Diffusivity of oxygen in aerobic granules

This work for the first time estimated apparent oxygen diffusivity (D(app)) of two types of aerobic granules, acetate-fed and phenol-fed, by probing the dissolved oxygen (DO) level at the granule center with a sudden change in the DO of the bulk liquid. With a high enough flow velocity across the granule to minimize the effects of external mass transfer resistance, the diffusivity coefficients of the two types of granules were estimated with reference to a one-dimensional diffusion model. The carbon source has a considerable effect on the granule diameter (d) and the oxygen diffusivity. The diffusivity coefficients were noted 1.24-2.28 x 10(-9) m2/s of 1.28-2.50 mm acetate-fed granules, and 2.50-7.65 x 10(-10) m2/s of 0.42-0.78 mm phenol-fed granules. Oxygen diffusivity declined with decreasing granule diameter, in particular, the diffusivity of acetate-fed granules is proportional to the size, whereas the diffusivity of phenol-fed granules is proportional to the square of granule diameter. The existence of large pores in granule, evidenced by FISH-CLSM imaging, was proposed to correspond to the noted size-dependent oxygen diffusivity. The phenol-fed granules exhibited a higher excellular polymer (ECP) content than the acetate-fed granules, hence yielding a lower oxygen diffusivity.     

Distribution of extracellular polymeric substances in aerobic granules

Extracellular matrix provides an architectural structure and mechanical stability for aerobic granules. Distributions of cells and extracellular polymeric substances (EPS), including proteins, α- and β-d-glucopyranose polysaccharides, in acetate-fed granules and phenol-fed granules were probed using a novel quadruple staining scheme. In acetate-fed granules, protein and β-d-glucopyranose polysaccharides formed the core, whereas, the cells and α-d-glucopyranose polysaccharides accumulated in the granule outer layers. Based on these experimental findings, this study indicated that different conclusions can be obtained regarding EPS distributions when granules were stained differently. The core of phenol-fed granules, conversely, was formed principally by proteins; whereas, the cells and α- and β-d-glucopyranose polysaccharides were accumulated at an outer filamentous layer. Using a series of confocal laser scanning microscope (CLSM) images whose threshold values were determined via Otsu’s scheme, the three-dimensional distributions of cells and EPS were produced using a polygonal surface model. Structural information extracted can be applied in further development of comprehensive granule models.     

Seeded Growth Route to Noble Calcium Carbonate Nanocrystal

A solution-phase route has been considered as the most promising route to synthesize noble nanostructures. A majority of their synthesis approaches of calcium carbonate (CaCO₃) are based on either using fungi or the CO₂ bubbling methods. Here, we approached the preparation of nano-precipitated calcium carbonate single crystal from salmacis sphaeroides in the presence of zwitterionic or cationic biosurfactants without external source of CO₂. The calcium carbonate crystals were rhombohedron structure and regularly shaped with side dimension ranging from 33–41 nm. The high degree of morphological control of CaCO₃ nanocrystals suggested that surfactants are capable of strongly interacting with the CaCO₃ surface and control the nucleation and growth direction of calcium carbonate nanocrystals. Finally, the mechanism of formation of nanocrystals in light of proposed routes was also discussed.     

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.     

Influence of substrate surface loading on the kinetic behaviour of aerobic granules

In the aerobic granular sludge reactor, the substrate loading is related to the size of the aerobic granules cultivated. This study investigated the influence of substrate surface loading on the growth and substrate-utilization kinetics of aerobic granules. Results showed that microbial surface growth rate and surface biodegradation rate are fairly related to the substrate surface loading by the Monod-type equation. In this study, both the theoretical maximum growth yield and the Pirt maintenance coefficient were determined. It was found that the estimated theoretical maximum growth yield of aerobic granules was as low as 0.2 g biomass g^–1 chemical oxygen demand (COD) and 10–40% of input substrate-COD was consumed through the maintenance metabolism, while experimental results further showed that the unit oxygen uptake by aerobic granules was 0.68 g oxygen g^–1 COD, which was much higher than that reported in activated sludge processes. Based on the growth yield and unit oxygen uptake determined, an oxidative assimilation equation of acetate-fed aerobic granules was derived; and this was confirmed by respirometric tests. In aerobic granular culture, about 74% of the input substrate-carbon was converted to carbon dioxide. The growth yield of aerobic granules was three times lower than that of activated sludge. It is likely that high carbon dioxide production is the main cause of the low growth yield of aerobic granules, indicating a possible energy uncoupling in aerobic granular culture.     

