Canti

G. N. Langworthy

M. E. Hodson

M. R. Lee

Morgan

Robertson

English

Lee, M.R.

Hodson, M.E.

Langworthy, G.N.

Central Archive at the University of Reading

Crystallisation of calcite from amorphous calcium carbonate: earthworms show the way

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Crystallization of calcite from amorphous calcium carbonate: earthworms show the way

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       Lee, M.R. and Hodson, M.E. and Langworthy, G.N. (2008) Crystallization of calcite from amorphous calcium carbonate: earthworms show the way. Mineralogical Magazine, 72 (1). pp. 257-261. ISSN 0026-461X  http://eprints.gla.ac.uk/24416/  Deposited on: 06 January 2010   Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk Crystallization of calcite from amorphous calcium carbonate:earthworms show the wayM. R. LEE1,*, M. E. HODSON2 AND G. N. LANGWORTHY2,31 Department of Geographical and Earth Sciences, Gregory Building, Lilybank Gardens, University of Glasgow,Glasgow, G12 8QQ, UK2 Department of Soil Science, School of Human and Environmental Science, University of Reading, RG6 6DW, UK3 ERM, Eaton House, Walbrook Court, North Hinksey Lane, Oxford OX2 0QS, UKIntroductionDARWIN (1881) was one of the ﬁrst scientists toformally record that earthworms excrete calciumcarbonate granules, with some up to 2 mm indiameter. However, since Darwin’s studies theyhave received relatively little attention. Thefunction of the granules remains a mystery, butmay be related to regulation of Ca, CO2, pH orsome other as yet undiscovered metabolic process(Robertson, 1936; Piearce, 1972). There hasrecently been an increase in research activityfocussed on earthworm calcium carbonate gran-ules driven by the work of Canti (Canti, 1998,2007; Canti and Piearce, 2003) and largelyrelating to their potential use in archaeology.Canti determined granule production rates of2.2 mg calcite day1 worm1 which translateinto C processing rates of 60 kg C ha1 y1 viaearthworm carbonate formation. These valuesindicate that granule production could play asmall but signiﬁcant role in the soil C cycle.Gago-Duport et al. (2008) have recentlypublished the ﬁrst detailed mineralogical studyof the calcium carbonate granules and have tracedtheir evolution from amorphous calcium carbo-nate (ACC) to calcite using scanning andtransmission electron microscopy (SEM andTEM), Fourier transform infrared (FTIR) spectro-scopy and X-ray diffraction (XRD). The aim ofour complementary study has been to investigatethe microstructures of the granules in order todetermine the mechanisms by which they growwithin the earthworm and subsequently crystallizeto calcite.MethodsClitellate Lumbricus terrestris (purchased fromBlades Biological Ltd. Cowden, Edenbridge Kent,TN8 7DX, UK) were cultivated in soil collectedfrom New Mayridge Farm (grid referenceSU618703) which was sieved to <250 mm priorto use. Individual earthworms were cultivated in 5cm diameter, 50 cm long polypropylene pipeseach containing 415 g of moist soil and were fed 5g of horse manure per week. After 28 days the soilwas removed from the columns and wet-sieved to250 mm. The >250 mm material was air-dried andstained with alizarin red to facilitate visualidentiﬁcation of the granules for hand-pickingfrom the mixture. The majority of the granuleswere digested in 5% nitric acid to determine theirtrace element composition; we also analysed thealizarin red solution used for staining. Theremaining granules were mounted in resin andpolished for high-resolution imaging andchemical analysis. The granules in cross-sectionwere imaged initially by optical cathodolumines-cence (optical-CL), then coated with a thin layerof carbon and imaged using backscatteredelectrons (BSE) in a FEI Quanta 200F ﬁeld-emission gun environmental scanning electronmicroscope (FEG-ESEM) operated at 20 kV.Following this initial characterization, high-resolution panchromatic CL images wereacquired using the FEG-ESEM (i.e. SEM-CLimages), which show net variations in emissionintensity over the ultra-violet