Morphology and the chemical make-up of the inorganic components of black corals

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

http://www.elsevier.com/copyright

Author's personal copy

Morphology and the chemical make-up of the inorganic components of black corals

D. Nowak a, M. Florek a, J. Nowak a, W. Kwiatek b, J. Lekki b, P. Chevallier c, A. Hacura d, R. Wrzalik d,B. Ben-Nissan e, R. Van Grieken f, A. Kuczumow a,⁎a Department of Chemistry, John Paul II Catholic University of Lublin, 20-718 Lublin, Polandb Institute of Nuclear Physics, Department of Nuclear Spectroscopy, 31-342 Krakow, Polandc former LPS, CEN Saclay et LURE, Université Paris-Sud, Bat 209D, F-91405 Orsay, Franced Institute of Physics, Silesian University, 40-007 Katowice, Polande Department of Chemistry, Materials and Forensic Sciences, University of Technology, Sydney, PO Box 123, Broadway 2007, NSW, Australiaf Department of Chemistry, University of Antwerp, B-2610 Antwerp, Belgium

a b s t r a c ta r t i c l e i n f o

Article history:Received 8 April 2008Received in revised form 22 August 2008Accepted 24 August 2008Available online 9 September 2008

Keywords:Black coralsChitinIodineLow- and medium-Z elementsElectron and proton microanalysis

Black corals (Cnidaria, Antipatharia) from three different sources were investigated with the aim of detectinginorganic components and their morphology. In general, the skeleton of black corals was composed of thechitin fibrils admixed with peptides and the chitin presence was confirmed by the X-ray diffraction (XRD),Fourier Transformed Infrared Spectrometry (FTIR) and microRaman Microscopy, the latter giving theopportunity of tracing single fibrils and their location. The composition and concentrations of the inorganiccomponents of the black corals were measured, using a scanning electron microprobe and micro-ParticleInduced X-ray Emission (µ-PIXE). The application of such instruments enabled the estimation of theconstituent distributions in a microscale. The mapping option was the most useful technique of makinganalyses in these studies, just to reveal the composition of chamber-like cells. Analysis of the morphologyand microstructure showed that there were three distinct regions within the coral: a core and the cellsencircled with adjacent interface gluing strips. The majority of the elements analyzed were selectivelydistributed and segregated in a striking way in mentioned distinctive zones of the skeleton and it wasdetected for the first time. The core area was characterized by the relatively elevated concentrations of Ca.The measurements gave extremely clear images of the distribution of particular elements in the skeletaltissue, with I, Ca, K and Fe much more concentrated in the gluing zones, while C, N, Na and Mg present in theinteriors of particular skeletal cells. The distribution of some elements (Mg, Fe) and some compounds(chitin) and functional groups (S–S, C–I) allows differentiating the biological and mechanical functions ofparticular fragments of the rods. The kinds of elements and their concentrations measured were essentiallyin compliance with rare data available in the literature. The Raman technique gave the additional qualitativeinformation about the structure of gluing zone and the chitin fibrils and surrounding matrix inside the cellinterior.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Black corals are common species living in all oceans around theworld. However, they are much less known in comparison with theirred counterparts. Although the deep-water environments of tropicaland subtropical ocean are their preferred locations, nevertheless,some of them live in the unfavorable environment of far northern orsouthern seas and fjords. Their habitat is very different from theknown shallow environment of the coral reef that is characteristic forthe red corals. Very importantly, black corals are not symbiotic withalgae (zooxanthellae) and for that reason they are independent ofsunlit conditions.

Black corals can live in underwater caves, under ledges, in placesswept by sea currents and very often at deep locations in the ocean.Although some black corals have been found in shallow waters, inlocations up to 80 m deep, mainly on the slopes of the reefs, othershave been observed in very deep positions, reaching 6000 m. Coloniescan grow up to 1.8 m in size. Their abundance is dependent on theavailable space in favourable conditions [1]. The Antipatharia order(black coral) is divided into approximately 160 species of colonialorganisms and new species are still being discovered [2,3]. All speciesdemonstrate slow growth, compensated for by a long life and lownatural adult mortality. Their first reproduction is after about 10 years,which is much later than for other similar organisms. The outputof larvae (called planula) is small. The recovery occurs at a slow rate[4]. Particular fragments (polyps) reach dimensions ranging from0.5 to 5 mm, however, the majority is sized between 1 and 2 mm [5].Black corals obey life cycles regulated through asexual and sexual

Materials Science and Engineering C 29 (2009) 1029–1038

⁎ Corresponding author.E-mail address: [email protected] (A. Kuczumow).