Calcium accumulation characterization in the aerobic granules cultivated in a continuous-flow airlift bioreactor

Limited work has been done on the accumulation characterization of Ca²⁺ in aerobic granules that are cultivated in a continuous-flow bioreactor. In this work, the contribution of Ca²⁺ to the biogranulation in a continuous flow airlift fluidized bed (CAFB) reactor has been studied. The spatial distribution and form of calcium in the granules were investigated by scanning electron microscopy-mapping, energy dispersive X-ray and X-ray diffraction (XRD). Calcium was located throughout the Ca-rich granules, rather than accumulating in the center of the granules of the sequencing batch reactor. Furthermore, CaCO₃ was detected as the main crystalline mineral form of the calcium. Calcium augmentation of the inflow promoted the accumulation of magnesium in the granules in the CAFB. The magnesium was presented as Ca₇Mg₂P₆O₂₄ according to XRD analyses.     

The cationic composition and pH in the moulting fluid of Porcellio scaber (Crustacea, Isopoda) during calcium carbonate deposit formation and resorption

Before moulting, terrestrial isopods resorb calcium carbonate (CaCO₃) from the posterior cuticle and store it in sternal deposits. These consist mainly of amorphous calcium carbonate (ACC) spherules that develop within the ecdysial space between the anterior sternal epithelium and the old cuticle. Ions that occur in the moulting fluid, including those required for mineral deposition, are transported from the hemolymph into the ecdysial space by the anterior sternal epithelial cells. The cationic composition of the moulting fluid probably affects mineral deposition and may provide information on the ion-transport activity of the sternal epithelial cells. This study presents the concentrations of inorganic cations within the moulting fluid of the anterior sternites during the late premoult and intramoult stages. The most abundant cation is Na⁺ followed by Mg²⁺, Ca²⁺ and K⁺. The concentrations of these ions do not change significantly between the stages whereas the mean pH changed from 8.2 to 6.9 units between mineral deposition in late premoult, and resorption in intramoult, respectively. Measurements of the transepithelial potential show that there is little driving force for passive movements of calcium across the anterior sternal epithelium. The results suggest a possible role of magnesium ions in ACC formation, and a contribution of pH changes to CaCO₃ precipitation and dissolution.     

Maltobionic acid enhances intestinal absorption of calcium and magnesium in rats

ABSTRACT

In Experiment 1, the effects of calcium maltobionate (MBCa) on calcium and magnesium absorption were examined using male rats. Four diets were designed in which 25%, 50%, and 100% of calcium carbonate (CaCO₃, Control) were substituted with MBCa and were designated as MBCa-25, MBCa-50, and MBCa-100, respectively. The cecal concentration of short-chain fatty acids was significantly higher in groups MBCa-50 and MBCa-100; however, pH of cecal contents did not significantly differ among the groups. Retention rates of calcium and magnesium were significantly higher in all MBCa groups as compared to the Control. In Experiment 2, the efficiency of calcium absorption was compared using everted sacs of jejunum and ileum with CaCO₃ and MBCa as calcium sources. More calcium from MBCa was absorbed as the concentration of calcium increased in comparison to CaCO₃. It was concluded that MBCa is a better calcium source than CaCO₃ in terms of both calcium retention and absorption.

Abbreviations: ANOVA: analysis of variance; Ca: Calcium; CaCO_3: calcium carbonate; ICP-OES: Inductivity coupled plasma optical emission spectrometer; Mg: magnesium; MBCa: calcium maltobionate; OCPC: o-cresolphthalein complexone; SCFAs: short-chain fatty acids; SE: standard error; TRPM6: transient receptor potential melastatin 6.     

Encystment ofAzotobacter vinelandii in liquid culture

Cyst formation in liquid cultures ofAzotobacter vinelandii could be raised to 90% by including 0.6% CaCO₃ in the Burk's basal salts solution. The cysts were resistant to desiccation and possessed exine and intine capsular components when observed by electron microscopy of ultrathin sections. Cells grown in liquid medium containing no CaCO₃, but in which the pH was controlled by addition of 0.1m KOH, were not resistant to desiccation. The capsular material was loosely aggregated and not organized into the typical coat structure. The culture supernatant fluid became viscous and could be precipitated with CaCl₂, yielding a thick, polysaccharide-like gel. Calcium carbonate in the liquid encystment medium served both as a means of acid neutralization and as a source of calcium ions. Calcium ions appear to be structural units necessary for coordination of the coat components into the rigid cyst structure. Other metal ions examined could not substitute effectively for calcium in the encysting system. This investigation was supported by Public Health Service research grants AI-02924 and AI-05551 and by Public Health Service predoctoral fellowship 5-F1-GM-29, 322-02.     

