur limiting condition, enhanced the phosphate accumulation by Acinetobacter towneri and these condition can be simulated for the effective removal of phosphate from wastewater sources.
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paper_title: Accumulation of phosphate and polyphosphate by Cryptococcus humicola and Saccharomyces cerevisiae in the absence of nitrogen.

paper_content:

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 30 mM, respectively), and up to 80% upon addition of 5 mM 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.
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paper_title: Accumulation of inorganic polyphosphates in Saccharomyces cerevisiae under nitrogen deprivation: Stimulation by magnesium ions and peculiarities of localization

paper_content:

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.
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paper_title: Methods for Detection and Quantification of Polyphosphate and Polyphosphate Accumulating Microorganisms in Aquatic Sediments

paper_content:

It has been speculated that the microbial P pool is highly variable in the uppermost layer of various aquatic sediments, especially when an excessive P accumulation in form of polyphosphate (Poly-P) occurs. Poly-P storage is a universal feature of many different organisms and has been technically optimised in wastewater treatment plants (WWTP) with enhanced biological phosphorus removal (EBPR). In the recent past, new insights into mechanisms of P elimination in WWTP almost exclusively depended on the development and application of novel methods like 31P-NMR spectroscopy and molecular methods for identifying Poly-P accumulating microorganisms (PAO). The aim of the present review is to compile current methods potentially available for detection and quantification of Poly-P in sediments and to complement it with yet unpublished results to validate their application in natural sediments. The most powerful tool for reliable Poly-P quantification in sediments is the liquid 31P-NMR technique which has been successfully used for Poly-P measurements in a variety of aquatic sediments. But the microorganisms as well as mechanisms involved in Poly-P storage and cycling are largely unknown. Therefore, we also intend to stimulate future studies focusing on these encouraging topics in sediment research via the implementation of novel methods. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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paper_title: Advances in techniques for phosphorus analysis in biological sources.

paper_content:

In general, conventional P analysis methods suffer from not only the fastidious extraction and pre-treatment procedures required but also the generally low specificity and poor resolution regarding the P composition and its temporal and spatial dynamics. More powerful yet feasible P analysis tools are in demand to help elucidating the biochemistry nature, roles and dynamics of various phosphorus-containing molecules in vitro and in vivo. Recent advances in analytical chemistry, especially in molecular and atomic spectrometry such as NMR, Raman and X-ray techniques, have enabled unique capability of P analysis relevant to submicron scale biochemical processes in individual cell and in natural samples without introducing too complex and invasive pretreatment steps. Great potential still remains to be explored in wider and more combined and integrated requests of these techniques to allow for new possibilities and more powerful P analysis in biological systems. This review provides a comprehensive summary of the available methods and recent developments in analytical techniques and their applications for characterization and quantification of various forms of phosphorus, particularly polyphosphate, in different biological sources.
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paper_title: Identification of functionally relevant populations in enhanced biological phosphorus removal processes based on intracellular polymers profiles and insights into the metabolic diversity and heterogeneity.

paper_content:

This study proposed and demonstrated the application of a new Raman microscopy-based method for metabolic state-based identification and quantification of functionally relevant populations, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), in enhanced biological phosphorus removal (EBPR) system via simultaneous detection of multiple intracellular polymers including polyphosphate (polyP), glycogen, and polyhydroxybutyrate (PHB). The unique Raman spectrum of different combinations of intracellular polymers within a cell at a given stage of the EBPR cycle allowed for its identification as PAO, GAO, or neither. The abundance of total PAOs and GAOs determined by Raman method were consistent with those obtained with polyP staining and fluorescence in situ hybridization (FISH). Different combinations and quantities of intracellular polymer inclusions observed in single cells revealed the distribution of different sub-PAOs groups among the total PAO populations, which exhibit phenotypic and metabolic heterogeneity and diversity. These results also provided evidence for the hypothesis that different PAOs may employ different extents of combination of glycolysis and TCA cycle pathways for anaerobic reducing power and energy generation and it is possible that some PAOs may rely on TCA cycle solely without glycolysis. Sum of cellular level quantification of the internal polymers associated with different population groups showed differentiated and distributed trends of glycogen and PHB level between PAOs and GAOs, which could not be elucidated before with conventional bulk measurements of EBPR mixed cultures.
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paper_title: Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription

paper_content:

Background. In hematology there has recently been increasing interest in inorganic polyphosphate. This polymer accumulates in platelet granules and its functions include modulating various stages in blood coagulation, inducing angiogenesis, and provoking apoptosis of plasma cells. In this work, we evaluate 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. ::: ::: Design and Methods. We have developed a novel method for detecting levels of polyphosphate in cell populations using flow cytometry. We also have studied polyphosphate localization and characteristics, using confocal microscopy and enzymatic analysis experiments. ::: ::: Results. We have found that myeloma plasma cells present higher levels of intracellular polyphosphate than normal plasma cells and other B-cell populations. Localization experiments indicated that polyphosphate accumulates at high levels in the nucleolus of myeloma cells. As the principal function of the nucleolus involves the transcription of ribosomal DNA genes, we have found changes in the cellular distribution of polyphosphate after the inhibition of the nucleolar transcription. In addition, we have found that RNA polymerase I activity, responsible for transcription in the nucleolus, is also modulated by polyphosphate, in a dose-dependent manner. ::: ::: Conclusions. Our results show an unusual high accumulation of polyphosphate in the nucleoli of myeloma cells and a functional relationship of this polymer with the nucleolar transcription.
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paper_title: A high throughput method and culture medium for rapid screening of phosphate accumulating microorganisms

paper_content:

Abstract A novel PA Medium (PAM) for efficient screening of phosphate-accumulating organisms (PAOs) was developed taking Serratia marcescens NBRI1213 as model organism. The defined National Botanical Research Institute’s growth medium (NBRI) supplemented with 0.1% maltose, designed for quantitative estimation of phosphate accumulation was designated as PAM. Our work suggested usage of PAM for efficient qualitative screening and as a microbiological medium for preferential selection of PAOs on Petri-plates. For qualitative screening of PAOs, Toluidine blue-O dye (TBO) was supplemented in PAM, designated as PAM-TBO. Qualitative analysis of phosphate accumulated by various groups correlated well with grouping based upon quantitative analysis of PAOs, effect of carbon, nitrogen, salts, and phosphate accumulation-defective transposon mutants. For significantly increasing sample throughput, efficiency of screening PAOs was further enhanced by adaptation of PAM-TBO assay to microtiter plate based method. It is envisaged that usage of this medium will be salutary for quick screening of PAOs from environment.
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paper_title: Sensitive fluorescence detection of polyphosphate in polyacrylamide gels using 4′,6-diamidino-2-phenylindol

paper_content:

PAGE is commonly used to identify and resolve inorganic polyphosphates (polyP). We now report highly sensitive and specific staining methods for polyP in polyacrylamide gels based on the fluorescent dye, 4',6-diamidino-2-phenylindol (DAPI). DAPI bound to polyP in gels fluoresced yellow while DAPI bound to nucleic acids or glycosaminoglycans fluoresced blue. Inclusion of EDTA prevented staining of glycosaminoglycans by DAPI. We also identified conditions under which DAPI that was bound to polyP (but not nucleic acids or other anionic polymers) rapidly photobleached. This allowed us to develop an even more sensitive and specific negative staining method that distinguishes polyP from nucleic acids and glycosaminoglycans. The lower LOD using DAPI negative staining was 4 pmol (0.3 ng) phosphate per band, compared to conventional toluidine blue staining with a lower LOD of 250 pmol per band.
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paper_title: Accumulation of phosphate and polyphosphate by Cryptococcus humicola and Saccharomyces cerevisiae in the absence of nitrogen.

