Identification of organelles in bacteria similar to acidocalcisomes of unicellular eukaryotes

Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds. In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes. Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane. X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium. Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration. Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A. tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles. Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short- and long-chain polyphosphate, but lack of markers of the plasma membrane. The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide. Polyphosphate was also localized to the volutin granules by 4',6'-diamino-2-phenylindole staining. The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport. The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged.     

Formation of volutin granules in Corynebacterium glutamicum

Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted.     

Effect of single nutrient limitation of poly-beta-hydroxybutyrate molecular weight distribution in alcaligens europhus

The effect of magnesium and phosphate limitation on the molecular weight distribution of poly-beta-hydroxybutyrate (PHB) in Alcaligens europhus in cotinuons culture has been stuied. Conditions of nitrogen limitation both with glucose excess (above ca. 20 g/L) and without excess were investigated Under N-limitation and glucose excess, M(w) decreases when the magnesium content is decreased below 50% (19.7 mg/L) of the basal medium content; this also results in a broadenng of molecular weight distribution (M(w)/M(n)) from 2 to 5 and a decrease in M(w) fron 2 x 10(6) to 0.9 x 10(6). Below 20% of the basal content of magnesium (7.9 mg/L) these two trends were reversed. This behaviour was not observed in the absence of glucose excess, phshate had virtually no effect on PHB M(w) or its distribution, whereas wih no (or little) glucose excess M(w) of the PHB decreased with phosphate concentrations below 50% of the basal level (0.705 g/L). Hence, in continuous or fed-batch cultures, in addition to nitrogen limitation to alklow for PHB accumulation, it is necesary to control both the addition of glucose (no excess) and also to maintain magnesium limitation (ca. 25% of basal medium level, 9.9 mg/L) and phosphate above 50% of he basal level (0.705 g/L). Thus, when broadening of molecular weight destribution (increase in M(w)/M(n)) is observed at the end of fed-batch culture it is probably caused by phosphate limitation and/or glucose excess. (c) 1995 John Wiley & Sons, Inc.     

Microbial population dynamics during sludge granulation in an anaerobic-aerobic biological phosphorus removal system

The evolution of a microbial community was investigated during sludge granulation using a wide range of micro-scale and molecular biology techniques. Experimental results demonstrate that polyphosphate-accumulating granules were successfully cultured during the anaerobic/aerobic cycle. Improvement in sludge sedimentation performance occurred prior to the formation of granular sludge and was not affected by change in granule size. Rod-shaped and filamentous bacteria appeared to initiate granule formation and generate the structures that supported further granule growth. It was observed that mature granules supported microbial populations that differed from nascent granules and were predominantly packed with coccoid bacteria. It was further observed that the diversity of the granular microbial community increased as the granules grew. Accumulibacter, Nitrosospira and Thauera were mainly responsible for nutrient removal while microorganisms such as Rhodocyclus and Hyphomicrobiaceae appeared to be primarily responsible for forming and maintaining the granule structure.     

Isolation of intact chains of polyphosphate from "Propionibacterium shermanii" grown on glucose or lactate

A procedure is presented for the isolation of intact polyphosphate (poly P) from "Propionibacterium shermanii." It is demonstrated, by including [32P]poly P during the extraction, that this procedure does not hydrolyze the poly P, and it is shown that two other widely used procedures do cause breakdown of the poly P. The procedure presented allows isolation of three fractions, short-chain poly P which is soluble in trichloroacetic acid, long-chain poly P which is soluble at neutral pH, and long-chain poly P which is present in volutin granules. Cells which had been grown on lactate did not contain short-chain poly P but did contain a high amount of long-chain poly P, which accumulated to 3% of the cell dry weight. At least 70% of this poly P was present in volutin granules. The poly P ranged in length from 250 to 725 phosphate residues and was the same average size as that synthesized in vitro by the poly P kinase from "P. shermanii". This indicates that the poly P kinase is responsible for catalyzing the synthesis of the poly P. In contrast to cells grown on lactate, those which had been grown on glucose did not contain volutin granules, did contain short-chain poly P and had 100-fold less long-chain poly P than lactate-grown cells. We propose that during the fermentation of glucose, the amount of poly P is lower than during growth on lactate because it is continuously utilized as a substrate in the phosphorylation of glucose.     