0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.msec.2008.08.028

Contents lists available at ScienceDirect

Materials Science and Engineering C

j ourna l homepage: www.e lsev ie r.com/ locate /msec


reproduction. In the context of our investigations, the asexual re-production is more important. In this kind of reproduction, livingtissue is added and, in consequence, the skeleton is secreted as well.The skeleton is arranged in the shape of a clear ring structure, whichcan be used for the estimation of the age of corals. Growth rings in theskeletons of Antipatharian corals have indeed been correlated withage [4,6–8]. This suggests that, as in the case of some red corals,additional information potentially stored in the rings (e.g., on thechronology, the water temperature, salinity) can be extracted anddeciphered.

Unlike the skeletons of their red and white relatives, the skeletonof black corals is composedmainly of organicmatter. The order's namecomes from the brilliant substance called antipathin, which iscomposed mainly of chitin and proteins [9,10]. From the chemicalpoint of view, this material is very similar to the insect cuticle [11,12].The skeletons of Antipatharians are more elastic and less rigid thansome other biomaterials selected and used in the nature as structuralcomponents, including wood, bone, mollusc shell and the insect cu-ticle. At the same time, the density of antipathin is higher than wood,lower than shell or bone, but nearly the same as insect cuticle. Highstiffness is often a bonus, since it enables the construction of strongand light skeletons. The hardness of the skeleton (Antipathes fiordensis

and Antipathes salix) on the Mohs scale is 3. It is sufficiently hard toscratch calcite [13]. It is a little strange that organisms living inconditions that provide them with only a small chance to obtain a lotof energy have decided to form such a refined and energy-consumingwhile constructed skeleton of chitin instead of the simple, aragonite-based and nearly self-sedimented inorganic skeleton. The probableexplanation is that the organic skeleton, due to its elasticity, obeys thenatural wave movements of the water streams. This makes huntingstrategies easier. In addition, after the initial expending of materialand energy on construction of the skeleton, the organism saves theenergy later on, with a total large surplus over the scale of a life time(~40 years for colonies reaching a maximum of 1.8 m in height).

From the morphological and mechanical point of view, black coralskeletons are laminated composites, composed primarily of chitinfibrils and non-fibrous proteins. In a composite structure, the fibrilscan be expected to stiffen the more deformable matrix by reinforcingit. Skeletal layers consist of light and dark bands, forming the ringstructures mentioned earlier.

All the species of black corals have a considerable chitin content,ranging from 6 to 18% of the total mass of the skeleton [9,10,14]. In themost complete experimental study, published by Goldberg et al. [15],it was estimated that the chitin represents 14.5% of the skeletal massin Antipathes salix. However, the protein fraction of the skeleton ismuch higher. Although nearly all common amino acidswere identifiedin this substance, several of them clearly prevailed, comprising morethan two thirds of the total protein content. The majority (over 5% ofthe total contents) of the amino acids in Antipathes salix are: thyroxine14.8%, alanine 12.7%, glycine 10.8%, aspartic acid 9.2%, histidine 7.5%,threonine 6.8%, and valine 6.7%. The environmental protein concen-tration seems not to influence the ratio of amino acids in the skeletonof the black corals mentioned [15]. Among the amino acids, the veryhigh content of histidine is striking, reaching up to 18% of the totalamino acids' mass [9,16,17], specifically for the species. A comparisonof protein content and amino acid composition shows a rather con-stant composition for Antipathes fiordensis and Antipathes salix andonly a small difference between the tip and base locations inside thesame species of black corals [15]. The really important difference inthe amino acid composition is linked to the content of the gluingzone between the skeletal cells: the amino acid composition [18] may

Table 1Contents of the detected elements in black corals.

Element AAS (authors'results) [ppm]

PIXE (authors'results) [ppm]

PIXE ((Goldberget al., 1994)results) [ppm]

XPS ((Kaczorowskaet al., 2003)results) [ppm]

Mg 700–800 1500–3500Ca 350–1300 60–200 1500–4500Fe 80–250 15–80 100–1100Na 1400–2400 2850–29,000 1600K 100–400 30 850Si 10–260 200 23,900Al 700–1400 450S 240–300 1300–4300 1100Cu 25–50 300Cl 300–470 4300–23,000 1500Br 500–800 5700–10,000I 2300–5000 40,000 1800

Fig. 1. Diffractograms of black coral, sample no. 1, analysed in the interior cellular position (dotted line) and in the wall position (dash-dot-dot line) — both as synchrotron-basedmeasurements at LURE, compared with the conventional diffractogram of standard α-chitin (solid line), measured by the use of the tabletop instrument. Synchrotron-based 2θvalues are recalculated to the conventional values from the tabletop instruments with Cu tube.

