Intracellular Polyphosphate Granules Research Articles

Characterization of the intracellular polyphosphate granules of the phototrophic green sulfur bacterium Chlorobaculum tepidum

The ability to generate polyphosphate (polyP) granules is important for survival for bacteria during resistance to diverse environmental stresses, however the genesis of polyP granules is poorly understood. Chlorobaculum tepidum (Cba tepidum) is a thermophilic green sulfur anoxygenic phototrophic bacterium which uses reduced sulfur compounds as electron donors. The presence of electron rich granules inside the Cba tepidum was reported, but no further information was provided. In this work we used cell thin sections at three different time points of cultivation to observe the biogenesis of the inclusions over time, and the in cell total phosphate concentration was monitored over time as well. Furthermore, the elemental analysis (EDS) of the electron rich inclusions showed the presence of phosphorus and oxygen. The existence of polyphosphate was demonstrated by 31P NMR spectroscopy of cell lysates. Finally, we show that the biogenesis of the phosphorus granules correlates with an abundance of proteins that are closely related to polyphosphate metabolism.

АДАПТАЦИЯ МИКРООРГАНИЗМОВ К ТЯЖЕЛЫМ МЕТАЛЛАМ

Many heavy metals (HM) (Zn, Cu, Mn, Co, etc.) take an active part in the most important processes of vital activity of microorganisms as microelements. However, at high concentrations they become toxic, and a number of metals (Pb, Hg, Cd, etc.) are highly toxic even at low concentrations. Microorganisms are able to resist the toxic effects of HMs due to the presence of various resistance mechanisms that are aimed at converting cations to a less toxic form or oxidation state, which makes them less mobile and bioavailable. The very first reaction of microorganisms to the toxic effects of metals is a change in cell morphology, their agglomeration, which leads to a decrease in the availability of binding sites for toxic metals. The mechanisms used by bacteria can be divided into biochemical and molecular. Bacterial cells have the ability to sorb metal cations with the help of metal-binding functional groups (carboxylic, sulfhydryl, hydroxyl, sulfate, phosphate, and amino groups) of the cell membrane,preventing their penetration into the cell. Bacteria have a variety of efflux systems for HM outflow from cells with the help of carrier proteins belonging to different families, which maintain a low concentration of HM inside the cell, protecting cellular components. Polysaccharides, biosurfactants, inorganic anions (phosphate, carbonate, and sulfide ions) and other metabolic products of microorganisms participate in extracellular detoxification, and gluta- thione, metal-binding proteins, intracellular polyphosphate granules, which bind HM cations into poorly soluble compounds, participate in intracellular sequestration. The reduction of HM ions with the help of enzymes leads to the formation of their less toxic forms. The genes responsible for bacterial resistance to toxic metals are localized on chromosomes or plasmids and can be transferred to closely related bacterial species, which plays an important role in the spread of HM resistance in nature. Microorganisms also demonstrate indirect mechanisms of HM tolerance aimed at maintaining cell integrity by protecting them from oxidative stress.

Influence of Uranium Concentration and pH on U-Phosphate Biomineralization by Caulobacter OR37

Uranium contamination of soils and groundwater in the United States represents a significant health risk and will require multiple remediation approaches. Microbial phosphatase activity coupled to the addition of an organic P source has recently been studied as a remediation strategy that provides an extended release of inorganic P (Pi) into U-contaminated sites, resulting in the precipitation of meta-autunite minerals. Previous laboratory- and field-based biomineralization studies have investigated environments with relatively high U concentrations (>20 μM). However, most contaminated sites have much lower U concentrations (<2 μM). The Environmental Protection Agency (EPA) limit for U in drinking water is 0.126 μM. Reaching this regulatory limit becomes challenging as U concentrations approach autunite solubility. We studied the precipitation of U(VI)-phosphate minerals by an environmental isolate of Caulobacter sp. (strain OR37) from an Oak Ridge, Tennessee, U-contaminated site. Abiotic U(VI) solubility experiments reveal that U(VI)-phosphate minerals do not form in the presence of excess Pi (500 μM) when U(VI) concentrations are <1 μM and pH is <5. When OR37 cells are reacted under the same conditions with Pi or glycerol-2-phosphate, U(VI)-phosphate mineral formation was observed, along with the formation of intracellular polyphosphate granules. These results show that bacteria provide supersaturated microenvironments needed for U(VI)-phosphate mineralization while hydrolyzing organic P sources. This provides a pathway to lower U concentrations to below EPA limits for drinking water.