The quorum-sensing effect of aerobic granules on bacterial adhesion, biofilm formation, and sludge granulation

Quorum sensing (QS) through signal chemical molecules is known to be essential to bacterial adhesion and biofilm formation. In this study, the QS ability of aerobic granules—a special form of biofilms used for biological wastewater treatment—was investigated and compared with that of conventional activated sludge flocs. A novel sectional membrane bioreactor was used together with a flow-cell to evaluate the possible influence of signal chemicals produced by the source sludge on the growth mode of bacterial cells. The results demonstrate the apparent production of QS chemicals from granules and its impact on initial cell attachment and granule formation. When granules were used as the signal-producing biomass, the attached-growth mode was dominant for the free cells, and the biofilm formation rate in the flow-cell was about ten times faster than in cases which used activated sludge as the signal source biomass. In addition, the intracellular extract from mature granules significantly accelerated the sludge granulation process. It is argued that the production and expression of QS signal chemicals from granules and granule precursors might have induced the gene expression of bacteria in suspension for attached growth rather than suspended growth, leading to granule formation and its stable structure.     

Polyphosphate metabolism in the blue-green alga Microcystis aeru-ginosa

The uptake of inorganic phosphorus was studied in an axenicstrain of phosphorus-starved cells of the blue-green alga Microcystisaeruginosa, an organism often causing blooms in freshwater bodies.Rates of growth and of cellular polyphosphate content as a functionof initial orthophosphate in the medium indicate the operationof the ‘phosphorus overplus’ phenomenon in M. aeruginosa,accompanied by formation of volutin granules. The granules wereisolated by a non-aqueous centrifugation method, and identifiedas polyphosphate bodies.     

Capacity for polyphosphate accumulation of predominant bacteria in activated sludge showing enhanced phosphate removal

The taxonomic identity of predominant bacteria in laboratory-scale anaerobic-aerobic activated sludge systems and their capacity for accumulating polyphosphate (polyP) were investigated. All bacterial isolates accumulated appreciable amounts of polyP under tested conditions, although only 7% of them were positive for the volutin stain. The isolates showing the most pronounced polyP accumulation and volutin formation were the unidentified Gram-positive bacteria containing menaquinone—10. Some other strains of Gram-positive bacteria and menaquinone-containing Gram-negative bacteria also formed volutin granules.     

An energy-based population-balance approach to model granule growth and breakage in high-shear wet granulation processes

A mathematical model of high shear wet granulation is proposed, where granule breakage, and not growth, is the dominant process. The energy required for granule breakage is assumed to be provided by the impact of granules between themselves and the granulator parts, and the extent of granule breakage determined by the balance between the impactenergy and the work of adhesion between the agglomerating particles. A specific volume of dry powder per unit crack surface area was allowed to reattach to the surface of broken granules to account for granule growth. To verify proposed model conditions, lactose monohydrate was granulated with a relatively low amount (6%) of the binder phase, polyvinyl-pyrrolidone and water, and was added to the powder before granulation. The trend in granule size distribution during the experiment closely follwed the predicted model with an initial increase in the weight fraction of the larger granules. This increase was possibly due to extensive breakage of weaker granules and less extensive breakage, as if by attrition, of stronger granules, accompanied by the attachment of dry powder to the cracked surfaces. Eventually, larger granules experience increased impact energy and break. When excess binder is added and, higher volumes of powder reattach to the crack surface, more large granules form leading to granule overgrowth. This model highlights the importance of the probability of impact per unit time interval (ie, the rate of impact), the strength of the granules and the volume of powder that could attach to the cracked surface in high shear granulation processes where significant granule breakage is encountered. Published: August 10, 2007     

Partial nitrifying granule stimulated by struvite carrier in treating pharmaceutical wastewater

Aerobic granule was successfully cultivated in SBR (sequencing batch reactor) by struvite carrier (magnesium ammonium phosphate, MgNH₄PO₄), which can increase polysaccharides to 42.2 mg/gMLVSS (mixed liquor volatile suspended solid) versus only 28.4 mg/gMLVSS of the sludge without it. Meanwhile, it was found that struvite play a positive role in initial granulation and bacterial group distribution in treating pharmaceutical wastewater, involving effect of solid surface and special contents of struvite. The results of fluorescence in situ hybridization technique indicate that ammonia-oxidizing bacteria can dominate over nitrite-oxidizing bacteria in mature granules. COD removal efficiency of 90 % and NO₂ ^?–N:(NO₂ ^?–N?+?NO₃ ^?–N) accumulation efficiency of 89 % were achieved in stable state. Emphasis is placed on that struvite addition can be applied as a new-type carrier to promote formation of partial nitrification granular sludge.     

Magnesium and iron addition to casein hydrolysate medium for production of staphylococcal enterotoxins A, B, and C.