Induction of Calcium Carbonate by Bacillus cereus

The purpose of this research was to study how the bacteria Bacillus cereus (DCB1) utilizes calcium ions in a culture medium with carbon dioxide (CO₂) to yield calcium carbonate (CaCO₃). The bacteria strain DCB1 was a dominant strain isolated from dolomitic surfaces in areas of Karst topographies. The experimental method was as follows: a modified beef extract-peptone medium (beef extract 3.0 g, peptone 10 g, NaCl 5.0 g, CaCl₂ 2.0 g, glass powder 2.0 g, distilled water 1 L, and a pH between 6.5 and 7.5) was inoculated with B. cereus to attempt to induce the synthesis of CaCO₃. The sample was then processed by centrifugation every 24 h during the 7-day cultivation period. The pH, carbonic anhydrase (CA) activity, and the concentrations of both HCO^- ₃ and Ca²⁺ in the supernatant fluid were measured. Subsequently, precipitation in the culture medium was analyzed to confirm, or otherwise, the presence and if present, the formation, of CaCO₃. Methods used included X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Energy Dispersive Spectroscopy (EDS). Meanwhile, the carbon source in the carbonate was classified by its isotope composition. Results showed that B. cereus can improve its pH value in this culture medium; concentrations of HCO^- ₃ and Ca²⁺ showed a significant decline over the duration of the cultivation period. CA activity reached its maximum during the second day; XRD, SEM, TEM, and isotope analysis all revealed the presence of CaCO₃ as a precipitate. Additionally, these results did not occur in an aseptic control group: no detectable level of CaCO₃ was produced therein. In conclusion: B. cereus can metabolize active materials, such as secretase, by its own growth and metabolism, and can either utilize atmospheric CO₂, or respire, to induce CaCO₃ production. Experimental evidence is offered for a concomitant CO₂ reduction and CaCO₃ induction by microorganisms.     

Evidences for Bioprecipitation of Pedogenic Calcite by Calcifying Bacteria from Three Different Soils of L'Aquila Basin (Abruzzi,Central Italy)

To provide further evidences on the role of bacterial soil species in the development of calcium carbonate deposits in soil, we isolated 36 heterotrophic bacterial strains from three soils of L'Aquila basin characterized by different CaCO₃ content and tested their ability to precipitate CaCO₃ when cultured on a Ca-rich medium. We found that the majority (63.89%) of these isolates could precipitate CaCO₃ minerals at 27°C. The aptitude to calcification (time and crystal amount) of each calcifying strains, morphology (SEM) and mineralogy of the formed bioliths were also investigated. X-ray diffraction confirmed the production of calcite. Crystal formation was not observed in the controls. Organic matter, total N and assimilable P, cation exchange capacity and exchangeable Ca²⁺, Mg²⁺, K⁺, Na⁺, pH, total and active calcium carbonate content, electric conductivity, skeleton, sand, silt and clay fractions of each soil sample were determined and related with its microbiological parameters. We found that the CaCO₃ content of soil was significatively related, in particular, to the percentage of calcifying bacterial strains (r = 0.95) and to the heterotrophic bacterial density (r = 0.98), which was found significatively related also with Ca²⁺ content of soil (r = ?0.97) and its CEC (r = ?0.97).     

Immunolocalization of an Acid Phosphatase from Pearl Oyster (<Emphasis Type="Italic">Pinctada fucata</Emphasis>) and Its In Vitro Effects on Calcium Carbonate Crystal Formation

Distribution of an acid phosphatase, AcPase I, from pearl oyster (Pinctada fucata) in different tissues was investigated via enzyme activity determination and immunohistochemistry. Positive reactions were observed in sections of digestive gland, base of gill filaments, and epithelia of the outer side of the middle fold and the inner side of the outer fold, which indicated AcPase I might participate in processes besides immune defense, such as calcium metabolism or shell formation. Its effects on CaCO₃ crystal formation were studied in vitro. Results revealed that AcPase I inhibited CaCO₃ precipitation in a dose-dependent manner and had no affinity for calcium. CaCO₃ crystals induced by AcPase I exhibited a cluster needle-like morphology, which proved to be aragonite. The morphology and size of the aragonites varied with different concentrations of AcPase I. Our observations described here may provide important clues to further understanding of the correlations between mineralization and immune defense in the oyster.     