Fluviibacter phosphoraccumulans gen. nov., sp. nov., a polyphosphate-accumulating bacterium of Fluviibacteraceae fam. nov., isolated from surface river water.

Three aerobic, Gram-stain-negative, non-motile, rod-shaped bacteria, designated as strains SHINM1T, ICHIJ1 and ICHIAU1, were isolated from surface river water (Saitama Prefecture, Japan). Phylogenetic analyses based on 16S rRNA and 40 marker gene sequences revealed that the strains formed a distinct phylogenetic lineage within the order Rhodocyclales. The three strains shared 100 % 16S rRNA gene similarity. Growth occurred at 15-30 °C and pH 6.0-9.5, but not in the presence of ≥1.0 % (w/v) NaCl. The isolates stained positive for intracellular polyphosphate granules. The major cellular fatty acids were C16 : 0, summed feature 2 (C12 : 1 aldehyde and/or iso-C16 : 1 I and/or C14 : 0 3-OH), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The major polar lipids were phosphatidylethanolamine and an unidentified phospholipid. The predominant quinone system of strain SHINM1T was ubiquinone-8 and its DNA G+C content was 56.7 mol%. Genome sequencing of the three isolates revealed a genome size of 2.29-2.43 Mbp and average nucleotide identity by orthology values of ≥98.9 %. Based on the results of phenotypic and phylogenetic analyses, strains SHINM1T, ICHIJ1 and ICHIAU1 represent a novel species of a new genus, for which the name Fluviibacter phosphoraccumulans gen. nov., sp. nov. is proposed, within a new family, Fluviibacteraceae fam. nov. of the order Rhodocyclales. The type strain is SHINM1T (=JCM 32071T=NCIMB 15105T).

Characteristics of intracellular polyphosphate granules and phosphorus-absorption of a marine polyphosphate-accumulating bacterium, Halomonas sp. YSR-3

Halomonas sp. YSR-3 was isolated from the Yellow Sea and identified as a polyphosphate-accumulating bacterium and the characteristics of its intracellular polyphosphate (polyP) granules and phosphorus absorption were studied. Most YSR-3 cells stored one or two polyP granules in regular appearance and high-density. The diameter of the granules was about 400 nm measuring by a transmission electron microscope (TEM). After stained with 4,6-diamidino-2-phenylindole (DAPI) and visualized by a fluorescence microscope, the cells turned blue and the granules were bright yellow. The composition of granules includes P (major ingredient), Mg, S, K, and Ca as detected by an energy dispersive X-ray spectrometer (EDS). When inorganic phosphorus (PO3−4) and ferric ion (Fe 3+) were added into media, the biomass increased and the cells formed intracellular polyP granules owing to the phosphorus assimilation from media. The YSR-3 obtained higher biomass by adding 0.02 g/L FePO 4 than 0.005 g/L and 0.01 g/L FePO 4; however, the phosphorus absorption was higher with 0.01 g/L FePO 4 than 0.005 g/L and 0.02 g/L FePO 4 The optical density at wavelength 480 nm (OD 480 nm) was 0.79 and 100% cells could form intracellular polyP granules. These results show that strain YSR-3 is able to acquire higher biomass and absorb more inorganic phosphorus when 0.01 g/L FePO 4 is added. The characteristics of absorbing and storing phosphorus as intracellular inorganic polyP granules have a potential for application in high-efficiency phosphorus removal in wastewater treatment.

