Biomineralisation by earthworms: an investigation into the stability and distribution of amorphous calcium carbonate

Background Many biominerals form from amorphous calcium carbonate (ACC), but this phase is highly unstable when synthesised in its pure form inorganically. Several species of earthworm secrete calcium carbonate granules which contain highly stable ACC. We analysed the milky fluid from which granules form and solid granules for amino acid (by liquid chromatography) and functional group (by Fourier transform infrared (FTIR) spectroscopy) compositions. Granule elemental composition was determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and electron microprobe analysis (EMPA). Mass of ACC present in solid granules was quantified using FTIR and compared to granule elemental and amino acid compositions. Bulk analysis of granules was of powdered bulk material. Spatially resolved analysis was of thin sections of granules using synchrotron-based μ-FTIR and EMPA electron microprobe analysis. Results The milky fluid from which granules form is amino acid-rich (≤ 136 ± 3 nmol mg−1 (n = 3; ± std dev) per individual amino acid); the CaCO3 phase present is ACC. Even four years after production, granules contain ACC. No correlation exists between mass of ACC present and granule elemental composition. Granule amino acid concentrations correlate well with ACC content (r ≥ 0.7, p ≤ 0.05) consistent with a role for amino acids (or the proteins they make up) in ACC stabilisation. Intra-granule variation in ACC (RSD = 16%) and amino acid concentration (RSD = 22–35%) was high for granules produced by the same earthworm. Maps of ACC distribution produced using synchrotron-based μ-FTIR mapping of granule thin sections and the relative intensity of the ν2: ν4 peak ratio, cluster analysis and component regression using ACC and calcite standards showed similar spatial distributions of likely ACC-rich and calcite-rich areas. We could not identify organic peaks in the μ-FTIR spectra and thus could not determine whether ACC-rich domains also had relatively high amino acid concentrations. No correlation exists between ACC distribution and elemental concentrations determined by EMPA. Conclusions ACC present in earthworm CaCO3 granules is highly stable. Our results suggest a role for amino acids (or proteins) in this stability. We see no evidence for stabilisation of ACC by incorporation of inorganic components.

Calcium removal from milk by ion exchange

Calcium removal, using Duolite C433 ion exchange resin, was faster from permeate than from milk. Almost all calcium could be removed, suggesting a fairly rapid conversion from both soluble calcium phosphate and from micellar calcium to ionic calcium. Calcium reduction from milk is accompanied by an increase in pH, a reduction in ionic calcium, an increase in ethanol stability and an increase in the rennet coagulation time. There is a gradual increase in the average casein micelle size with calcium removal, up to a point where the micelle size increases dramatically. Zeta potential becomes more negative with calcium removal. At higher levels of calcium removal, the changes are not reversible, on reducing pH to its original value. For goat's milk, over the range 0-20% total calcium removal, relatively small reductions in total calcium gave rise to proportionally larger reductions in ionic calcium in a ratio of about 1:3.2.

The role of gypsum in the reactions of phosphate with soils

The aim of this study was to examine the mechanisms by which gypsum increases the sorption of fertilizer-P in soils of and and semi-arid regions. Either gypsum or soil (Usher from the UK; pH 7.8, 7% organic matter, 21% CaCO3: Yasouj from Iran; pH 8.2, 1.4% OM, 18% CaCO3: Ghanimeh from Saudi Arabia; pH 7.8, 1% OM, 26% CaCO3, 13% gypsum) was shaken for 24 It with KH2PO4 solutions in 10 mM CaCl2. With gypsum, grinding increased sorption by a factor of about 3, and increase in pH from 5.6 to 7.5 greatly increased sorption. Scanning electron micrographs (SEM) and EDX quantitative analysis showed that small crystals of gypsum disappeared and roughly spherical particles of dicalcium phosphate (DCPD) were formed. Analysis of equilibrium Solutions showed, using GEOCHEM, that octa-calcium phosphate (OCP) coated the DCPD. For the soils, sorption was in the order Ghanimeh > Yasouj > Usher. Removal of gypsum from Ghanimeh reduced sorption, with precipitated gypsum having a greater effect than gypsum mixed physically with the soil. Addition to Usher had no effect. SEM and EDX could not be used in the soil matrix, but solubility analysis again showed that solutions were close to equilibrium with OCP. Usher was unresponsive to added gypsum, presumably because of its small sorption capacity and high organic matter content. In Ghanimeh and Yasouj soils, gypsum increased sorption by being a source of readily available Ca2+ (c) 2005 Elsevier B.V. All rights reserved.