The effect of carbonic anhydrase,urea and urease on the calcium carbonate deposition in the shell-repair membrane of the snail,Helix pomatia L.

Summary The effect of carbonic anhydrase (CA), urea and urease on the CaCO₃ deposition in the shell-repair membrane of the snail, Helix pomatia, was studied by injection of CA separately or in combination with urease. This treatment resulted in increased deposits of CaCO₃ and apparent crystal formation within the shell-repair membranes compared with those of the controls. The reactions to CA combined with urea were not uniform. Formation of organic crystalline structures and dendritic spherulites was observed in some of these membranes, whereas the deposition of CaCO₃ crystals was suppressed. Administration of urea alone inhibited the formation of large CaCO₃ crystals, whereas urease stimulated this process. The reaction of young snails was greater compared to adults. The membranes of young snails contained tighly packed, small CaCO₃ crystals and organic crystalline structures, which indicated increase of the calcifying centra and their successive mineralization. The results support the assumption that carbonic anhydrase and urease enhance the rate of calcium carbonate deposition and crystal formation in Helix pomatia.     

Effects of different calcium salts on calcium carbonate crystal formation by Sporosarcina pasteurii KCTC 3558

We examined the effectiveness of using different calcium salts for bioconsolidation. Four calcium salts were chosen based on their applicability and solubility. Initial experiments demonstrated that the addition of any calcium salt had a negative effect on the urease activity of S. pasteurii. Microscopic examinations elucidated the morphological and structural differences of the calcium carbonate (CaCO₃) crystals induced. Calcite and vaterite are the prominent forms of CaCO₃ detected according to X-ray diffraction (XRD) analysis. Bioconsolidated sand samples were able to significantly resist water flow through a column compared to the non-treated samples. Also, in a tightness test, the differences in the ability to retain water within columns were observed among the samples tested. Moreover, despite the differences, the calcium salts tested still bound the sand together to form blocks. Our results further explain the influence of multiple factors in crystal formation and sand bioconsolidation effectiveness.     

Exopolymeric substances of sulfate-reducing bacteria: Interactions with calcium at alkaline pH and implication for formation of carbonate minerals

Sulfate‐reducing bacteria (SRB) have been recognized as key players in the precipitation of calcium carbonate in lithifying microbial communities. These bacteria increase the alkalinity by reducing sulfate ions, and consuming organic acids. SRB also produce copious amounts of exopolymeric substances (EPS). All of these processes influence the morphology and mineralogy of the carbonate minerals. Interactions of EPS with metals, calcium in particular, are believed to be the main processes through which the extracellular matrix controls the precipitation of the carbonate minerals. SRB exopolymers were purified from lithifying mat and type cultures, and their potential role in CaCO₃ precipitation was determined from acid‐base titrations and calcium‐binding experiments. Major EPS characteristics were established using infrared spectroscopy and gas chromatography to characterize the chemical functional groups and the sugar monomers composition. Our results demonstrate that all of the three SRB strains tested were able to produce large amounts of EPS. This EPS exhibited three main buffering capacities, which correspond to carboxylic acids (pK_a = 3.0), sulfur‐containing groups (thiols, sulfonic and sulfinic acids – pK_a = 7.0–7.1) and amino groups (pK_a = 8.4–9.2). The calcium‐binding capacity of these exopolymers in solution at pH 9.0 ranged from 0.12g_Ca g_EPS^?1–0.15 g_Ca g_EPS^?1. These results suggest that SRB could play a critical role in the formation of CaCO₃ in lithifying microbial mats. The unusually high sulfur content, which has not been reported for EPS before, indicates a possible strong interaction with iron. In addition to changing the saturation index through metabolic activity, our results imply that SRB affect the rock record through EPS production and its effect on the CaCO₃ precipitation. Furthermore, EPS produced by SRB may account for the incorporation of metals (e.g. Sr, Fe, Mg) associated with carbonate minerals in the rock record.     

The effect of adenosine triphosphate,magnesium chloride and phospholipids on crystal formation in the demineralized shell-repair membrane of the snail,Helix pomatia L.