Combined genomic and structural analyses of a cultured magnetotactic bacterium reveals its niche adaptation to a dynamic environment.

BackgroundMagnetotactic bacteria (MTB) are a unique group of prokaryotes that have a potentially high impact on global geochemical cycling of significant primary elements because of their metabolic plasticity and the ability to biomineralize iron-rich magnetic particles called magnetosomes. Understanding the genetic composition of the few cultivated MTB along with the unique morphological features of this group of bacteria may provide an important framework for discerning their potential biogeochemical roles in natural environments.ResultsGenomic and ultrastructural analyses were combined to characterize the cultivated magnetotactic coccus Magnetofaba australis strain IT-1. Cells of this species synthesize a single chain of elongated, cuboctahedral magnetite (Fe3O4) magnetosomes that cause them to align along magnetic field lines while they swim being propelled by two bundles of flagella at velocities up to 300 μm s−1. High-speed microscopy imaging showed the cells move in a straight line rather than in the helical trajectory described for other magnetotactic cocci. Specific genes within the genome of Mf. australis strain IT-1 suggest the strain is capable of nitrogen fixation, sulfur reduction and oxidation, synthesis of intracellular polyphosphate granules and transporting iron with low and high affinity. Mf. australis strain IT-1 and Magnetococcus marinus strain MC-1 are closely related phylogenetically although similarity values between their homologous proteins are not very high.Conclusion Mf. australis strain IT-1 inhabits a constantly changing environment and its complete genome sequence reveals a great metabolic plasticity to deal with these changes. Aside from its chemoautotrophic and chemoheterotrophic metabolism, genomic data indicate the cells are capable of nitrogen fixation, possess high and low affinity iron transporters, and might be capable of reducing and oxidizing a number of sulfur compounds. The relatively large number of genes encoding transporters as well as chemotaxis receptors in the genome of Mf. australis strain IT-1 combined with its rapid swimming velocities, indicate that cells respond rapidly to environmental changes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3064-9) contains supplementary material, which is available to authorized users.

Short-term effect of temperature variation on the competition between PAOs and GAOs during acclimation period of an EBPR system

Sequencing batch reactor (SBR) for enhanced biological phosphorus removal (EBPR) processes was used to investigate the impact of the temperature shock on the competition between phosphorus-accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) in start-up stage. During the 34 days operation, SBR was set with temperature variation(0–5 d, 22±1°C; 6–13 d, 29±1°C; 14–34 d, 14±1°C). PAOs and GAOs were analyzed by fluorescent in situ hybridization (FISH), and intracellular polyphosphate granules were stained by Neisser-stain. The results showed that the influence of temperature shock on PAOs’ abundance was more serious than that on GAOs in the enriching process. Under sudden and substantially temperature variation, from 22±1°C to 29±1°C and then to 14±1°C, the domination of PAOs was deteriorated. After temperature shock, PAOs’ competitive advantages at low temperature that concluded in other study did not appear in our study. As mesophilic, GAOs (indicated by Alphaproteobacteria and Gammaproteobacteria) were more temperature adaptive and better grew and took the domination at 14±1°C in the end. In the competition process, organisms of tetrad forming organisms (TFOs)-like shape which were considered as typical GAOs, were observed. With the evidence of poly-P granules containing by Neisser-straining and result of FISH, these organisms of TFOs-like shape were better to be assumed as adaption state or a special self-protecting shape of PAOs.