paper_content:

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 30 mM, respectively), and up to 80% upon addition of 5 mM 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.
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paper_title: Accumulation of inorganic polyphosphates in Saccharomyces cerevisiae under nitrogen deprivation: Stimulation by magnesium ions and peculiarities of localization

paper_content:

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

paper_content:

Background. In hematology there has recently been increasing interest in inorganic polyphosphate. This polymer accumulates in platelet granules and its functions include modulating various stages in blood coagulation, inducing angiogenesis, and provoking apoptosis of plasma cells. In this work, we evaluate 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. ::: ::: Design and Methods. We have developed a novel method for detecting levels of polyphosphate in cell populations using flow cytometry. We also have studied polyphosphate localization and characteristics, using confocal microscopy and enzymatic analysis experiments. ::: ::: Results. We have found that myeloma plasma cells present higher levels of intracellular polyphosphate than normal plasma cells and other B-cell populations. Localization experiments indicated that polyphosphate accumulates at high levels in the nucleolus of myeloma cells. As the principal function of the nucleolus involves the transcription of ribosomal DNA genes, we have found changes in the cellular distribution of polyphosphate after the inhibition of the nucleolar transcription. In addition, we have found that RNA polymerase I activity, responsible for transcription in the nucleolus, is also modulated by polyphosphate, in a dose-dependent manner. ::: ::: Conclusions. Our results show an unusual high accumulation of polyphosphate in the nucleoli of myeloma cells and a functional relationship of this polymer with the nucleolar transcription.
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paper_title: Accumulation of inorganic polyphosphates in Saccharomyces cerevisiae under nitrogen deprivation: Stimulation by magnesium ions and peculiarities of localization

paper_content:

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.
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paper_title: Biological phosphorus removal from abattoir wastewater at very short sludge ages mediated by novel PAO clade Comamonadaceae.

paper_content:

Recent increases in global phosphorus costs, together with the need to remove phosphorus from wastewater to comply with water discharge regulations, make phosphorus recovery from wastewater economically and environmentally attractive. Biological phosphorus (Bio-P) removal process can effectively capture the phosphorus from wastewater and concentrate it in a form that is easily amendable for recovery in contrast to traditional (chemical) phosphorus removal processes. However, Bio-P removal processes have historically been operated at medium to long solids retention times (SRTs, 10-20 days typically), which inherently increases the energy consumption while reducing the recoverable carbon fraction and hence makes it incompatible with the drive towards energy self-sufficient wastewater treatment plants. In this study, a novel high-rate Bio-P removal process has been developed as an energy efficient alternative for phosphorus removal from wastewater through operation at an SRT of less than 4 days. The process was most effective at an SRT of 2-2.5 days, achieving >90% phosphate removal. Further reducing the SRT to 1.7 days resulted in a loss of Bio-P activity. 16S pyrotag sequencing showed the community changed considerably with changes in the SRT, but that Comamonadaceae was consistently abundant when the Bio-P activity was evident. FISH analysis combined with DAPI staining confirmed that bacterial cells of Comamonadaceae arranged in tetrads contained polyphosphate, identifying them as the key polyphosphate accumulating organisms at these low SRT conditions. Overall, this paper demonstrates a novel, high-rate phosphorus removal process that can be effectively integrated with short SRT, energy-efficient carbon removal and recovery processes.
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paper_title: Isolation and Phylogenetic Analysis of Polyphosphate Accumulating Organisms in Water and Sludge of Intensive Catfish Ponds in the Mekong Delta, Vietnam

paper_content:

Polyphosphate accumulating organisms were isolated from water and sludge samples of intensive catfish ponds in the Mekong Delta, Vietnam. The Results of estimation of intracellular polyphosphate concentration conducted on each of monocultures indicated that the content of intracellular polyphosphate varied from 2 mg/l to 148.1 mg/l after 6 days of incubation in the medium. Of 191 isolates, twenty-one have uptake and store intracellular phosphate from 19.6 to 148.1 mg/l. They have shaped like a rods and short rods or cocci, a few of them were slightly curved or straight or curved rods. The majority of them are gram-positive (76.2%) and the remains are gram-negative. The partial 16S rRNA genes of these isolates were sequenced and compared with bacterial 16S rRNA genes in Genbank using BlastN Program. Phylogenetic tree was constructed on the basic 16S rRNA gene sequences demonstrating the population of high phosphate accumulating bacteria obtained from samples of catfish ponds were affiliated with four major bacterial lineages. Twenty-one bacteria isolates from samples of catfish ponds included in four classes: Bacilli, Actinobacteria, Beta-proteobacteria, Gamma-proteobacteria. The majority of the strains showed excess phosphate accumulation. Strains related to Bacillus sp. were dominant bacteria group constituted up to 52.4% of all identified isolates, but high phosphate accumulating bacteria are Burkholderia vietnamiensis TVT003L within class Beta-proteobacteria, Acinetobacter radioresistens TGT013L within Gamma-proteobacteria and Arthrobacter protophomiae VLT002L within class Actinobacteria. Methyl blue Loeffler’s staning and electron microscopy examination confirmed that the bacteria had stored polyphosphate granules intracellularly.
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paper_title: Advances in techniques for phosphorus analysis in biological sources.

paper_content:

In general, conventional P analysis methods suffer from not only the fastidious extraction and pre-treatment procedures required but also the generally low specificity and poor resolution regarding the P composition and its temporal and spatial dynamics. More powerful yet feasible P analysis tools are in demand to help elucidating the biochemistry nature, roles and dynamics of various phosphorus-containing molecules in vitro and in vivo. Recent advances in analytical chemistry, especially in molecular and atomic spectrometry such as NMR, Raman and X-ray techniques, have enabled unique capability of P analysis relevant to submicron scale biochemical processes in individual cell and in natural samples without introducing too complex and invasive pretreatment steps. Great potential still remains to be explored in wider and more combined and integrated requests of these techniques to allow for new possibilities and more powerful P analysis in biological systems. This review provides a comprehensive summary of the available methods and recent developments in analytical techniques and their applications for characterization and quantification of various forms of phosphorus, particularly polyphosphate, in different biological sources.
---
paper_title: Identification of functionally relevant populations in enhanced biological phosphorus removal processes based on intracellular polymers profiles and insights into the metabolic diversity and heterogeneity.

paper_content:

This study proposed and demonstrated the application of a new Raman microscopy-based method for metabolic state-based identification and quantification of functionally relevant populations, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), in enhanced biological phosphorus removal (EBPR) system via simultaneous detection of multiple intracellular polymers including polyphosphate (polyP), glycogen, and polyhydroxybutyrate (PHB). The unique Raman spectrum of different combinations of intracellular polymers within a cell at a given stage of the EBPR cycle allowed for its identification as PAO, GAO, or neither. The abundance of total PAOs and GAOs determined by Raman method were consistent with those obtained with polyP staining and fluorescence in situ hybridization (FISH). Different combinations and quantities of intracellular polymer inclusions observed in single cells revealed the distribution of different sub-PAOs groups among the total PAO populations, which exhibit phenotypic and metabolic heterogeneity and diversity. These results also provided evidence for the hypothesis that different PAOs may employ different extents of combination of glycolysis and TCA cycle pathways for anaerobic reducing power and energy generation and it is possible that some PAOs may rely on TCA cycle solely without glycolysis. Sum of cellular level quantification of the internal polymers associated with different population groups showed differentiated and distributed trends of glycogen and PHB level between PAOs and GAOs, which could not be elucidated before with conventional bulk measurements of EBPR mixed cultures.
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paper_title: Copper ions stimulate polyphosphate degradation and phosphate efflux in Acidithiobacillus ferrooxidans.

paper_content:

For some bacteria and algae, it has been proposed that inorganic polyphosphates and transport of metal-phosphate complexes could participate in heavy metal tolerance. To test for this possibility in Acidithiobacillus ferrooxidans, a microorganism with a high level of resistance to heavy metals, the polyphosphate levels were determined when the bacterium was grown in or shifted to the presence of a high copper concentration (100 mM). Under these conditions, cells showed a rapid decrease in polyphosphate levels with a concomitant increase in exopolyphosphatase activity and a stimulation of phosphate efflux. Copper in the range of 1 to 2 μM greatly stimulated exopolyphosphatase activity in cell extracts from A. ferrooxidans. The same was seen to a lesser extent with cadmium and zinc. Bioinformatic analysis of the available A. ferrooxidans ATCC 23270 genomic sequence did not show a putative pit gene for phosphate efflux but rather an open reading frame similar in primary and secondary structure to that of the Saccharomyces cerevisiae phosphate transporter that is functional at acidic pH (Pho84). Our results support a model for metal detoxification in which heavy metals stimulate polyphosphate hydrolysis and the metal-phosphate complexes formed are transported out of the cell as part of a possibly functional heavy metal tolerance mechanism in A. ferrooxidans.
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paper_title: Microscopy evidence of bacterial microfossils in phosphorite crusts of the Peruvian shelf: Implications for phosphogenesis mechanisms

paper_content:

Abstract Phosphorites are sedimentary formations enriched in Ca-phosphate minerals. The precipitation of these minerals is thought to be partly mediated by the activity of microorganisms. The vast majority of studies on phosphorites have focused on a petrological and geochemical characterization of these rocks. However, detailed descriptions are needed at the sub-micrometer scale at which crucial information can be retrieved about traces of past or modern microbial activities. Here, scanning electron microscopy (SEM) analyses of a recent phosphorite crust from the upwelling-style phosphogenesis area off Peru revealed that it contained a great number of rod-like and coccus-like shaped micrometer-sized (~ 1.1 μm and 0.5 μm, respectively) objects, referred to as biomorphs. Some of these biomorphs were filled with carbonate fluoroapatite (CFA, a calcium-phosphate phase common in phosphorites); some were empty; some were surrounded by one or two layers of pyrite. Transmission electron microscopy (TEM) and energy dispersive X-ray spectrometry (EDXS) analyses were performed on focused ion beam (FIB) milled ultrathin foils to characterize the texture of CFA and pyrite in these biomorphs at the few nanometer scale. Non-pyritized phosphatic biomorphs were surrounded by a thin (5–15 nm thick) rim appearing as a void on TEM images. Bundles of CFA crystals sharing the same crystallographic orientations (aligned along their c -axis) were found in the interior of some biomorphs. Pyrite formed a thick (~ 35–115 nm) layer with closely packed crystals surrounding the pyritized biomorphs, whereas pyrite crystals at distance from the biomorphs were smaller and distributed more sparsely. Scanning transmission X-ray microscopy (STXM) analyses performed at the C K-edge provided maps of organic and inorganic carbon in the samples. Inorganic C, mainly present as carbonate groups in the CFA lattice, was homogeneously distributed, whereas organic C was concentrated in the rims of the phosphatic biomorphs. Finally, STXM analyses at the Fe L 2,3 -edges together with TEM-EDXS analyses, revealed that some pyritized biomorphs experienced partial oxidation. The mineralogical features of these phosphatic biomorphs are very similar to those formed by bacteria having precipitated phosphate minerals intra- and extracellularly in laboratory experiments. Similarly, pyritized biomorphs resemble bacteria encrusted by pyrite. We therefore interpret phosphatic and pyritized biomorphs present in the Peruvian phosphorite crust as microorganisms fossilized near the boundary of zones of sulfate reduction. The implications of these observations are then discussed in the light of the different possible and non-exclusive microbially-driven phosphogenesis mechanisms that have been proposed in the past: (i) Organic matter mineralization, in particular mediated by iron reducing bacteria and/or sulfate-reducing bacteria (SRB), (ii) reduction of iron-(oxyhydr)oxides by iron-reducing bacteria and/or SRB, and (iii) polyphosphate metabolism in sulfide-oxidizing bacteria, possibly associated with SRB.
---
paper_title: Precipitation of Phosphate Minerals by Microorganisms Isolated from a Fixed-Biofilm Reactor Used for the Treatment of Domestic Wastewater

paper_content:

The ability of bacteria isolated from a fixed-film bioreactor to precipitate phosphate crystals for the treatment of domestic wastewater in both artificial and natural media was studied. When this was demonstrated in artificial solid media for crystal formation, precipitation took place rapidly, and crystal formation began 3 days after inoculation. The percentage of phosphate-forming bacteria was slightly higher than 75%. Twelve major colonies with phosphate precipitation capacity were the dominant heterotrophic platable bacteria growing aerobically in artificial media. According to their taxonomic affiliations (based on partial sequencing of the 16S rRNA), the 12 strains belonged to the following genera of Gram-negative bacteria: Rhodobacter, Pseudoxanthobacter, Escherichia, Alcaligenes, Roseobacter, Ochrobactrum, Agromyce, Sphingomonas and Paracoccus. The phylogenetic tree shows that most of the identified populations were evolutionarily related to the Alphaproteobacteria (91.66% of sequences). The minerals formed were studied by X-ray diffraction, scanning electron microscopy (SEM), and energy dispersive X-ray microanalysis (EDX). All of these strains formed phosphate crystals and precipitated struvite (MgNH4PO4·6H2O), bobierrite [Mg3(PO4)2·8H2O] and baricite [(MgFe)3(PO4)2·8H2O]. The results obtained in this study show that struvite and spherulite crystals did not show any cell marks. Moreover, phosphate precipitation was observed in the bacterial mass but also near the colonies. Our results suggest that the microbial population contributed to phosphate precipitation by changing the media as a consequence of their metabolic activity. Moreover, the results of this research suggest that bacteria play an active role in the mineral precipitation of soluble phosphate from urban wastewater in submerged fixed-film bioreactors.
---
paper_title: Accumulation of inorganic polyphosphates in Saccharomyces cerevisiae under nitrogen deprivation: Stimulation by magnesium ions and peculiarities of localization

paper_content:

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.
---
paper_title: Biological phosphorus removal from abattoir wastewater at very short sludge ages mediated by novel PAO clade Comamonadaceae.