1030 D. Nowak et al. / Materials Science and Engineering C 29 (2009) 1029–1038


suggest an electrostatic binding function (i.e., an interlaminar glue) ifone takes into account the excess of carboxyamino acids relative to thewhole skeleton.

Extractable lipids, supplementary to the chitin and proteins arepresent at rather low and variable concentrations in the mentionedspecies [14]. Using the Nuclear Magnetic Resonance (NMR) technique,the organic content of the skeleton base of Antipathes fiordensis wasestimated as 70% protein, 10% chitin, 15% diphenol and 5% lipid [19].The composition of the skeleton of Antipathes salix is somewhat dif-ferent, with 54% of protein and 15% of chitin and small amounts (~4%)of o-diphenols, as 3,4-dihydroxyphenylalanine (DOPA). The lipoidalcontent of the skeleton seems to be associated with the gluing cement[15]. In general, the gluing zone should have the elevated amounts ofthe carboxyamino acids and lipids in comparison with the interior ofthe cells.

Much less is known about the inorganic content of black corals,especially if one compares it with Goldberg's findings in the range oforganic components. Although the organic character of the skeleton inblack corals is beyond dispute, the uncertainties in the content of the

inorganic species are easily noticeable. The report by Goldberg et al. [15]cites the Proton Induced X-ray Emission (PIXE) analysis of the skeletonsof Antipathes salix and Antipathes fiordensis. It shows the presence of 22elements. Some of them occur irregularly and are in rather negligibleamounts (Cr, Mn, Ni, Sr, As and Pb), while others (Al, Si, Ti and P) al-though in trace amounts, occur nearly always. On the opposite side, Na,Cl, Br and I are present inmuchhigher concentrations, of the orderof oneto several weight per cent. A strict correlation between the levels of Naand Cl has been established, and this probably results from their uptakefrom the seawater as sodium chloride. The I levels are variable, with anincrease in the tip in comparison towithin the base. Taken together, the Iand Br concentrations reach 5% of the skeletal weight inAntipathes salix.Otherwise, one can estimate theupper limit of the I contents in the blackcoral matrix: if the content of proteins is ~70%, the thyroxine in theproteins reaches 15% and iodine in the thyroxine is 65%, then the iodineconcentration can be estimated as reaching even 6.8%. This is inreasonable agreement with Goldberg's analysis [15], (see also Table 1).

Mg, Ca, S, Fe and K are the common components, but their levelsare of the order of tens of percentage. It was reported [20] that the

Fig. 2. Fig. 2. FTIR spectra of black coral sample no.1 in our measurements (black solid line): A) as compared with the FTIR spectrum of pure α-chitin (dotted line) and B) with themeasurements on Antipathes salix (grey solid line) and with the results by Opresco [33] – all other lines.

1031D. Nowak et al. / Materials Science and Engineering C 29 (2009) 1029–1038


spectral bands, characteristic for calcite and silica, can be detected inthe Fourier-Transform InfraRed (FTIR) spectra of black coral fromCuba. This claim can be enforced per analogy, since calcium carbonateis detected in the somewhat similar shells of crabs and shrimps.

As stated earlier, although the organic components of the blackcorals (especially chitin [21]) are well established, the morphology andspatial distribution of their elemental structure within the matrix andtheir influence on the growth structure are all unknown. The studiesand understanding of the role of inorganic compounds in theconstruction and function of the skeleton are in the very early stages.Several elements (Ca, I, Mg) seem to be of potential anatomical andphysiological importance. The aim of this paper is to recognize thissituation.