Halomonas vilamensis sp. nov., isolated from high-altitude Andean lakes

A Gram-stain-negative, aerobic, rod-shaped, non-spore-forming bacterium (SV325(T)) was isolated from the sediment of a hypersaline lake located 4600 m above sea level (Laguna Vilama, Argentina). Strain SV325(T) formed cream to pink colonies, was motile and moderately halophilic, and tolerated NaCl concentrations of 1-25 % (w/v) with an optimum of 5-10 % (w/v). Growth occurred at 5-40 °C (optimum around 30 °C) and at pH 5.0-10.0 (optimum 7.0-8.0). The bacterium did not produce exopolysaccharides and stained positively for intracellular polyphosphate granules but not for poly-β-hydroxyalkanoates. It produced catalase and oxidase, reduced nitrate to nitrite, hydrolysed gelatin, did not produce acids from sugars and utilized a limited range of substrates as carbon and energy sources: acetate, caproate, fumarate, dl-β-hydroxybutyrate, malate, maleate, malonate and succinate. The predominant ubiquinones were Q-9 (92.5 %) and Q-8 (7.5 %), the major fatty acids were C(19 : 0) cyclo ω8c, C(16 : 0), C(17 : 0) cyclo and C(16 : 1)ω7c/iso-C(15:0) 2-OH, and the DNA G+C content was 55.0 mol%. Phylogenetic analyses based on the 16S rRNA gene indicated that strain SV325(T) belongs to the genus Halomonas in the class Gammaproteobacteria. Physiological and biochemical tests allowed phenotypic differentiation of strain SV325(T) from closely related species with validly published names. We therefore propose a novel species, Halomonas vilamensis sp. nov., with type strain SV325(T) ( = DSM 21020(T) = LMG 24332(T)).

Copper isotope fractionation in acid mine drainage

We measured the Cu isotopic composition of primary minerals and stream water affected by acid mine drainage in a mineralized watershed (Colorado, USA). The δ 65Cu values (based on 65Cu/ 63Cu) of enargite (δ 65Cu = −0.01 ± 0.10‰; 2 σ) and chalcopyrite (δ 65Cu = 0.16 ± 0.10‰) are within the range of reported values for terrestrial primary Cu sulfides (−1‰ < δ 65Cu < 1‰). These mineral samples show lower δ 65Cu values than stream waters (1.38‰ ⩽ δ 65Cu ⩽ 1.69‰). The average isotopic fractionation (Δ aq-min = δ 65Cu aq − δ 65Cu min, where the latter is measured on mineral samples from the field system), equals 1.43 ± 0.14‰ and 1.60 ± 0.14‰ for chalcopyrite and enargite, respectively. To interpret this field survey, we leached chalcopyrite and enargite in batch experiments and found that, as in the field, the leachate is enriched in 65Cu relative to chalcopyrite (1.37 ± 0.14‰) and enargite (0.98 ± 0.14‰) when microorganisms are absent. Leaching of minerals in the presence of Acidithiobacillus ferrooxidans results in smaller average fractionation in the opposite direction for chalcopyrite ( Δ aq-min o = - 0.57 ± 0.14 ‰ , where min o refers to the starting mineral) and no apparent fractionation for enargite ( Δ aq-min o = 0.14 ± 0.14 ‰ ). Abiotic fractionation is attributed to preferential oxidation of 65Cu + at the interface of the isotopically homogeneous mineral and the surface oxidized layer, followed by solubilization. When microorganisms are present, the abiotic fractionation is most likely not seen due to preferential association of 65Cu aq with A. ferrooxidans cells and related precipitates. In the biotic experiments, Cu was observed under TEM to occur in precipitates around bacteria and in intracellular polyphosphate granules. Thus, the values of δ 65Cu in the field and laboratory systems are presumably determined by the balance of Cu released abiotically and Cu that interacts with cells and related precipitates. Such isotopic signatures resulting from Cu sulfide dissolution should be useful for acid mine drainage remediation and ore prospecting purposes.