Summary The effect of adenosine triphosphate (ATP), magnesium chloride (MgCl₂) and phospholipids on the calcium-binding activity and crystal formation within the decalcified shell-repair membrane of the snail, Helix pomatia, was studied in vitro. The application of ATP produced a characteristic dual effect on calcification: (1) It strongly inhibited the formation of inorganic calcium carbonate (CaCO₃) crystals. (2) It stimulated the development of organic crystalline bodies and induced deposition of amorphous calcium carbonate. The demineralized shell-repair membranes became white and rigid after incubation for 7 days in the medium containing 1.0mM ATP. The inhibitory effect of Mg²⁺ on CaCO₃ crystal formation was diminished by reduction of the concentration of MgCl₂ in the incubation solution. Thus, after incubation for only 24h, 1.0mM MgCl₂ promoted the formation of birefringent CaCO₃ crystals within the repair membranes. The principal effect of phospholipids on the demineralized shell-repair membrane was stimulatory, but after application of phospholipids to the medium, the formation of crystals proceeded slowly. The very large, composite crystals that were formed within the repair membranes showed strong birefringence. In all cases the development of the crystals and the organic crystalline bodies occurred in close vicinity to the amoebocytes. The role of ATP, MgCl₂ and phospholipids in the recalcification of shell-repair membrane is discussed.The author wishes to thank Mrs. E. Hellmén for valuable technical assistance     

Bacterial Calcium Carbonate Precipitation in Cave Environments: A Function of Calcium Homeostasis

To determine if microbial species play an active role in the development of calcium carbonate (CaCO ₃ ) deposits (speleothems) in cave environments, we isolated 51 culturable bacteria from a coralloid speleothem and tested their ability to dissolve and precipitate CaCO ₃ . The majority of these isolates could precipitate CaCO ₃ minerals; scanning electron microscopy and X-ray diffractrometry demonstrated that aragonite, calcite and vaterite were produced in this process. Due to the inability of dead cells to precipitate these minerals, this suggested that calcification requires metabolic activity. Given growth of these species on calcium acetate, but the toxicity of Ca ²⁺ ions to bacteria, we created a loss-of-function gene knock-out in the Ca ²⁺ ion efflux protein ChaA. The loss of this protein inhibited growth on media containing calcium, suggesting that the need to remove Ca ²⁺ ions from the cell may drive calcification. With no carbonate in the media used in the calcification studies, we used stable isotope probing with C ¹³ O ₂ to determine whether atmospheric CO ₂ could be the source of these ions. The resultant crystals were significantly enriched in this heavy isotope, suggesting that extracellular CO ₂ does indeed contribute to the mineral structure. The physiological adaptation of removing toxic Ca ²⁺ ions by calcification, while useful in numerous environments, would be particularly beneficial to bacteria in Ca ²⁺ -rich cave environments. Such activity may also create the initial crystal nucleation sites that contribute to the formation of secondary CaCO ₃ deposits within caves.     

Strain-Specific Ureolytic Microbial Calcium Carbonate Precipitation

During a study of ureolytic microbial calcium carbonate (CaCO₃) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO₃ crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (K_m) and maximum hydrolysis rates (V_max) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium.     

Meeting reports

Production of calcium carbonate by moderately halophilic bacteria was investigated. The crystalline structures were identified by x‐ray diffraction analysis. The results showed that the CaCO₃ formation was a general phenomenon for moderately halophilic bacteria and the different salt concentrations in the growth medium were an important factor in the characteristics of crystals formed.     

Aragonite crystallization in a Christmas-tree model microfluidic device with the addition of magnesium ions

Aragonite is an important dimorph of calcium carbonate, industrially and biologically. However, aragonite is so thermodynamically unstable that it is difficult to understand its formation mechanism. A continuous microfluidic system was employed, in which crystallization was induced only by diffusion in a micron-scale channel. Calcium carbonate (CaCO₃) formed by liquid-liquid reaction and magnesium ions (Mg²⁺) were used as additives. To assess the influence of Mg²⁺ concentration, the Mg²⁺/Ca²⁺ molar ratio was set to 1, 3, and 5. Laminar streams flowed in the detection channel with different concentration gradients. The initial crystallization time (t_I.C) increased exponentially and the density of crystals decreased as the Mg²⁺ ion concentration increased. Following transformation of all particles into snowman or sphere shapes, they became spinose sphere-shaped crystals, which was the final form in this study.