Mineral partitioning in milk and milk permeates at high temperature

The soluble phase of milk was separated at 20 and 80°C using ultrafiltration. The resulting permeates were then subjected to further ultrafiltration and dialysis at close to these two temperatures. It was found that pH, Ca2+ and soluble Ca decreased as the separation temperature increased both in original UF permeates and in dialysates obtained from these permeates, but P decreased only slightly. The major reason for these changes was due to the precipitation of calcium phosphate/citrate complexes onto the casein micelle with concomitant release of H+. The pH of both permeates and dialysates from milk at 20°C were slightly higher than for milk. When UF permeates collected at 20 and 80°C, were each dialysed at both these temperatures, the dialysate collected at 80°C showed much less temperature dependence for pH and ionic calcium compared with that collected at 20°C. This is in contrast to milk, which shows considerable temperature dependence for pH and ionic calcium. Further experiments revealed that the pH and Ca2+ concentration of permeates showed high temperature dependence above the temperature at which they were separated, but a much lower temperature dependence below that temperature. These findings suggest that dialysis and UF of milk at high temperature provide the best means yet for estimating the pH and ionic calcium of milk at that temperature.

Naturally occurring radioactive material (NORM) from a former phosphoric acid processing plant

In recent years there has been an increasing awareness of the radiological impact of non-nuclear industries that extract and/or process ores and minerals containing naturally occurring radioactive material (NORM). These industrial activities may result in significant radioactive contamination of (by-) products, wastes and plant installations. In this study, scale samples were collected from a decommissioned phosphoric acid processing plant. To determine the nature and concentration of NORM retained in pipe-work and associated process plant, four main areas of the site were investigated: (1) the 'Green Acid Plant', where crude acid was concentrated; (2) the green acid storage tanks; (3) the Purified White Acid (PWA) plant, where inorganic impurities were removed; and (4) the solid waste, disposed of on-site as landfill. The scale samples predominantly comprise the following: fluorides (e.g. ralstonite); calcium sulphate (e.g. gypsum); and an assemblage of mixed fluorides and phosphates (e.g. iron fluoride hydrate, calcium phosphate), respectively. The radioactive inventory is dominated by U-238 and its decay chain products, and significant fractionation along the series occurs. Compared to the feedstock ore, elevated concentrations (<= 8.8 Bq/g) of U-238 Were found to be retained in installations where the process stream was rich in fluorides and phosphates. In addition, enriched levels (<= 11 Bq/g) of Ra-226 were found in association with precipitates of calcium sulphate. Water extraction tests indicate that many of the scales and waste contain significantly soluble materials and readily release radioactivity into solution. (c) 2005 Elsevier Ltd. All rights reserved.

Earthworms produce granules of intricately zoned calcite

Earthworms of the family Lumbricidae, which includes many common species, produce and secrete up to millimeter-sized calcite granules, and the intricate fine-scale zoning of their constituent crystals is unique for a biomineral. Granule calcite is produced by crystallization of amorphous calcium carbonate (ACC) that initially precipitates within the earthworm calciferous glands, then forms protogranules by accretion on quartz grain cores. Crystallization of ACC is mediated by migrating fluid films and is largely complete within 24 11 of ACC production and before granules leave the earthworm. Variations in the density of defects formed as a byproduct of trace element incorporation during calcite crystall growth have generated zoning that can be resolved by cathodoluminescence imaging at ultraviolet to blue wavelengths and using the novel technique of scanning electron microscope charge contrast imaging. Mapping of calcite crystal orientations by electron backscatter diffraction reveals an approximate radial fabric to the granules that reflects crystal growth from internal nucleation sites toward their margins. The survival within granules of ACC inclusions for months after they enter soils indicates that they crystallize only within the earthworm and in the presence of fluids containing biochemical catalysts. The earthworm probably promotes crystallization of ACC in order to prevent remobilization of the calcium carbonate by dissolution. Calcite granules vividly illustrate the role of transient precursors in biomineralization, but the underlying question of why earth-worms produce granules in volumes sufficient to have a measurable impact on soil carbon cycling remains to be answered.