APPEARANCE OF NOVEL G‐BACTERIA BELONGING TO ACIDOBACTERIA IN A DAIRY WASTEWATER TREATMENT PLANT

A dairy wastewater treatment plant operates a sequencing batch reactor (SBR) and stimulates enhanced biological phosphorus removal (EBPR) process with alternating anaerobic and aerobic cycles. Occasionally, the plant suffers from a high suspended solids problem in the supernatant. Interestingly, the occurrence of high suspended solids coincided with times when the EBPR process failed to remove phosphorus. To find out if there was a relationship between the EBPR failure and the high suspended solids problem, effluent samples were collected from the site during the period of poor phosphorus removal and examined microscopically. It was found that cocci‐shaped bacteria (3–4 µm in diameter) were abundant in the effluent samples and they were clustered in tetrads. These were believed to be G‐bacteria and results of both Gram and Neisser staining tests were negative, suggesting that they had few intracellular polyphosphate granules. Using polymerase chain reaction (PCR), cloning and deoxyribonucleic acid (DNA) sequence analysis, the phylogenetic information of in situ G‐bacteria was obtained. It was found that all of the recovered clones were clustered in the phylum of Acidobacteria.

Granulicoccus phenolivorans gen. nov., sp. nov., a Gram-positive, phenol-degrading coccus isolated from phenol-degrading aerobic granules

A Gram-positive bacterium, designated strain PG-02(T), was isolated by serial dilution from aerobic granules obtained from a laboratory-scale sequencing batch reactor for bioremediation of phenolic wastewater. Strain PG-02(T) grew axenically as cocci and is an oxidase-negative and catalase-positive, non-motile facultative anaerobe. It does not reduce nitrate and grows between 15 and 37 degrees C, with an optimum temperature of 30 degrees C. The pH range for growth is between 5.0 and 8.5, with an optimum pH of 7.0. Strain PG-02(T) contains type A3gamma peptidoglycan (ll-A(2)pm<--Gly with alanine at position 1 of the peptide subunit). The G+C content of the DNA is 69 mol%. Menaquinone MK-9(H(4)) was the major isoprenoid quinone. The polar lipids included diphosphatidylglycerol and phosphatidylglycerol, while 13-methyltetradecanoic acid (i-C(15 : 0)) and 1,1-dimethoxy-iso-pentadecane (i-C(15 : 0) DMA) were the major components in whole-cell methanolysates. PG-02(T) stained positively for intracellular polyphosphate granules but not poly-beta-hydroxyalkanoates. It produces capsular material and possesses an autoaggregation capability. Phenotypic and 16S rRNA gene sequence analyses showed that PG-02(T) differed from its closest phylogenetic relatives, namely members of the suborder Propionibacterineae, which includes the genera Tessaracoccus, Microlunatus, Luteococcus, Micropruina, Propionibacterium, Propioniferax, Nocardioides, Friedmanniella and Aeromicrobium, and that it should be placed in a new genus and species as Granulicoccus phenolivorans gen. nov., sp. nov. The type strain of Granulicoccus phenolivorans is PG-02(T) (=ATCC BAA-1292(T)=DSM 17626(T)).

Defluvicoccus vanus gen. nov., sp. nov., a novel Gram-negative coccus/coccobacillus in the ‘Alphaproteobacteria’ from activated sludge

A novel Gram-negative coccus/coccobacillus, strain Ben 114(T), growing in tetrads, clusters or aggregates, was isolated from activated sludge by micromanipulation. 16S rRNA gene sequence analysis revealed that it belonged to the 'Alphaproteobacteria', with no close relatives among cultured bacterial isolates. On the basis of phylogenetic data, this organism is considered to belong to a new genus, Defluvicoccus, represented by the species Defluvicoccus vanus sp. nov., a name chosen because of the distinctive staining properties of this organism; only the cell wall stained strongly with a wide range of stains, giving the cell a hollow and empty appearance. No intracellular polyphosphate granules could be detected after staining, but poly-beta-hydroxyalkanoate inclusions were detected using Nile blue A staining. Because of its taxonomic distance from its closest relatives among the 'Alphaproteobacteria', namely members of the genera Azospirillum, Phaeospirillum, Rhodospirillum, Rhodocista, Magnetospirillum and Rhodospira, D. vanus is considered to represent a new phylogenetic lineage within subgroup 1 of the 'Alphaproteobacteria', the D. vanus subgroup. The type strain is Ben 114(T) (=NCIMB 13612(T)=CIP 107350(T)).