to infra-redwavelength range (Lee et al., 2005). Afterremoval of the carbon coating, charge contrast(CC) images were acquired from the granuleswith the FEG-ESEM operated in ‘low vacuum’mode (chamber pressure ~60 Pa). These imagesare formed using secondary electrons and recordvariations in the degree of charge accumulationover the scanned area. The properties of the* E-mail: Martin.Lee@ges.gla.ac.ukDOI: 10.1180/minmag.2008.072.1.257Mineralogical Magazine, February 2008, Vol. 72(1), pp. 257–261# 2008 The Mineralogical Societycrystals that control the local degree of chargeaccumulation are poorly known, but probablyrelate to defect densities (Cuthbert and Buckman,2005). Granule microstructures were character-ized by electron backscatter diffraction (EBSD)employing an EDAX-TSL system attached to theFEG-ESEM, again in low vacuum mode. TheEBSD generates backscatter Kikuchi patterns thatcan be indexed in order to determine thecrystallographic orientation of the diffractingcrystal and can also be used to distinguishcrystalline from amorphous material by thepresence and absence, respectively, of Kikuchibands. The CL emission spectra spanningwavelengths 350850 nm range were acquiredusing a Cameca SX100 electron probe operated at20 kV and with a 2 mm spot (Lee et al., 2005).Results and discussionThe trace element composition of the granules isas follows. Because this is the ﬁrst detailedcompositional study of calcium carbonate gran-ules to be published, every element present in ourinductively coupled plasma optical emissionspectroscopy (ICP-OES) multi-element standardhas been included; values are the mean of threereplicate batches of calcite granules with standarderrors: 0.9 (N0.1) ppm Ag, 61 (N8) ppm Al,47 (N3) ppm Ba, 3 (N0.4) ppm Cu, 153 (N32) ppmFe, 7 (N1) ppm In, 352 (N23) ppm Mg,174 (N18) ppm Mn, 55 (N6) ppm P, 5 (N1) ppmPb, 345 (N23) ppm Sr and 10 (N2) ppm Zn.Elements that were analysed for but were belowdetection were (detection limits in brackets): B (3ppm), Bi (11 ppm), Cd (0.1 ppm), Co (0.2 ppm),Cr (0.1 ppm), Ga (0.1 ppm), K (113 ppm), Li (0.5ppm), Ni (0.1 ppm), Tl (0.1 ppm). Analysis of thealizarin red solution used to stain the granulesgave the following concentrations, recalculated tothe mass of the solid in the alizarin red solution:160 ppm B, 6 ppm Co, 93822 ppm Fe, 23 ppmGa, 195662 ppm K, 4 ppm Mg, 2 ppm Sr and 13ppm Zn. On the basis of elements detected in thealizarin red solution, elements below detectionlimits in the calcite granules and calculateddetection limits, the maximum concentrations ofelements contributed to granules from the stainwere: 8149 ppm Fe, 0.4 ppm Mg, 0.2 ppm Sr and1 ppm Zn. Thus the majority, if not all the Fedetected in the granules is probably stain-derivedbut the Mg, Sr and Zn values most likelyrepresent true concentrations in the granules.The BSE images of polished cross-sections ofthe granules are shown in Fig. 1. Unlike those ofCanti and Piearce (2003), the edges of thesegranules appear etched, which is due to slightdissolution during staining. The granulescomprise an agglomeration of grains, predomi-nantly calcium carbonate, and have complexinternal structures; we identiﬁed two varieties,compact and concentrically laminated (Fig. 1a)and porous and structureless (Fig. 1b). Allgranules are circular to oval in cross sectionwith aspect ratios of 1.01.7 (Fig. 1). They arecomposed predominantly of calcium carbonatebut three of the 21 granules imaged had quartzcores 50300 mm in diameter (Fig. 1a). Thispercentage of quartz cores (14%) is consistentwith all the granules having quartz cores, howeverFIG. 