2. Materials and methods

2.1. Materials

Three samples of black corals (Cnidaria, Antipatharia) wereanalysed. The first was collected from the Chinese Coast close to

Hong Kong (~1 cm diameter). The second belonged to the Antipathessalix species and was much thinner (diameter ~4 mm). This samplewas supplied by Professor Goldberg andwas collected in the Caribbeanregion close to the Bahamas. The third sample came from theunidentified location in the Gulf of Mexico (~4 mm diameter). Forthe investigation, thin transverse cross-sections were prepared afterremoving the soft tissue. Some thin sections were cut from two sides.The cutting was in direction perpendicular to the main growth axis ofthe branch. It was carried out with water-cooled diamond saw. Next,slices were carefully polished from one side only to obtain smoothsurfaces. The final thickness was approximately 150 µm. The sampleswere rinsed with distilled water and dried in the room temperature.They were initially examined by the optical microscope and next bySEM (scanning electron microscope). All the studies were carried outwithin two days after sample preparation. It left the samplesundamaged by the time of studies.

To avoid the random findings, the samples were prepared in theduplicates; also, themeasurementswere repeated.Moreover, the resultsfrom different methods were intercompared and compared with theoptical images. All this confirmed the repeatability of the results. The

Fig. 3. The transverse cross section of black coral (sample no.2) as observed in backscattered electrons (magnification 900×) in SEM and consecutive elemental mappings from theelectron microprobe for C; Na; O; N; Mg; Br; I; Ca; S. All images cover the same location 425×316 µm.



remains of the material are preserved to be potentially used in otherfurther studies if we have in the future access to other useful methods,e.g. EXAFS (Extended X-ray Absorption Fine Structure) or XANES (X-rayAbsorption Near Edge Structure).

2.2. Instrumentation

Two different microprobes were used for the analysis of thedistribution of the inorganic components in the skeleton of the blackcoral: electron (Electron Probe MicroAnalyser - EPMA) and proton(micro-Particle-Induced X-ray Emission - µ-PIXE) ones.

The electron microscope is applied to obtain the backscatteredelectron (optionally secondary electron) images that supplement theelemental mappings and help to relate the chemical information to

the topographical details on the sample. Each time, the relevantbackscatter electron image of the analysed location was stored. Tovisualize the images better, the image processing program Micro-Image v.4.0 (Olympus) was applied. A scanning electron microscopeLEO 1430 VP (manufactured by LEO, Austria) at the Catholic Universityof Lublin) was used in this study. The electron microscope wasequipped with an Si(Li) detector (Röntec) with an energy resolutionof 180 eV, as measured for MnKα line. All the experimental work wascarried out with the accelerating voltage set at 20 keV and the beamcurrent at 0.7 nA. Such a selection of parameters provided the op-portunity to detect both medium- and low-Z element, includingcarbon. Due to the fact that 2D distributions of the elements on thetransverse cross-sections of corals were important in this study, wechose the mapping option.

Fig. 4. Chemical mappings of black coral made by µ−PIXE method (sample no.1). Successively, the maps of Cl, K, Al, I, S, Si, Ca, Fe and Cr, Si are presented. The first image is from theoptical microscope. All images concern the same location 250×250 µm.



The µ-PIXEmeasurementswere performed at theNiewodniczańskiInstitute of Nuclear Physics, Cracow, Poland. This method was selectedsince the detection limits for the elements are much lower here thanin the electron microprobe, due to small Bremsstrahlung backgroundwith proton excitation. It enables the better detection of trace ele-ments. The Cracow proton microprobe belongs to the category ofrelatively short (total length 230 cm) instruments. The Van der Graafaccelerator was a base for the device. The particle-focusing optics ofthe instrument was arranged using two doublets of magnetic quad-rupole lenses, named the “Divided Russian Quadruplet”. The demag-nification factor was 17.7 for an appliedworking distance of 15 cm. Thebeam spot on the target was selected as below 6 µm×6 µm if using theobject aperture of 10 µm. The beam current on the target was ~1 pA.The applied protons with energy of 2.5 MeV were deflected by±125 µm in two dimensions on the sample by a magnetic scanningsystem. This system was installed in between the focusing opticalsystem and the sample-holder. The construction and working para-meters of the Cracow PIXE device have been described in more detailby Lebed et al. [22].