Quadrisphaera granulorum gen. nov., sp. nov., a Gram-positive polyphosphate-accumulating coccus in tetrads or aggregates isolated from aerobic granules

A Gram-positive bacterium, designated strain AG019(T), was isolated by micromanipulation from aerobic granules obtained from a laboratory-scale sequencing batch reactor. This isolate grew axenically as cocci clustered predominantly in tetrads, and was morphologically similar to the dominant organisms observed in the biomass. The morphology also resembled that of the tetrad-forming organisms commonly seen in activated sludge samples. Strain AG019(T) was found to be an oxidase-negative, catalase-positive, non-motile aerobe that does not reduce nitrate and grows at temperatures between 15 and 40 degrees C, with an optimum at 37 degrees C. The pH range for growth was 5.0-9.0, with an optimum at pH 7.5. Strain AG019(T) contained a peptidoglycan with directly cross-linked meso-diaminopimelic acid (type A1gamma) and lacked mycolic acids. The G+C content of the DNA was 75 mol%. Menaquinone MK-8(H(2)) was the major isoprenoid quinone. The bacterium stained positively for intracellular polyphosphate granules but not for poly-beta-hydroxyalkanoates. It produced capsular material and showed autoaggregation ability. Phenotypic and 16S rRNA gene analyses showed that the bacterium differed sufficiently from its closest phylogenetic relatives, namely members of the suborder Frankineae, which includes the genera Geodermatophilus, Blastococcus, Frankia, Sporichthya, Acidothermus and Microsphaera, that it is proposed that it be placed in a novel genus, Quadrisphaera, as Quadrisphaera granulorum gen. nov., sp. nov. The type strain is AG019(T) (=ATCC BAA-1104(T)=DSM 44889(T)).

Chemical and Microbial Composition of Sediments in Reservoirs with Different Trophic State

AbstractFor the assessment of the metabolic potential of a sediment, the upper 5 cm layer should preferentially be regarded, since this is the horizon with the highest biochemical activity in the entire reservoir. Sediment cores from four different reservoirs were examined. These represent a broad range of trophic conditions (Neunzehnhain – oligotrophic; Muldenberg – oligotrophic, acidic, dystrophic; Saidenbach – mesotrophic; Quitzdorf – highly eutrophic). The vertical concentration gradients in the interstitial water of the examined sediments showed the tendencies characteristic of the trophic state of the reservoirs. The gradients increased with the trophic state, which affected the release of dissolved substances. The internal P load was substantial in all cases, in Quitzdorf it even exceeded the external P load. The total cell numbers of the bacteria in the sediment were only slightly influenced by the trophic state, all being in the same order of magnitude. The reservoir Quitzdorf displayed very high P release rates from the sediment and mass growths of Microcystis with a high content of intracellular polyphosphate granules in the summer months.

Effects of manganese on element distribution and structure in thalli of the epiphytic lichens Hypogymnia physodes and Lecanora conizaeoides