From comparisons of 4% N-Z Amine NAK made with distilled water, naturally hard water, and synthetic salt solutions, it appeared that magnesium and, to a lesser extent, iron were limiting factors in the production of staphylococcal enterotoxins B and C but not A. Maximum enterotoxin production with NAK medium was achieved by the addition of 5 mg of Mg2/ per liter (for a total of 9 mg of Mg2+ per liter) and 0.5 mg of Fe2+ per liter. Higher levels of magnesium were not inhibitory. Supplementing NAK with commonly used complex components, which added Mg2+ above the 9-mg/liter level, did not result in maximum yields of enterotoxin. Variability in the ability of different lots of NAK to support enterotoxin production may be minimized by supplementing NAK medium with magnesium and iron.     

Magnesium and iron addition to casein hydrolysate medium for production of staphylococcal enterotoxins A, B, and C.

From comparisons of 4% N-Z Amine NAK made with distilled water, naturally hard water, and synthetic salt solutions, it appeared that magnesium and, to a lesser extent, iron were limiting factors in the production of staphylococcal enterotoxins B and C but not A. Maximum enterotoxin production with NAK medium was achieved by the addition of 5 mg of Mg2/ per liter (for a total of 9 mg of Mg2+ per liter) and 0.5 mg of Fe2+ per liter. Higher levels of magnesium were not inhibitory. Supplementing NAK with commonly used complex components, which added Mg2+ above the 9-mg/liter level, did not result in maximum yields of enterotoxin. Variability in the ability of different lots of NAK to support enterotoxin production may be minimized by supplementing NAK medium with magnesium and iron.     

Effects of Cycle Time on Properties of Aerobic Granules in Sequencing Batch Airlift Reactors

Summary The granulation and properties of aerobic sludge were studied in two sequencing batch airlift reactors (SBARs). The synthetic wastewater in the two reactors had initially different levels of COD (400 mg l⁻¹ in R1 and 1600 mg l⁻¹ in R2). A hydraulic cycle time of 3 and 12 h was conducted in the reactors R1 and R2, respectively and the process of granulation was observed by optical microscopy. It was found that the course of granulation at a cycle time of 3 h in R1 was shorter than that at cycle time of 12 h in R2 and the properties of aerobic granules were distinct in the reactors due to the different hydraulic cycle time. Under a cycle time of 3 h, granule diameter was around 1.0–2.0 mm, VSS ratio was 92.08% with stronger granule strength; under a cycle time of 12 h, granule diameter was around 0.5–1.0 mm, VSS ratio was 83.92% with weaker granule strength. In addition, the morphology of microorganisms in granules was obviously dissimilar when the hydraulic cycle time was different. It was concluded that the hydraulic cycle time plays a crucial role in the granulation and properties of aerobic granules. It is expected that the experimental findings will provide useful information on factors affecting aerobic granulation.     

Ultrastructural investigation of acidocalcisomes and ATPase activity in yeast Candida guilliermondii NP-4 as ‘complementary’ stress-targets

As a result of electron microscopic studies of morphogenesis in yeast Candida guilliermondii NP-4, the formation of new structures of volutin acidocalcisomes has been established within the cell cytoplasm. Under influence of X-irradiation, the changes in morphometric and electron-dense properties of yeast cells were identified: in yeast cytoplasm, the electron-dense volutin granules were increased up to 400 nm in size. After 24-h post-irradiation incubation of yeasts, the large volutin pellets are fragmented into smaller number particles in size up to 25–150 nm. The ATPase activity in yeast mitochondria was changed under X-irradiation. In latent phase of growth, ATPase activity was decreased 1·35-fold in comparison with non-irradiated yeasts. In logarithmic phase of growth, ATPase activity was three times higher than in latent phase, and in stationary phase of growth it has a value similar to the latent phase. Probably, the cells receive the necessary energy from alternative energy sources, such as volutin. Electron microscopy of volutin granule changes might serve as convenient method for evaluation of damages and repair processes in cells under influence of different environmental stress-factors.     

Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate.     

Aerobic granular sludge: recent advances

Aerobic granulation, a novel environmental biotechnological process, was increasingly drawing interest of researchers engaging in work in the area of biological wastewater treatment. Developed about one decade ago, it was exciting research work that explored beyond the limits of aerobic wastewater treatment such as treatment of high strength organic wastewaters, bioremediation of toxic aromatic pollutants including phenol, toluene, pyridine and textile dyes, removal of nitrogen, phosphate, sulphate and nuclear waste and adsorption of heavy metals. Despite this intensive research the mechanisms responsible for aerobic granulation and the strategy to expedite the formation of granular sludge, and effects of different operational and environmental factors have not yet been clearly described. This paper provides an up-to-date review on recent research development in aerobic biogranulation technology and applications in treating toxic industrial and municipal wastewaters. Factors affecting granulation, granule characterization, granulation hypotheses, effects of different operational parameters on aerobic granulation, response of aerobic granules to different environmental conditions, their applications in bioremediations, and possible future trends were delineated. The review attempts to shed light on the fundamental understanding in aerobic granulation by newly employed confocal laser scanning microscopic techniques and microscopic observations of granules.     