X-ray microdiffraction experiments were carried out at Laboratoirepour l'Utilisation du Rayonnement Électromagnétique, Orsay - LURE(Orsay, Paris) — now closed. The device was an end station at the D15beamline at the DCI storage ring. Chevallier et al. [23] and Dillman et al.[24] have describedboth the construction and functioningof this stationin detail. The primary polychromatic beam was focused and mono-chromatized by the use of theBragg FresnelMultilayer Lens (BFML). Theselected energy of photonswas about 15 keV. Next, the beampassed theinput pinhole that reduced the diameter of the beam to 15 µm. Itdetermined the size of the details that could be traced on the surface ofthe samples. This size matched or was less than the width of the gluinglayer in the black corals. The settingof the location on the sample againstthe beam was executed by the computer-controlled work of four stepmotors. The transmission mode of operation was applied for X-raymicrodiffraction. Themeasurements were performed both on the chitinstandard and on the samples at the locations belonging both to thegluing zone and to the cell interior. The diffraction images were re-gistered on 2D-CCD (Charge Coupled Detector) imaging plate, made byFuji. Such a plate collected the diffraction imagewithin 40° of 2θ if it wasplaced ~10 cm outside the sample, perpendicular to the originaldirection of X-ray beam. The time of measurement was selected alwaysas 45 min. This provided sufficient efficiency of exposition and goodstatistical repeatability. The hidden imagewas scanned from a plate by aMolecular Dynamic Scanner and transformed to the image-type datafile. The grey scale levels of the diffraction rings were read and integra-ted from the data in the file by the use of FIT2D software [25]. Theapplication of the imaging plates in diffraction measurements has beendescribed by Ermrich et al. [26]. Next, the 2θ values of peaks were read,recalculated on apparent excitation by the copper tube and comparedwith standards from the JCPDF (International Centre for DiffractionData) database. The DIFFRAC plus software ensured the automaticcomparison and identification of the diffraction spectra.

Due to the fact that both electron microprobe and PIXE resultswere only semiquantified, the atomic absorption method was appliedto check the concentrations, on a macroscale only. The PolarizedZeeman Atomic Absorption Spectrophotometer [AAS], Model Z-8200(made by Hitachi, Japan), working in a flame mode, was applied. Thesmall fragments of the samples were dissolved in microwave digesterMaxidigest MX 4350, (made by Prolabo, France). The samples weredissolved in two cycles of digestion with 35–38% HCl p.p.a. at 60 °C,were then diluted to the assumed volume and analysed. The methodof the multiple standard additions was applied for quantification.

For the micro-Raman studies, a inVia Reflex (made by Renishaw,England) spectrometer was applied. A stereoscopy microscope (pro-duced by Leica, Germany) with attached CCD camera was associatedwith the main instrument to enable the strict location of the excitedareas. The laser diode emitting the radiation of thewavelength 785 nmwas applied for excitation. The power usedwas 300mW. TheRenishawSynchroScan mode was from 400 to 3200 cm−1 with a spectral re-solution about 2 cm−1 and spatial resolution of ~1 µm.

For the analysis of the total organic contents in a macroscale, 2400CHN (Carbon–Hydrogen–Nitrogen) Elemental Analyzer (made byPerkin-Elmer, USA) was applied. This equipment uses a combustionmethod in a pure oxygen environment to convert the accuratelyweighed sample into the simple gases: CO2, H2O, andN2. After reductionthrough the pure Cu, the resulting C-, H- and N-containing gases werecontrolled to exact conditions of pressure, temperature and volume. Thesystem used a steady-state wave-front approach to separate thecontrolled gases. This approach involved separating a continuoushomogenized mixture of gases through a chromatographic column.

The additional analysis of the coral matrix was performed by anMB-120 FTIR spectrophotometer (manufactured by Bomem). Thisanalysis was on a macro-scale and covered the large fragments of thesample.

All the samples of black corals were observed by the use of theEclipse E400 optical microscope and the selected images werephotographed by the use of an attached Coolpix 950 digital camera(Nikon Europe B.V., The Netherlands). The images were collected asthe numerical files. The analysis of images was performed using theimage-processing program LUCIA G. The images could be comparedwith the results of chemical mappings from the electron microprobeor PIXE.When the function of the linear profile extractionwas used forphotos, the resulting optical scan could be easily compared with thechemical scans [27].

3. Results

3.1. Matrix

The analysis of the CHN content of black coral resulted in thefollowing atomic ratios: C:H:N=1.00 : 1.67 : 0.30. The results werefairly similar to the data extracted fromGoldberg's [14] estimations: C:H:N=1.00 : 1.83 : 0.26. For comparison, the ratio for pure chitin wasobtained as: 1.00:1.64:0.125. Our results revealed a small surplus of Nand moderate deficit of H in comparison with Goldberg's data,meaning either a slightly higher proportion of proteins composed oflightest or multinitrogen aminoacids, or a smaller proportion of chi-tin in our samples or both. The lower H content observed can beattributed to the different level of drying between the two studies andis less informative. The water is always present in the skeleton, inquantities close to 20% in Antipathes salix [13].