Thallus pieces of the Mn-sensitive epiphytic lichen Hypogymnia physodes and of the Mn-resistant Lecanora conizaeoides were incubated in 5 mM MnCl 2 for 1 h. Element concentrations and thallus structure were subsequently studied with scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray microanalysis. Mn concentrations both in fungal and algal cell walls and cell lumina were much lower in L. conizaeoides than in H. physodes, because the former immobilised Mn in the thallus (e.g. in polyphosphate granules) and in apothecia. Within the apothecia, Mn was deposited with phosphate in the hypothecium and in an unknown form in the asci. Effective immobilisation could cause the high Mn tolerance of L. conizaeoides. H. physodes also immobilised some Mn in extracellular particles in the upper cortex and in intracellular polyphosphate granules in the lower cortex. However, extra- and intra-cellular Mn concentrations in H. physodes increased much more during incubation with Mn compared with L. conizaeoides. The highest Mn concentrations were found in the upper and the lower cortex (i.e. in the cell walls and in the interhyphal polysaccharide matrix). The photobiont of H. physodes took up considerably less Mn than the mycobiont; this suggests that the latter is capable of protecting the photobiont to a certain extent from Mn invasion. Mn uptake released much Ca and Mg from H. physodes, especially from cortical cell walls and polysaccharide matrices. In the medulla, Mn was incorporated in Ca oxalate crystals especially on the surface of young growing hyphae. On a long-term basis, this is suspected to affect the integrity of the crystals, which fulfil important structural and physiological functions. Mn exposure decreased the Fe/Mn ratio more in H. physodes than in L. conizaeoides. As Fe is known to alleviate Mn toxicity in H. physodes, this could be a mechanism causing the higher Mn sensitivity of this species. Si/Mn ratios decreased in all thallus layers of H. physodes, but not of L. conizaeoides. Previous studies with soredia of H. physodes suggested possible alleviating effects of Si on Mn toxicity in lichens. Structural changes were observed in neither the mycobiont nor the photobiont of either lichen species.

Manganese toxicity in epiphytic lichens: chlorophyll degradation and interaction with iron and phosphorus

Concentrations of chlorophylls a and b decreased with increasing MnCl 2 supply in the epiphytic lichen Hypogymnia physodes, but not in Lecanora conizaeoides. The reduction of chlorophyll concentrations in H. physodes was as strong (chlorophyll a) or even stronger (chlorophyll b) as in samples treated with CuCl 2. FeCl 3 compensated for MnCl 2-induced chlorophyll degradation in H. physodes. Furthermore, MnCl 2-induced growth inhibition of soredia cultivated on agar plates was alleviated by FeCl 3. These results suggest that Mn causes intracellular Fe deficiency in H. physodes. A soredia growth test with MnCl 2 and KCl in combination proved that mitigating effects of FeCl 3 were not just caused by reduced chemical activity of Mn 2+ due to the addition of another salt. Furthermore, the test showed that Cl − did not inhibit soredia growth. High FeCl 3 concentrations applied alone or in combination with MnCl 2 were even more detrimental to H. physodes than MnCl 2. MnCl 2 did not affect the concentrations of ATP, ADP and AMP in H. physodes. This suggests that Mn uptake does not induce intracellular P deficiency in H. physodes despite that Mn is known to be immobilized with P in H. physodes in intracellular polyphosphate granules and in extracellular encrustations.

Tetrasphaera elongata sp. nov., a polyphosphate-accumulating bacterium isolated from activated sludge.

A new meso-diaminopimelic acid-containing, gram-positive bacterium was isolated from an activated sludge reactor showing enhanced biological phosphorus removal activity. The isolate was an asporogenous oval to rod-shaped bacterium, but occasionally formed clumps. The Neisser staining was positive, suggesting intracellular polyphosphate granules. The isolate was an aerobic chemoheterotroph which was capable of utilizing various sugars, sugar alcohols and organic acids. It contained anteiso-C15:0, iso-C15:0, iso-C14:0 and C16:0 as the major cellular fatty acids, and menaquinone-8(H4) as the major quinone. The G+C content of the genomic DNA was 69.6 mol%. Analysis of the 16S rDNA sequence revealed that the isolate is a new member of the family Intrasporangiaceae. The closest relatives were Tetrasphaera species. On the basis of the phenotypic and phylogenetic distinctiveness of the isolate, it was concluded that the organism represents a new species in the genus Tetrasphaera, for which the name Tetrasphaera elongata sp. nov. is proposed. The type strain is strain Lp2T (= JCM 11141T = DSM 14184T).

Tessaracoccus bendigoensis gen. nov., sp. nov., a gram-positive coccus occurring in regular packages or tetrads, isolated from activated sludge biomass.