Heterogeneous Distribution of Microbial Activity in Methanogenic Aggregates: pH and Glucose Microprofiles

Methanogenic aggregates, harvested from an upflow anaerobic sludge blanket reactor treating potato starch wastewater, were acclimatized to either glucose or a mixture of sugars and organic nitrogen compounds (i.e., diluted molasses). Both types of granules exhibited internal pH and substrate concentration gradients in mineral medium (pH 7.0, 30°C) as was measured with microelectrodes. Glucose-acclimatized granules suspended in a mineral medium lacking glucose exhibited a distinct internal pH decrease of about 1 U within the granule, suggesting strong metabolism by the acidogenic bacteria. Molasses-acclimatized and aged granules suspended in mineral medium did not exhibit such a pH decrease, suggesting the importance of the metabolic state of these acidogens. The pH gradient did not occur in deactivated granules and was not observable in strongly buffered media (mineral medium containing 33 mM phosphate or reactor liquid). When glucose (0.5 to 5.0 mM) was added to the mineral medium, granules exhibited a convex pH profile. Glucose consumption was located exclusively in the outer 200 to 300 μm of the aggregates (mean diameter = 1.5 mm). The addition of 20 mM 2-bromoethanesulfonic acid to the mineral medium indicated that the higher pH levels in the centre of the granule appeared to be related to the activity of methanogens. It is suggested that acidogenic activity occurs predominantly in the outer 200 to 300 μm of the aggregate and methanogenic activity occurs predominantly in the center of the investigated granules.     

A Comparison of Granules Produced by High-Shear and Fluidized-Bed Granulation Methods

Placebo granules were manufactured by both wet high-shear and fluidized-bed techniques. The granules were compared based on size, shape, surface morphology, and a variety of different flowability measurements. This comparison showed that granule formation and growth were different, with induction growth for high-shear granulation and steady growth for fluidized-bed granulation. Final granules from high-shear granulation were more spherical and dense compared with the irregular granules from fluidized-bed granulation. The high-shear granules demonstrated better overall flow properties.     

Rubratoxin production by penicillium rubrum when grown in a synthetic medium containing different sources of carbon and nitrogen

A sterile mineral salts broth was fortified with different additives, inoculated with conidia ofPenicillium rubrum P-13, and incubated quiescently for 14 days or with shaking for 3 to 5 days. Maximal fungal growth and rubratoxin production occurred when the broth contained 20% sucrose. Broth with 10% glucose, 10% fructose, 5% maltose, or 1% asparagine supported formation of substantial amounts of rubratoxin (52.9–78.5 mg/100 ml). When the broth was fortified with glucose plus lysine, arginine aspartic acid, cystine, ammonium citrate, or ammonium phosphate, moderate amounts (27.5–39.5 mg/100 ml) of rubratoxin and mycelium (0.1–1.5 g/100 ml) were produced. Presence in the broth of 5% galactose or starch resulted in accumulation of small amounts (22.2 and 24.6 mg/100 ml, respectively) of rubratoxin and mold tissue (0.70 and 0.5 g/ 100 ml, respectively). Whereas some toxin was recovered from mineral salts broth fortified with lactose or ribose, toxin was not recovered when the mold grew in broth containing mannitol or fumarate. With the exception of gluconate which supported some growth and toxin formation and ethanol which permitted formation of small amounts of toxin, other carbon sources resulted in little or no fungal growth and no toxin formation. Yields of rubratoxin decreased with an increase in amount of agitation or length of incubation ofP. rubrum cultures. Mold growth increased and toxin formation decreased with an increase in volume of culture.     

Kinetics of methyl t-butyl ether cometabolism at low concentrations by pure cultures of butane-degrading bacteria

Butane-oxidizing Arthrobacter (ATCC 27778) bacteria were shown to degrade low concentrations of methyl t-butyl ether (MTBE; range, 100 to 800 microg/liter) with an apparent half-saturation concentration (K(s)) of 2.14 mg/liter and a maximum substrate utilization rate (k(c)) of 0.43 mg/mg of total suspended solids per day. Arthrobacter bacteria demonstrated MTBE degradation activity when grown on butane but not when grown on glucose, butanol, or tryptose phosphate broth. The presence of butane, tert-butyl alcohol, or acetylene had a negative impact on the MTBE degradation rate. Neither Methylosinus trichosporium OB3b nor Streptomyces griseus was able to cometabolize MTBE.