1. BSE images of granules. (a) A compact granule with a quartz (Q) core and concentric internal structure; and(b) a porous and structureless granule containing numerous small quartz inclusions (arrowed).258M. R. LEE ET AL.they were only observed in granules cross-sectioned close to their centre. Quartz cores toearthworm calcium carbonate granules have notbeen reported previously and are likely to be ofsigniﬁcance in their formation.The EBSD mapping indicates that granules cancontain both calcite and amorphous calciumcarbonate (ACC). The calcite is most abundantand crystals are euhedral and interlocking.Granule calcite is a homogeneous orange inoptical-CL, whereas higher resolution CC andSEM-CL imaging reveals intricate and ﬁne-scalezoning (zone widths of ~2002000 nm) in allcrystals (Fig. 2a,b). The CL spectroscopy showsthat the calcite has two luminescence emissionpeaks, at ~400 nm (ultra-violet to blue) and ~600nm (orange) (Fig. 2c). The longer wavelengthpeak is indicative of Mn2+ activation whereas theultra-violet to blue peak is the ‘intrinsic’luminescence of calcite and likely to be activatedby defects, possibly including those associatedwith Fe2+ substitution (Lee et al., 2005). Thezoning recorded by SEM-CL corresponds closelywith variations in intensity of the ~400 nm peak.The pattern of zoning recorded by SEM-CL iscomparable to that in CC images (Fig. 2a,b),which is consistent with contrast in the CC imagesalso being determined by intracrystalline differ-ences in defect density. The boundaries betweenzones are oriented parallel to calcite crystalterminations and traces of their {1014} planes,thus demonstrating that they represent the formerposition of crystal faces. The EBSD mappingshows that granule calcite crystals, which tend tobe elongate, are oriented with their long axesnormal to the quartz core and granule edge(Fig. 3a). Calcite crystals also show a weakpreferred orientation, with poles to {0001}planes lying at a low angle to the granulemargin (Fig. 3b).The ACC can be identiﬁed readily within thegranules by its ‘stromatolitic’ appearance in BSEimages (Fig. 4a) and the absence of diffractionbands in Kikuchi patterns (Fig. 4b,c). The ACCbodies are ~30300 mm in size, usually enclosedby calcite, and comprise up to 20% of any onegranule. The interface between calcite and ACC issharp but irregular and the calcite cross-cuts theradial and concentric internal structure of theACC. In contrast to Gago-Duport et al. (2008), wedetected no aragonite or vaterite within thegranules using EBSD.Our observations suggest that the calciumcarbonate spherulites previously observed toFIG. 2. (a) CC image of calcite close to a granule edge showing pervasive and very ﬁne-scale zoning. (b) SEM-CLimage of the boxed area in (a). Note the close correspondence in zoning patterns revealed by the two imagingtechniques. (c) CL emission spectra from two wide zones identiﬁed by SEM-CL, one bright and one dull. Thesespectra indicate that the contrast in the SEM-CL image is due to variations in intensity of the ~400 nm emission bandwhereas the ~600 nm orange band shows little intensity variation.EARTHWORMS AND CALCITE CRYSTALLIZATION259form in the earthworm calciferous gland (Morgan,1981; Gago-Duport et al., 2008) produce granulesby adhering to large soil particles. The presenceof small quartz and feldspar inclusions withingranules indicates that they grew gradually and byaccretion of ACC spherulites in an environmentFIG. 3. Results of EBSD mapping of a granule. (a) Image quality map showing a large quartz core (Q) away fromwhich radiate elongate calcite crystals. (b) Contoured {0001} equal angle pole ﬁgure of calcite crystals in (a)showing that the majority of crystals are oriented with {0001} at a low angle to the granule margin (i.e. close to theequator of the ﬁgure).FIG. 4. (a) BSE image of an inclusion of ACC within a calcite granule displaying the very ﬁne-scale ‘stromatolitic’internal structure. (b) Backscatter Kikuchi pattern of the ACC illustrating its amorphous nature by lack of diffractionbands. (c) Backscatter Kikuchi pattern of granule calcite showing well-deﬁned diffraction bands indicative ofcrystallinity.260M. R. LEE ET AL.that also contained ingested soil. In agreementwith the ﬁndings of Gago-Duport et al. (2008),our results suggest that following accretion, theACC crystallized to calcite, although we found noevidence for a vaterite precursor. The pattern ofzoning in calcite indicates that crystallization tookplace by dissolution-reprecipitation as ﬂuid ﬁlmson