Mineralised organic remains from cesspits at the Roman town of Silchester: Processes and preservation

Mineralised organic remains (including apple pips and cereal grains) collected during the ongoing excavations of Insula IX at the Roman town of Silchester, Hampshire have been analysed by a combination of SEM-EDX, powder XRD and IR spectroscopy. The experiments included mapping experiments using spatially resolved versions of each technique. IR and powder XRD mapping have been carried out utilising the synchrotron source at The Daresbury Laboratory oil stations 11.1 and 9.6. It is concluded that these samples are preserved by rapid mineralisation in the carbonate-substituted calcium phosphate mineral, dahllite. The rapid mineralisation leads to excellent preservation of the samples and a small crystal size. The value of IR spectroscopy in studying materials like this where the crystal size is small is demonstrated. A comparison is made between the excellent preservation seen in this context and the much poorer preservation of mineralised remains seen in Context 5276 or Cesspit 5251. Comments on the possible mechanism of mineralisation of these samples are made. (C) 2008 Elsevier B.V.. All rights reserved.

A study by high energy transfer inelastic neutron scattering spectroscopy of the mineral fraction of ox femur bone

We have used high energy transfer (HET) inelastic neutron scattering spectroscopy to measure the vibrational modes in the spectra of hydroxyapatite, bone and brushite to confirm our earlier work that only a fraction of the hydroxyl groups in bone mineral are substituted. The HET spectra are better observed due to the higher scattering cross section of hydrogen compared with the other elements in the calcium phosphate compounds. (C) 2003 Elsevier Science B.V. All rights reserved.

Mineralised organic remains from cesspits at the Roman town of Silchester: processes and preservation

Mineralised organic remains (including apple pips and cereal grains) collected during the ongoing excavations of Insula IX at the Roman town of Silchester, Hampshire have been analysed by a combination of SEM-EDX, powder XRD and IR spectroscopy. The experiments included mapping experiments using spatially resolved versions of each technique. IR and powder XRD mapping have been carried out utilising the synchrotron source at The Daresbury Laboratory oil stations 11.1 and 9.6. It is concluded that these samples are preserved by rapid mineralisation in the carbonate-substituted calcium phosphate mineral, dahllite. The rapid mineralisation leads to excellent preservation of the samples and a small crystal size. The value of IR spectroscopy in studying materials like this where the crystal size is small is demonstrated. A comparison is made between the excellent preservation seen in this context and the much poorer preservation of mineralised remains seen in Context 5276 or Cesspit 5251. Comments on the possible mechanism of mineralisation of these samples are made. (C) 2008 Elsevier B.V.. All rights reserved.

A study by high energy transfer inelastic neutron scattering spectroscopy of the mineral fraction of ox femur bone

We have used high energy transfer (HET) inelastic neutron scattering spectroscopy to measure the vibrational modes in the spectra of hydroxyapatite, bone and brushite to confirm our earlier work that only a fraction of the hydroxyl groups in bone mineral are substituted. The HET spectra are better observed due to the higher scattering cross section of hydrogen compared with the other elements in the calcium phosphate compounds. (C) 2003 Elsevier Science B.V. All rights reserved.

