title: Biomineralisation by earthworms - an investigation into the stability and distribution of amorphous calcium carbonate.
abstract: 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 CaCO3phase 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: ν4peak 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 CaCO3granules 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. Graphical abstractSynchrotron-based μ-FTIR mapping was used to determine the spatial distribution of amorphous calcium carbonate in earthworm-produced CaCO3granules.
authors.affiliation: Environment Department, University of York, YO10 5DD York, UK.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Hodson
authors.affiliation: Cohen Laboratories, School of Earth and Environment, University of Leeds, LS2 9JT Leeds, UK ; GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Telegrafenberg, 14473 Potsdam, Germany.
authors.forename: Liane G
authors.initials: LG
authors.lastname: Benning
authors.affiliation: BioArCh, Departments of Chemistry and Archaeology, University of York, York, UK.
authors.forename: Bea
authors.initials: B
authors.lastname: Demarchi
authors.affiliation: BioArCh, Departments of Chemistry and Archaeology, University of York, York, UK.
authors.forename: Kirsty E H
authors.initials: KE
authors.lastname: Penkman
authors.affiliation: Cohen Laboratories, School of Earth and Environment, University of Leeds, LS2 9JT Leeds, UK ; Nano-Science Center, Department of Chemistry, University of Copenhagen, 2100 Copenhagen, Denmark.
authors.forename: Juan D
authors.initials: JD
authors.lastname: Rodriguez-Blanco
authors.affiliation: Mineral and Planetary Sciences, Department of Earth Sciences, Natural History Museum, London, SW7 5BD UK.
authors.forename: Paul F
authors.initials: PF
authors.lastname: Schofield
authors.affiliation: Soil Research Centre, Department of Geography and Environmental Science, School of Archaeology, Geography and Environmental Science, University of Reading, Wokingham, RG6 6DW UK ; NASA Jet Propulsion Laboratory, California Institute of Technology, 4800 Oak Grove Drive, Pasadena, CA 91109 USA.
authors.forename: Emma A A
authors.initials: EA
authors.lastname: Versteegh
keywords: ACC; Amino acids; CaCO3; Calcite; Earthworms; FTIR; Stability; Synchrotron