calcite crystal faces migrated through the ACCand the fabric within granules revealed by EBSDindicates that crystals grew approximatelyradially outwards from the quartz nuclei. Thecalcite zoning has probably developed in responseto regular ﬂuctuations in trace-element concentra-tions of the ﬂuids due to cyclical incorporation,depletion and replacement of solutes. Weconclude that earthworm granules provide anunprecedented opportunity for detailed study ofthe processes and products of crystallization ofACC, which have important implications forunderstanding biomineralization throughout theanimal kingdom.AcknowledgementsThis work was supported by NERC grantNE/F009623/1 and a NERC studentship awardedto Graham Langworthy by the University ofReading Soils and Environmental PollutionMasters programme. We thank Sir WilliamBenyon for permission to collect soil on theEngleﬁeld Estate and Mr John Jacks for makingthe calcite resin blocks.ReferencesCanti, M.G. (1998) Origin of calcium carbonategranules found in buried soils and Quaternarydeposits. Boreas, 27, 275288.Canti, M.G. (2007) Deposition and taphonomy ofearthworm granules in relation to their interpretativepotential in Quaternary stratigraphy. Journal ofQuaternary Science, 22, 111118.Canti, M.G. and Piearce, T.G. (2003) Morphology anddynamics of calcium carbonate granules produced bydifferent earthworm species. Pedobiologia, 47,511521.Cuthbert, S.J. and Buckman, J.O. (2005) Charge contrastimaging of ﬁne-scale microstructure and composi-tional variation in garnet using the environmentalscanning elec tron microscope. AmericanMineralogist, 90, 701707.Darwin, C. (1881) The Formation of Vegetable Mould,Through the Action of Worms, with Observations ontheir Habits. John Murray, London.Gago-Duport, L., Briones, M.J.I., Rodriguez, J.B. andCovelo, B. (2008) Amorphous calcium carbonatebiomineralisation in the earthworm’s calciferousgland: pathways to the formation of crystallinephases. Journal of Structural Biology, (in press).Lee, M.R., Martin, R.W., Trager-Cowan, C. andEdwards, P. R. (2005) Imaging of cathodolumines-cence zoning in calcite by scanning electronmicroscopy and hyperspectral mapping. Journal ofSedimentary Research, 75, 313322.Morgan, A.J. (1981) A morphological and electronmicroprobe study of the inorganic composition of themineralized secretory products of the calciferousgland and chloragogenous tissue of the earthwormLumbricus terrestris. L. Cell and Tissue Research,220, 829844.Piearce, T.G. (1972) The calcium relations of selectedLumbricidae. Journal of Animal Ecology, 41,167188.Robertson, J.D. (1936) The function of the calciferousglands of earthworms. Journal of ExperimentalBiology, 13, 279297.EARTHWORMS AND CALCITE CRYSTALLIZATION261

A morphological and electron microprobe study of the inorganic composition of the mineralized secretory products of the calciferous gland and chloragogenous tissue of the earthworm Lumbricus terrestris.

Amorphous calcium carbonate biomineralisation in the earthworm’s calciferous gland: pathways to the formation of crystalline phases.

Charge contrast imaging of ﬁne-scale microstructure and compositional variation in garnet using the environmental scanning electron microscope.

Deposition and taphonomy of earthworm granules in relation to their interpretative potential in Quaternary stratigraphy.

Imaging of cathodoluminescence zoning in calcite by scanning electron microscopy and hyperspectral mapping.

Morphology and dynamics of calcium carbonate granules produced by different earthworm species.

Origin of calcium carbonate granules found in buried soils and Quaternary deposits.

The calcium relations of selected Lumbricidae. J o u r n a lo fA n i m a lE c o l o g y

The Formation of Vegetable Mould, Through the Action of Worms, with Observations on their Habits.

The function of the calciferous glands of earthworms.

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Crystallization of calcite from amorphous calcium carbonate: earthworms show the way

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