Root distributions of Australian herbaceous perennial legumes in response to phosphorus placement

Many Australian plant species have specific root adaptations for growth in phosphorus-impoverished soils, and are often sensitive to high external P concentrations. The growth responses of native Australian legumes in agricultural soils with elevated P availability in the surface horizons are unknown. The aim of these experiments was to test the hypothesis that increased P concentration in surface soil would reduce root proliferation at depth in native legumes. The effect of P placement on root distribution was assessed for two Australian legumes, Kennedia prorepens F. Muell. and Lotus australis Andrews, and the exotic Medicago sativa L. Three treatments were established in a low-P loam soil: amendment of 0.15 g mono-calcium phosphate in either (i) the top 50 mm (120 µg P g–1) or (ii) the top 500 mm (12 µg P g–1) of soil, and an unamended control. In the unamended soil M. sativa was shallow rooted, with 58% of the root length of in the top 50 mm. K. prorepens and L. australis had a more even distribution down the pot length, with only 4 and 22% of their roots in the 0–50 mm pot section, respectively. When exposed to amendment of P in the top 50 mm, root length in the top 50 mm increased 4-fold for K. prorepens and 10-fold for M. sativa, although the pattern of root distribution did not change for M. sativa. L. australis was relatively unresponsive to P additions and had an even distribution of roots down the pot. Shoot P concentrations differed according to species but not treatment (K. prorepens 2.1 mg g–1, L. australis 2.4 mg g–1, M. sativa 3.2 mg g–1). Total shoot P content was higher for K. prorepens than for the other species in all treatments. In a second experiment, mono-ester phosphatases were analysed from 1-mm slices of soil collected directly adjacent to the rhizosphere. All species exuded phosphatases into the rhizosphere, but addition of P to soil reduced phosphatase activity only for K. prorepens. Overall, high P concentration in the surface soil altered root distribution, but did not reduce root proliferation at depth. Furthermore, the Australian herbaceous perennial legumes had root distributions that enhanced P acquisition from low-P soils.

The effect of calcium removal from milk on casein micelle stability and structure

The total calcium level of raw skimmed milk was reduced by 10, 19, 29, 40 and 51% using Duolite® ion-exchange resin. The products were examined for concentrations of ionic calcium, sodium and potassium and the pH, ethanol stability, micelle diameter and ζ-potential were also measured. Ionic calcium decreased with removal of calcium and pH increased. Calcium removal resulted in an increase in the ethanol stability from 88% to above 100%. Casein micelle diameter increased as calcium was removed. The ζ-potential of the skimmed bulk milk was -24.4 mV, gradually becoming more negative with calcium removal to -30.6 mV after 51% calcium removal. The milk became more translucent as calcium was removed. To investigate the reversibility of this process, calcium chloride was added back to the depleted samples to restore their original total calcium content. At 51% removal, restoration of the total calcium level resulted in formation of clots. At levels of 10 and 19% calcium removal, the ethanol stability remained above 100%, but at higher levels of calcium removal the alcohol stability was adversely affected when the calcium was added back. Adding back calcium resulted in partial restoration of the original casein micelle diameter.

Effects of stabiliser addition and in-container sterilisation on selected properties of milk related to casein micelle stability

Different stabilising salts and calcium chloride were added to raw milk to evaluate changes in pH, ionic calcium, ethanol stability, casein micelle size and zeta potential. These milk samples were then sterilised at 121 °C for 15 min and stored for 6 months to determine how these properties changed. Addition of tri-sodium citrate (TSC) and di-sodium hydrogen phosphate (DSHP) to milk reduced ionic calcium, increased pH and increased ethanol stability in a concentration-dependent fashion. There was relatively little change in casein micelle size and a slight decrease in zeta potential. Sodium hexametaphosphate (SHMP) also reduced ionic calcium considerably, but its effect on pH was less noticeable. In contrast, sodium dihydrogen phosphate (SDHP) reduced pH but had little effect on ionic calcium. In-container sterilisation of these samples reduced pH, increased ethanol stability and increased casein micelle size, but had variable effects on ionic calcium; for DSHP and SDHP, ionic calcium decreased after sterilisation but, for SHMP, it remained little changed or increased. Milk containing 3.2 mM SHMP and more than 4.5 mM CaCl2 coagulated upon sterilisation. All other samples were stable but there were differences in browning, which increased in intensity as milk pH increased. Heat-induced sediment was not directly related to ionic calcium concentration, so reducing ionic calcium was not the only consideration in terms of improving heat stability. After 6 months of storage, the most acceptable product, in appearance, was that containing SDHP, as this minimised browning during sterilisation and further development of browning during storage.