paper_content:

Recent increases in global phosphorus costs, together with the need to remove phosphorus from wastewater to comply with water discharge regulations, make phosphorus recovery from wastewater economically and environmentally attractive. Biological phosphorus (Bio-P) removal process can effectively capture the phosphorus from wastewater and concentrate it in a form that is easily amendable for recovery in contrast to traditional (chemical) phosphorus removal processes. However, Bio-P removal processes have historically been operated at medium to long solids retention times (SRTs, 10-20 days typically), which inherently increases the energy consumption while reducing the recoverable carbon fraction and hence makes it incompatible with the drive towards energy self-sufficient wastewater treatment plants. In this study, a novel high-rate Bio-P removal process has been developed as an energy efficient alternative for phosphorus removal from wastewater through operation at an SRT of less than 4 days. The process was most effective at an SRT of 2-2.5 days, achieving >90% phosphate removal. Further reducing the SRT to 1.7 days resulted in a loss of Bio-P activity. 16S pyrotag sequencing showed the community changed considerably with changes in the SRT, but that Comamonadaceae was consistently abundant when the Bio-P activity was evident. FISH analysis combined with DAPI staining confirmed that bacterial cells of Comamonadaceae arranged in tetrads contained polyphosphate, identifying them as the key polyphosphate accumulating organisms at these low SRT conditions. Overall, this paper demonstrates a novel, high-rate phosphorus removal process that can be effectively integrated with short SRT, energy-efficient carbon removal and recovery processes.
---
paper_title: Methods for Detection and Quantification of Polyphosphate and Polyphosphate Accumulating Microorganisms in Aquatic Sediments

paper_content:

It has been speculated that the microbial P pool is highly variable in the uppermost layer of various aquatic sediments, especially when an excessive P accumulation in form of polyphosphate (Poly-P) occurs. Poly-P storage is a universal feature of many different organisms and has been technically optimised in wastewater treatment plants (WWTP) with enhanced biological phosphorus removal (EBPR). In the recent past, new insights into mechanisms of P elimination in WWTP almost exclusively depended on the development and application of novel methods like 31P-NMR spectroscopy and molecular methods for identifying Poly-P accumulating microorganisms (PAO). The aim of the present review is to compile current methods potentially available for detection and quantification of Poly-P in sediments and to complement it with yet unpublished results to validate their application in natural sediments. The most powerful tool for reliable Poly-P quantification in sediments is the liquid 31P-NMR technique which has been successfully used for Poly-P measurements in a variety of aquatic sediments. But the microorganisms as well as mechanisms involved in Poly-P storage and cycling are largely unknown. Therefore, we also intend to stimulate future studies focusing on these encouraging topics in sediment research via the implementation of novel methods. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
---
paper_title: Advances in techniques for phosphorus analysis in biological sources.

paper_content:

In general, conventional P analysis methods suffer from not only the fastidious extraction and pre-treatment procedures required but also the generally low specificity and poor resolution regarding the P composition and its temporal and spatial dynamics. More powerful yet feasible P analysis tools are in demand to help elucidating the biochemistry nature, roles and dynamics of various phosphorus-containing molecules in vitro and in vivo. Recent advances in analytical chemistry, especially in molecular and atomic spectrometry such as NMR, Raman and X-ray techniques, have enabled unique capability of P analysis relevant to submicron scale biochemical processes in individual cell and in natural samples without introducing too complex and invasive pretreatment steps. Great potential still remains to be explored in wider and more combined and integrated requests of these techniques to allow for new possibilities and more powerful P analysis in biological systems. This review provides a comprehensive summary of the available methods and recent developments in analytical techniques and their applications for characterization and quantification of various forms of phosphorus, particularly polyphosphate, in different biological sources.
---
paper_title: Identification of functionally relevant populations in enhanced biological phosphorus removal processes based on intracellular polymers profiles and insights into the metabolic diversity and heterogeneity.

paper_content:

This study proposed and demonstrated the application of a new Raman microscopy-based method for metabolic state-based identification and quantification of functionally relevant populations, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), in enhanced biological phosphorus removal (EBPR) system via simultaneous detection of multiple intracellular polymers including polyphosphate (polyP), glycogen, and polyhydroxybutyrate (PHB). The unique Raman spectrum of different combinations of intracellular polymers within a cell at a given stage of the EBPR cycle allowed for its identification as PAO, GAO, or neither. The abundance of total PAOs and GAOs determined by Raman method were consistent with those obtained with polyP staining and fluorescence in situ hybridization (FISH). Different combinations and quantities of intracellular polymer inclusions observed in single cells revealed the distribution of different sub-PAOs groups among the total PAO populations, which exhibit phenotypic and metabolic heterogeneity and diversity. These results also provided evidence for the hypothesis that different PAOs may employ different extents of combination of glycolysis and TCA cycle pathways for anaerobic reducing power and energy generation and it is possible that some PAOs may rely on TCA cycle solely without glycolysis. Sum of cellular level quantification of the internal polymers associated with different population groups showed differentiated and distributed trends of glycogen and PHB level between PAOs and GAOs, which could not be elucidated before with conventional bulk measurements of EBPR mixed cultures.
---
paper_title: Copper ions stimulate polyphosphate degradation and phosphate efflux in Acidithiobacillus ferrooxidans.

paper_content:

For some bacteria and algae, it has been proposed that inorganic polyphosphates and transport of metal-phosphate complexes could participate in heavy metal tolerance. To test for this possibility in Acidithiobacillus ferrooxidans, a microorganism with a high level of resistance to heavy metals, the polyphosphate levels were determined when the bacterium was grown in or shifted to the presence of a high copper concentration (100 mM). Under these conditions, cells showed a rapid decrease in polyphosphate levels with a concomitant increase in exopolyphosphatase activity and a stimulation of phosphate efflux. Copper in the range of 1 to 2 μM greatly stimulated exopolyphosphatase activity in cell extracts from A. ferrooxidans. The same was seen to a lesser extent with cadmium and zinc. Bioinformatic analysis of the available A. ferrooxidans ATCC 23270 genomic sequence did not show a putative pit gene for phosphate efflux but rather an open reading frame similar in primary and secondary structure to that of the Saccharomyces cerevisiae phosphate transporter that is functional at acidic pH (Pho84). Our results support a model for metal detoxification in which heavy metals stimulate polyphosphate hydrolysis and the metal-phosphate complexes formed are transported out of the cell as part of a possibly functional heavy metal tolerance mechanism in A. ferrooxidans.
---
paper_title: Microscopy evidence of bacterial microfossils in phosphorite crusts of the Peruvian shelf: Implications for phosphogenesis mechanisms

paper_content:

Abstract Phosphorites are sedimentary formations enriched in Ca-phosphate minerals. The precipitation of these minerals is thought to be partly mediated by the activity of microorganisms. The vast majority of studies on phosphorites have focused on a petrological and geochemical characterization of these rocks. However, detailed descriptions are needed at the sub-micrometer scale at which crucial information can be retrieved about traces of past or modern microbial activities. Here, scanning electron microscopy (SEM) analyses of a recent phosphorite crust from the upwelling-style phosphogenesis area off Peru revealed that it contained a great number of rod-like and coccus-like shaped micrometer-sized (~ 1.1 μm and 0.5 μm, respectively) objects, referred to as biomorphs. Some of these biomorphs were filled with carbonate fluoroapatite (CFA, a calcium-phosphate phase common in phosphorites); some were empty; some were surrounded by one or two layers of pyrite. Transmission electron microscopy (TEM) and energy dispersive X-ray spectrometry (EDXS) analyses were performed on focused ion beam (FIB) milled ultrathin foils to characterize the texture of CFA and pyrite in these biomorphs at the few nanometer scale. Non-pyritized phosphatic biomorphs were surrounded by a thin (5–15 nm thick) rim appearing as a void on TEM images. Bundles of CFA crystals sharing the same crystallographic orientations (aligned along their c -axis) were found in the interior of some biomorphs. Pyrite formed a thick (~ 35–115 nm) layer with closely packed crystals surrounding the pyritized biomorphs, whereas pyrite crystals at distance from the biomorphs were smaller and distributed more sparsely. Scanning transmission X-ray microscopy (STXM) analyses performed at the C K-edge provided maps of organic and inorganic carbon in the samples. Inorganic C, mainly present as carbonate groups in the CFA lattice, was homogeneously distributed, whereas organic C was concentrated in the rims of the phosphatic biomorphs. Finally, STXM analyses at the Fe L 2,3 -edges together with TEM-EDXS analyses, revealed that some pyritized biomorphs experienced partial oxidation. The mineralogical features of these phosphatic biomorphs are very similar to those formed by bacteria having precipitated phosphate minerals intra- and extracellularly in laboratory experiments. Similarly, pyritized biomorphs resemble bacteria encrusted by pyrite. We therefore interpret phosphatic and pyritized biomorphs present in the Peruvian phosphorite crust as microorganisms fossilized near the boundary of zones of sulfate reduction. The implications of these observations are then discussed in the light of the different possible and non-exclusive microbially-driven phosphogenesis mechanisms that have been proposed in the past: (i) Organic matter mineralization, in particular mediated by iron reducing bacteria and/or sulfate-reducing bacteria (SRB), (ii) reduction of iron-(oxyhydr)oxides by iron-reducing bacteria and/or SRB, and (iii) polyphosphate metabolism in sulfide-oxidizing bacteria, possibly associated with SRB.
---
paper_title: Precipitation of Phosphate Minerals by Microorganisms Isolated from a Fixed-Biofilm Reactor Used for the Treatment of Domestic Wastewater

paper_content:

The ability of bacteria isolated from a fixed-film bioreactor to precipitate phosphate crystals for the treatment of domestic wastewater in both artificial and natural media was studied. When this was demonstrated in artificial solid media for crystal formation, precipitation took place rapidly, and crystal formation began 3 days after inoculation. The percentage of phosphate-forming bacteria was slightly higher than 75%. Twelve major colonies with phosphate precipitation capacity were the dominant heterotrophic platable bacteria growing aerobically in artificial media. According to their taxonomic affiliations (based on partial sequencing of the 16S rRNA), the 12 strains belonged to the following genera of Gram-negative bacteria: Rhodobacter, Pseudoxanthobacter, Escherichia, Alcaligenes, Roseobacter, Ochrobactrum, Agromyce, Sphingomonas and Paracoccus. The phylogenetic tree shows that most of the identified populations were evolutionarily related to the Alphaproteobacteria (91.66% of sequences). The minerals formed were studied by X-ray diffraction, scanning electron microscopy (SEM), and energy dispersive X-ray microanalysis (EDX). All of these strains formed phosphate crystals and precipitated struvite (MgNH4PO4·6H2O), bobierrite [Mg3(PO4)2·8H2O] and baricite [(MgFe)3(PO4)2·8H2O]. The results obtained in this study show that struvite and spherulite crystals did not show any cell marks. Moreover, phosphate precipitation was observed in the bacterial mass but also near the colonies. Our results suggest that the microbial population contributed to phosphate precipitation by changing the media as a consequence of their metabolic activity. Moreover, the results of this research suggest that bacteria play an active role in the mineral precipitation of soluble phosphate from urban wastewater in submerged fixed-film bioreactors.
---
paper_title: Inorganic polyphosphate: essential for growth and survival.

paper_content:

Inorganic polyphosphate (Poly P) is a polymer of tens to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds as in ATP. Found in abundance in all cells in nature, it is unique in its likely role in the origin and survival of species. Here, we present extensive evidence that the remarkable properties of Poly P as a polyanion have made it suited for a crucial role in the emergence of cells on earth. Beyond that, Poly P has proved in a variety of ways to be essential for growth of cells, their responses to stresses and stringencies, and the virulence of pathogens. In this review, we pay particular attention to the enzyme, polyphosphate kinase 1 (Poly P kinase 1 or PPK1), responsible for Poly P synthesis and highly conserved in many bacterial species, including 20 or more of the major pathogens. Mutants lacking PPK1 are defective in motility, quorum sensing, biofilm formation, and virulence. Structural studies are cited that reveal the conserved ATP-binding site of PPK1 at atomic resolution and reveal that the site can be blocked with minute concentrations of designed inhibitors. Another widely conserved enzyme is PPK2, which has distinctive kinetic properties and is also implicated in the virulence of some pathogens. Thus, these enzymes, absent in yeast and animals, are novel attractive targets for treatment of many microbial diseases. Still another enzyme featured in this review is one discovered in Dictyostelium discoideum that becomes an actin-like fiber concurrent with the synthesis, step by step, of a Poly P chain made from ATP. The Poly P-actin fiber complex, localized in the cell, lengthens and recedes in response to metabolic signals. Homologs of DdPPK2 are found in pathogenic protozoa and in the alga Chlamydomonas. Beyond the immediate relevance of Poly P as a target for anti-infective drugs, a large variety of cellular operations that rely on Poly P will be considered.
---
paper_title: Advances in techniques for phosphorus analysis in biological sources.

paper_content:

In general, conventional P analysis methods suffer from not only the fastidious extraction and pre-treatment procedures required but also the generally low specificity and poor resolution regarding the P composition and its temporal and spatial dynamics. More powerful yet feasible P analysis tools are in demand to help elucidating the biochemistry nature, roles and dynamics of various phosphorus-containing molecules in vitro and in vivo. Recent advances in analytical chemistry, especially in molecular and atomic spectrometry such as NMR, Raman and X-ray techniques, have enabled unique capability of P analysis relevant to submicron scale biochemical processes in individual cell and in natural samples without introducing too complex and invasive pretreatment steps. Great potential still remains to be explored in wider and more combined and integrated requests of these techniques to allow for new possibilities and more powerful P analysis in biological systems. This review provides a comprehensive summary of the available methods and recent developments in analytical techniques and their applications for characterization and quantification of various forms of phosphorus, particularly polyphosphate, in different biological sources.
---
paper_title: Recovery of phosphorus from dairy manure: a pilot-scale study.

paper_content:

Phosphorus was recovered from dairy manure via a microwave-enhanced advanced oxidation process (MW/H2O2-AOP) followed by struvite crystallization in a pilot-scale continuous flow operation. Soluble phosphorus in dairy manure increased by over 50% after the MW/H2O2-AOP, and the settleability of suspended solids was greatly improved. More than 50% of clear supernatant was obtained after microwave treatment, and the maximum volume of supernatant was obtained at a hydrogen peroxide dosage of 0.3% and pH 3.5. By adding oxalic acid into the supernatant, about 90% of calcium was removed, while more than 90% of magnesium was retained. As a result, the resulting solution was well suited for struvite crystallization. Nearly 95% of phosphorus in the treated supernatant was removed and recovered as struvite.
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paper_title: Determination of carbon, carbonate, nitrogen, and phosphorus in freshwater sediments by near-infrared reflectance spectroscopy: Rapid analysis and a check on conventional analytical methods

paper_content:

Sediments are typically analyzed for C, N, and P for characterization, sediment quality assessment, and in nutrient and contaminant studies. Cost and time required for analysis of these constituents by conventional chemical techniques can be limiting factors in these studies. Determination of these constituents by near-infrared reflectance spectroscopy (NIRS) may be a rapid, cost-effective method provided the technology can be applied generally across aquatic ecosystems. In this study, we explored the feasibility of using NIRS to predict total C, CO3−2 organic C, N, and P in deep-water sediment cores from four Canadian lakes varying over 19 degrees of latitude. Concentration ranges of constituents in the samples (dry weight basis) were total C, 12-55; CO3−2, 6-26; organic C, 7-31; N, 0.6-3.1; and P, 0.22-2.1 mg g−1. Coefficients of determination, r2, between results from conventional chemical analysis and NIR-predicted concentrations, based on calibrations across all the four lakes, were 0.97-0.99 for total C, organic C, and N. Prediction for CO3−2 was good for the hard water lake from a calibration across all four lakes, but this constituent in the three soft water lakes was better predicted by a calibration across the soft water lakes. The NIR calibration for P fell below acceptable levels for the technique, but proved useful in the identification of outliers from the chemical method that were later removed with the re-analysis of several samples. This study demonstrated that NIRS is useful for rapid, simultaneous, cost-effective analysis of total C, CO3−2, organic C, N, and P in dried sediments from lakes at widely varying latitudes. Also, this study showed that NIRS is an independent analytical tool useful for the identification of outliers that may be due to error during the analysis or to distinctive composition of the samples.
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paper_title: Phosphorus recovery from wastewater through microbial processes

paper_content:

Waste streams offer a compelling opportunity to recover phosphorus (P). 15–20% of world demand for phosphate rock could theoretically be satisfied by recovering phosphorus from domestic waste streams alone. For very dilute streams ( −1 ), including domestic wastewater, it is necessary to concentrate phosphorus in order to make recovery and reuse feasible. This review discusses enhanced biological phosphorus removal (EBPR) as a key technology to achieve this. EBPR relies on polyphosphate accumulating organisms (PAOs) to take up phosphorus from waste streams, so concentrating phosphorus in biomass. The P-rich biosolids can be either directly applied to land, or solubilized and phosphorus recovered as a mineral product. Direct application is effective, but the product is bulky and carries contaminant risks that need to be managed. Phosphorus release can be achieved using either thermochemical or biochemical methods, while recovery is generally by precipitation as struvite. We conclude that while EBPR technology is mature, the subsequent phosphorus release and recovery technologies need additional development.
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paper_title: Evaluation of Intracellular Polyphosphate Dynamics in Enhanced Biological Phosphorus Removal Process using Raman Microscopy

paper_content:

A Raman microscopy method was developed and successfully applied to evaluate the dynamics of intracellular polyphosphate in polyphosphate-accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) processes. Distinctive Raman spectra of polyphosphates allowed for both identification of PAOs and quantification of intracellular polyphosphate during various metabolic phases in a lab-scale EBPR process. Observation of polyphosphate at individual cell level indicated that there are distributed states of cells in terms of polyphosphate content at any given time, suggesting that agent-based distributive modeling would more accurately reflect the behavior of an EBPR process than the traditional average-state based modeling. The results, for the first time, showed that the polyphosphate depletion or replenishment observed at the overall population level were collective results from shifts/transition in the distribution of abundance of PAOs with different amounts of polyphosphate inclusions duri...
---
paper_title: Single-cell analysis of bacteria by Raman microscopy: spectral information on the chemical composition of cells and on the heterogeneity in a culture

paper_content:

In the acetone–butanol (ABE) fermentation process, the utilised organisms from the group of the solventogenic Clostridia go through a complex cell-cycle. The role of different cell types in product formation is not understood in detail yet. We aim to use Raman spectroscopy to characterise the population distribution in Clostridium cultures. Cell suspensions were dried on calcium fluoride carriers. Raman spectra of single cells were obtained using a confocal Raman microscope (Dilor, Lille, France). The laser beam was focused on individual cells through the microscope objective. Spectra with good signal-to-noise ratio were obtained. Cells of different morphology, but also apparently similar cells, showed different spectra. Several cell components could be detected and varied in quantity. Compared to other methods for single-cell analysis, the new method is much more time-consuming to analyse one individual cell. However, a large amount of chemical information is obtained from each single cell in a non-destructive, non-invasive way. Raman microscopy appears to be a suitable method for studying population distributions in bacterial cultures.
---
paper_title: Advances in techniques for phosphorus analysis in biological sources.

paper_content:

In general, conventional P analysis methods suffer from not only the fastidious extraction and pre-treatment procedures required but also the generally low specificity and poor resolution regarding the P composition and its temporal and spatial dynamics. More powerful yet feasible P analysis tools are in demand to help elucidating the biochemistry nature, roles and dynamics of various phosphorus-containing molecules in vitro and in vivo. Recent advances in analytical chemistry, especially in molecular and atomic spectrometry such as NMR, Raman and X-ray techniques, have enabled unique capability of P analysis relevant to submicron scale biochemical processes in individual cell and in natural samples without introducing too complex and invasive pretreatment steps. Great potential still remains to be explored in wider and more combined and integrated requests of these techniques to allow for new possibilities and more powerful P analysis in biological systems. This review provides a comprehensive summary of the available methods and recent developments in analytical techniques and their applications for characterization and quantification of various forms of phosphorus, particularly polyphosphate, in different biological sources.
---
paper_title: Identification of functionally relevant populations in enhanced biological phosphorus removal processes based on intracellular polymers profiles and insights into the metabolic diversity and heterogeneity.

paper_content:

This study proposed and demonstrated the application of a new Raman microscopy-based method for metabolic state-based identification and quantification of functionally relevant populations, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), in enhanced biological phosphorus removal (EBPR) system via simultaneous detection of multiple intracellular polymers including polyphosphate (polyP), glycogen, and polyhydroxybutyrate (PHB). The unique Raman spectrum of different combinations of intracellular polymers within a cell at a given stage of the EBPR cycle allowed for its identification as PAO, GAO, or neither. The abundance of total PAOs and GAOs determined by Raman method were consistent with those obtained with polyP staining and fluorescence in situ hybridization (FISH). Different combinations and quantities of intracellular polymer inclusions observed in single cells revealed the distribution of different sub-PAOs groups among the total PAO populations, which exhibit phenotypic and metabolic heterogeneity and diversity. These results also provided evidence for the hypothesis that different PAOs may employ different extents of combination of glycolysis and TCA cycle pathways for anaerobic reducing power and energy generation and it is possible that some PAOs may rely on TCA cycle solely without glycolysis. Sum of cellular level quantification of the internal polymers associated with different population groups showed differentiated and distributed trends of glycogen and PHB level between PAOs and GAOs, which could not be elucidated before with conventional bulk measurements of EBPR mixed cultures.
---
paper_title: Application of Raman Microscopy for Simultaneous and Quantitative Evaluation of Multiple Intracellular Polymers Dynamics Functionally Relevant to Enhanced Biological Phosphorus Removal Processes

paper_content:

Polyphosphate (poly-P), polyhydroxyalkanoates (PHAs), and glycogen are the key functionally relevant intracellular polymers involved in the enhanced biological phosphorus removal (EBPR) process. Further understanding of the mechanisms of EBPR has been hampered by the lack of cellular level quantification tools to accurately measure the dynamics of these polymers during the EBPR process. In this study, we developed a novel Raman microscopy method for simultaneous identification and quantification of poly-P, PHB, and glycogen abundance in each individual cell and their distribution among the populations in EBPR. Validation of the method was demonstrated via a batch phosphorus uptake and release test, in which the total intracellular polymers abundance determined via Raman approach correlated well with those measured via conventional bulk chemical analysis (correlation coefficient r = 0.8 for poly-P, r = 0.94 for PHB, and r = 0.7 for glycogen). Raman results, for the first time, clearly showed the distributions of microbial cells containing different abundance levels of the three intracellular polymers under the same environmental conditions (at a given time point), indicating population heterogeneity exists. The results revealed the intracellular distribution and dynamics of the functionally relevant polymers in different metabolic stages of the EBPR process and elucidated the association of cellular metabolic state with the fate of these polymers during various substrates availability conditions.
---
paper_title: Inorganic polyphosphate: essential for growth and survival.

paper_content:

Inorganic polyphosphate (Poly P) is a polymer of tens to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds as in ATP. Found in abundance in all cells in nature, it is unique in its likely role in the origin and survival of species. Here, we present extensive evidence that the remarkable properties of Poly P as a polyanion have made it suited for a crucial role in the emergence of cells on earth. Beyond that, Poly P has proved in a variety of ways to be essential for growth of cells, their responses to stresses and stringencies, and the virulence of pathogens. In this review, we pay particular attention to the enzyme, polyphosphate kinase 1 (Poly P kinase 1 or PPK1), responsible for Poly P synthesis and highly conserved in many bacterial species, including 20 or more of the major pathogens. Mutants lacking PPK1 are defective in motility, quorum sensing, biofilm formation, and virulence. Structural studies are cited that reveal the conserved ATP-binding site of PPK1 at atomic resolution and reveal that the site can be blocked with minute concentrations of designed inhibitors. Another widely conserved enzyme is PPK2, which has distinctive kinetic properties and is also implicated in the virulence of some pathogens. Thus, these enzymes, absent in yeast and animals, are novel attractive targets for treatment of many microbial diseases. Still another enzyme featured in this review is one discovered in Dictyostelium discoideum that becomes an actin-like fiber concurrent with the synthesis, step by step, of a Poly P chain made from ATP. The Poly P-actin fiber complex, localized in the cell, lengthens and recedes in response to metabolic signals. Homologs of DdPPK2 are found in pathogenic protozoa and in the alga Chlamydomonas. Beyond the immediate relevance of Poly P as a target for anti-infective drugs, a large variety of cellular operations that rely on Poly P will be considered.
---
paper_title: Methods for Detection and Quantification of Polyphosphate and Polyphosphate Accumulating Microorganisms in Aquatic Sediments

paper_content:

It has been speculated that the microbial P pool is highly variable in the uppermost layer of various aquatic sediments, especially when an excessive P accumulation in form of polyphosphate (Poly-P) occurs. Poly-P storage is a universal feature of many different organisms and has been technically optimised in wastewater treatment plants (WWTP) with enhanced biological phosphorus removal (EBPR). In the recent past, new insights into mechanisms of P elimination in WWTP almost exclusively depended on the development and application of novel methods like 31P-NMR spectroscopy and molecular methods for identifying Poly-P accumulating microorganisms (PAO). The aim of the present review is to compile current methods potentially available for detection and quantification of Poly-P in sediments and to complement it with yet unpublished results to validate their application in natural sediments. The most powerful tool for reliable Poly-P quantification in sediments is the liquid 31P-NMR technique which has been successfully used for Poly-P measurements in a variety of aquatic sediments. But the microorganisms as well as mechanisms involved in Poly-P storage and cycling are largely unknown. Therefore, we also intend to stimulate future studies focusing on these encouraging topics in sediment research via the implementation of novel methods. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
---
paper_title: Copper ions stimulate polyphosphate degradation and phosphate efflux in Acidithiobacillus ferrooxidans.

paper_content:

For some bacteria and algae, it has been proposed that inorganic polyphosphates and transport of metal-phosphate complexes could participate in heavy metal tolerance. To test for this possibility in Acidithiobacillus ferrooxidans, a microorganism with a high level of resistance to heavy metals, the polyphosphate levels were determined when the bacterium was grown in or shifted to the presence of a high copper concentration (100 mM). Under these conditions, cells showed a rapid decrease in polyphosphate levels with a concomitant increase in exopolyphosphatase activity and a stimulation of phosphate efflux. Copper in the range of 1 to 2 μM greatly stimulated exopolyphosphatase activity in cell extracts from A. ferrooxidans. The same was seen to a lesser extent with cadmium and zinc. Bioinformatic analysis of the available A. ferrooxidans ATCC 23270 genomic sequence did not show a putative pit gene for phosphate efflux but rather an open reading frame similar in primary and secondary structure to that of the Saccharomyces cerevisiae phosphate transporter that is functional at acidic pH (Pho84). Our results support a model for metal detoxification in which heavy metals stimulate polyphosphate hydrolysis and the metal-phosphate complexes formed are transported out of the cell as part of a possibly functional heavy metal tolerance mechanism in A. ferrooxidans.
---
paper_title: Characterization of intact subcellular bodies in whole bacteria by cryo-electron tomography and spectroscopic imaging.

paper_content:

We illustrate the combined use of cryo-electron tomography and spectroscopic difference imaging in the study of subcellular structure and subcellular bodies in whole bacteria. We limited our goal and focus to bodies with a distinct elemental composition that was in a sufficiently high concentration to provide the necessary signal-to-noise level at the relatively large sample thicknesses of the intact cell. This combination proved very powerful, as demonstrated by the identification of a phosphorus-rich body in Caulobacter crescentus. We also confirmed the presence of a body rich in carbon, demonstrated that these two types of bodies are readily recognized and distinguished from each other, and provided, for the first time to our knowledge, structural information about them in their intact state. In addition, we also showed the presence of a similar type of phosphorus-rich body in Deinococcus grandis, a member of a completely unrelated bacteria genus. Cryo-electron microscopy and tomography allowed the study of the biogenesis and morphology of these bodies at resolutions better than 10 nm, whereas spectroscopic difference imaging provided a direct identification of their chemical composition.
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paper_title: Inorganic polyphosphate: essential for growth and survival.

paper_content:

Inorganic polyphosphate (Poly P) is a polymer of tens to hundreds of phosphate residues linked by "high-energy" phosphoanhydride bonds as in ATP. Found in abundance in all cells in nature, it is unique in its likely role in the origin and survival of species. Here, we present extensive evidence that the remarkable properties of Poly P as a polyanion have made it suited for a crucial role in the emergence of cells on earth. Beyond that, Poly P has proved in a variety of ways to be essential for growth of cells, their responses to stresses and stringencies, and the virulence of pathogens. In this review, we pay particular attention to the enzyme, polyphosphate kinase 1 (Poly P kinase 1 or PPK1), responsible for Poly P synthesis and highly conserved in many bacterial species, including 20 or more of the major pathogens. Mutants lacking PPK1 are defective in motility, quorum sensing, biofilm formation, and virulence. Structural studies are cited that reveal the conserved ATP-binding site of PPK1 at atomic resolution and reveal that the site can be blocked with minute concentrations of designed inhibitors. Another widely conserved enzyme is PPK2, which has distinctive kinetic properties and is also implicated in the virulence of some pathogens. Thus, these enzymes, absent in yeast and animals, are novel attractive targets for treatment of many microbial diseases. Still another enzyme featured in this review is one discovered in Dictyostelium discoideum that becomes an actin-like fiber concurrent with the synthesis, step by step, of a Poly P chain made from ATP. The Poly P-actin fiber complex, localized in the cell, lengthens and recedes in response to metabolic signals. Homologs of DdPPK2 are found in pathogenic protozoa and in the alga Chlamydomonas. Beyond the immediate relevance of Poly P as a target for anti-infective drugs, a large variety of cellular operations that rely on Poly P will be considered.
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paper_title: Direct Labeling of Polyphosphate at the Ultrastructural Level in Saccharomyces cerevisiae by Using the Affinity of the Polyphosphate Binding Domain of Escherichia coli Exopolyphosphatase

paper_content:

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate and has many biological functions in prokaryotic and eukaryotic organisms. To investigate polyP localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to polyP. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain polyP and its weak affinity for short-chain polyP and nucleic acids. To directly demonstrate polyP localization in Saccharomyces cerevisiae on resin sections prepared by rapid freezing and freeze-substitution, specimens were labeled with PPBD containing an epitope tag and then the epitope tag was detected by an indirect immunocytochemical method. A goat anti-mouse immunoglobulin G antibody conjugated with Alexa 488 for laser confocal microscopy or with colloidal gold for transmission electron microscopy was used. When the S. cerevisiae was cultured in yeast extract-peptone-dextrose medium (10 mM phosphate) for 10 h, polyP was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few polyP signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions, polyP granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. The method directly demonstrated the localization of polyP at the electron microscopic level for the first time and enabled the visualization of polyP localization with much higher specificity and resolution than with other conventional methods.
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paper_title: Polyphosphate kinase from activated sludge performing enhanced biological phosphorus removal

paper_content:

A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like beta-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg(2+), and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.
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paper_title: Radiolabelled proteomics to determine differential functioning ofAccumulibacterduring the anaerobic and aerobic phases of a bioreactor operating for enhanced biological phosphorus removal

paper_content:

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.
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paper_title: Metaproteomics Provides Functional Insight into Activated Sludge Wastewater Treatment

paper_content:

BACKGROUND ::: Through identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR). ::: ::: ::: METHODOLOGY/PRINCIPAL FINDINGS ::: A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism "Candidatus Accumulibacter phosphatis". When EBPR failed, the sludge was dominated by tetrad-forming alpha-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from "Candidatus Accumulibacter phosphatis" and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid beta oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected. ::: ::: ::: CONCLUSIONS/SIGNIFICANCE ::: Importantly, this study provides direct evidence linking the metabolic activities of "Accumulibacter" to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models.
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Title: Characterisation of Phosphate Accumulating Organisms and Techniques for Polyphosphate Detection: A Review

Section 1: Introduction
Description 1: Outline the importance of phosphorus in food production, current sources of phosphorus, geopolitical considerations, and the rationale for researching phosphate-accumulating organisms (PAOs) and polyphosphate (Poly-P) detection techniques.

Section 2: Isolation of PAOs
Description 2: Discuss the various forms of phosphate accumulation, the isolation attempts of PAOs, and the conditions affecting phosphate uptake and release in different microorganisms.

Section 3: Poly-P Detection and Identification of PAOs
Description 3: Provide an overview of the methods used to identify and quantify Poly-P in microbial cells, focusing on different detection techniques and the specific information they offer.

Section 4: Staining Techniques-Light and Epifluorescence Microscopy (LEM)
Description 4: Describe the staining techniques used for detecting Poly-P through light and epifluorescence microscopy, including the specific dyes and protocols used.

Section 5: Flow Cytometry (FC)
Description 5: Detail the application of flow cytometry in Poly-P detection, including the staining procedures, data acquisition, analysis, and separation of cells.

Section 6: Fluorescence in Situ Hybridization (FISH) Analysis
Description 6: Explain the use of FISH in identifying PAOs, studying gene expression, and targeting specific bacterial clades in environmental samples.

Section 7: Extraction Procedures and Polyphosphate Quantification (EXT)
Description 7: Summarize the methods for extracting Poly-P from microbial cells and the subsequent quantification techniques.

Section 8: Polyacrylamide Gel Electrophoresis (PAGE)
Description 8: Discuss the use of gel electrophoresis in determining the degree of polymerization of Poly-P, including extraction and staining methods.

Section 9: Electron Microscopy (EM)
Description 9: Explain the role of electron microscopy in visualizing Poly-P granules in cells and its combination with other analytical techniques.

Section 10: X-Ray Analysis Techniques (X-RAY)
Description 10: Describe how X-ray analysis provides information about the composition of Poly-P granules and its application in combination with microscopy.

Section 11: Nuclear Magnetic Resonance Spectroscopy (NMRS)
Description 11: Outline the use of NMR spectroscopy to study the physiology of PAOs and the presence of Poly-P in different sample types.

Section 12: RAMAN Spectromicroscopy (RAM)
Description 12: Detail the application of RAMAN spectromicroscopy in identifying and quantifying Poly-P within individual cells and examining microbiota composition.

Section 13: Enzyme Assays (EA)
Description 13: Discuss enzyme-based methods for detecting Poly-P, highlighting the enzymatic reactions and the analysis of resulting products.

Section 14: Cryoelectron Tomography and Spectroscopic Imaging (CTSI)
Description 14: Describe the technique of cryoelectron tomography combined with spectroscopic imaging for studying the structure of PAOs and Poly-P.

Section 15: Mass Spectrometry (MS)
Description 15: Provide an overview of the use of mass spectrometry in analyzing Poly-P and related compounds in microbial samples.

Section 16: Proteic Affinity (PA)
Description 16: Explain the technique based on protein affinity for detecting and locating Poly-P granules within cells.

Section 17: "Omics" Techniques (OMICS)
Description 17: Summarize the use of metagenomics, metatranscriptomics, and metaproteomics in studying PAOs and their roles in the EBPR process.

Section 18: Conclusions
Description 18: Highlight the diversity of techniques available for isolating and detecting Poly-P, the need for further research on PAO culture, and potential alternative approaches to phosphate recovery.