An isolate of a Gram-positive bacterium, designated strain Ben 106T, was obtained in pure culture by micromanipulation of a biomass sample obtained from a laboratory-scale sequencing batch reactor. This isolate grew axenically as cocci or clusters of cocci arranged in regular tetrads and was morphologically similar to the dominant organism observed in the biomass. This morphology resembled that of some Gram-positive and -negative bacteria and the so-called 'G-bacteria' commonly seen in activated sludge samples. Strain Ben 106T is a non-motile, facultative anaerobe. It is oxidase-negative, catalase-positive and is capable of reducing nitrate. This organism can grow between 20 and 37 degrees C, with an optimum temperature of 25 degrees C. The pH range for growth is between 6.0 and 9.0, with an optimum pH of 7.5. The isolate stained positively for intracellular polyphosphate granules. The diagnostic diamino acid of the peptidoglycan is LL-diaminopimelic acid (LL-A2pm) with a glycine moiety at position 1 of the peptide subunit, which characterizes the presence of a rare peptidoglycan (type A3-gamma'). Two menaquinones, MK-9(H4) and MK-7(H4), are present and the main cellular fatty acid is 12-methyltetradecanoic acid. The G + C content is 74 mol%. From phenotypic characteristics and 16S rDNA sequence analysis, the isolate differed sufficiently from its closest phylogenetic relatives, namely Propionibacterium propionicum, Propioniferax innocua, Friedmanniella antarctica, Luteococcus japonicus and Microlunatus phosphovorus in the A1 subdivision of the Gram-positive bacteria (i.e. Firmicutes with a high G + C content), suborder Propionibacterineae, to be placed in a new genus, Tessaracoccus, as Tessaracoccus bendigoensis gen. nov., sp. nov. The type strain is Ben 106T (= ACM 5119T).

Utilization of PP i as an Energy Source by a Clostridium sp

The growth of an anaerobic, spore-forming rod we have isolated from the cockroach gut after enrichment on media containing PP(i) was stimulated by the presence of PP(i). The doubling time decreased and cell yield increased proportionately to PP(i) concentrations of up to 0.35%. A similar stimulation of the growth of Desulfotomaculum sp. by PP(i) has been reported. The PP(i)-stimulated Clostridium sp. fermented a number of sugars with the production of hydrogen, acetate, and butyrate, with smaller amounts of ethanol and butanol being produced from some substrates. The fermentation products were not qualitatively changed by the presence of PP(i), but significantly more hydrogen was produced. The organism contained several of the enzymes previously reported from Entamoeba sp. and Propionibacterium sp., in which PP(i) serves as a source of a high-energy bond in place of ATP. These include significant amounts of pyruvate-phosphate dikinase and phosphoenolpyruvate carboxytransphosphorylase. The activities of many of the catabolic enzymes of the organism, as well as of its phosphatases and pyrophosphatase, were similar whether it was grown in the presence or absence of PP(i). The organism did not accumulate intracellular polyphosphate granules but stored large amounts of glycogen.

Possible Biological Mechanism of Phosphorus Removal

This paper describes an investigation which was conducted to determine the location and nature of accumulated phosphorus in several enhanced phosphorus-removing activated sludges. Phosphorus accumulations were located by electron microscopy combined with the energy dispersive analysis of X-rays. In all the sludges phosphorus accumulations were located in a specific morphological cell-type, which was also the predominant organism in the sludges. These cells typically occurred in large cell-clusters and were identified as Acinetobacter spp. Quantitative analyses indicated that the intracellular polyphosphate granules contained in excess of 25 % (m/m) phosphorus and a Ca:P ratio between 0,15-0,36. These cells released o-P during anaerobiosis. Factors leading to accumulation were examined and it was found that accumulation was possible under continuous aerobic culture and was enhanced by unidentified fermentation products of raw sewage; these products also induced the accumulation of intracel1ular carbon reserves by the cells. It was concluded that enhanced biological phosphorus removal by activated sludge is dependent on the enrichment of certain Acinetobacter spp in the system.