The diffraction results confirmed the dominating role of theα-chitinin the structure of black corals. Fig.1 shows the comparison between theresults of the diffraction measurements carried out on the corals withthe standard α-chitin diffractogram, obtained by the traditional table-top diffractometer. The standard chitin diffractogram obtained in thisstudy was compared with the standards published by Carlström [28],Kato et al. [29], Zhang et al. [30] and Manoli et al. [31] and the result

Table 2Relative contents of elements in the growth ring, gluing zone and central core ofAntipathes salix, ratioed against the concentration in the growth ring.

Element Growth ring Gluing zone Central core

Mg 1 0.58 ndCa 1 1.6 5.86Sr 1 0.59 0.9Zn 1 1.18 6.45C 1 0.79 0.62P 1 0.46 ndS 1 0.73 0.8Br 1 0.76 0.62I 1 1.93 1.9

Results based on the averaged SEM measurements.



indicated an excellent agreement between these studies (here notshown). Hence, the result obtained in this study was always treated asthe reference spectrum (Fig. 1). The rather restricted value of the so-called camera constant at LURE instrument translated into the narrowrange of 2θ angles and only a few reflexes in the spectra were availablefor comparison. One of the representative measurements presented inthis work was made inside the cell structure of the black coral whileanother was in the location of the gluing zone. In the latter case, theexciting beam covered not only the ring structure but a part of theinterior of the cell as well. Nevertheless, the clear structure of α-chitin,with the presence of an unknown organic phase mainly in the interiorpart of the black coral, was observed on both diffractograms. Thespreading of the peaks in the synchrotron measurements resulted notonly from the worse spectral resolution of the technique used in LUREbut also from the worse crystallinity of chitin in black corals than inchitin standard. In thewall, the separate organic phasewith the spacing

1.04 nmwas discovered. In the cell, another organic phase with spacing0.74 nm, adhering to the chitin 0.516 nm spacing ([002] reflexion of theα-chitin) was detected.

The FTIR measurements provided very clear images of the phases.At first, the comparison of the black coral spectrum with the spec-trum of pure chitin was made. Except the spectral regions containedbetween 1100 and 1300 cm−1, and forwave numbers below 800 cm−1

where some number of differing details was observed in both spec-tra, the remaining parts of the spectra are strikingly similar (Fig. 2A).Undoubtedly, the matrix of the black coral is based on chitin, althoughthe chitin content represents only some one-seventh of the totalmass of the skeleton. This was confirmed by comparing our resultswith the classic set of results by Opresco (Fig. 2B). Using the threewavenumbers in the differing parts of the spectra asmentioned above,one can probably construct the Gibbs diagram for recognition of thespecies.

Fig. 5. Raman microimaging (sample no. 2): A) optical image of the cell fragment; B) C–I — symmetric stretching vibration of ring (190 cm−1); C) amide I (1656 cm−1); D) S–Sstretching deformation (500 cm−1); E) C–Br (650 cm−1); F) chitin (1108 cm−1).



3.2. Inorganic constituents

The quantitative analyses of inorganic components in the blackcorals were tabulated and are shown in the second and third columnof Table 1. The values were obtained frommeasurements by the use ofAAS and PIXE. For comparison, the results by Goldberg et al. [15] andKaczorowska et al. [20] were also cited and tabulated in the last twocolumns in Table 1.

In this study, 15 elements were detected with the use of electronmicroprobe measurements: C, N, O, Na, Mg, Si, P, S, Cl, K, Ca, Zn, Br, Sr, I(the results from the electron microprobe are not shown in Table 1).PIXE measurements detected 15 elements: Mg, Al, Si, P, S, Cl, K, Ca, Ti,Cr, Fe, Cu, Br, I, Ba. Although many elements were detected using bothmethods, very light elements such as C, N, O and Na could be detectedonly using the electron microprobe, while trace, medium-Z elementssuch as Ti, Cr, Fe and Cu were detectable using PIXE.

Mappings made by the use of the electron microprobe supple-mented thosemade by PIXE. Specific technical abilities of themethodsresulted in a difference in the dimensions of the images being pro-duced, for instance in the EPMA the images were in the order of mm2

while in the second method they were set as 250 µm×250 µm.Mappings collected by the electron microprobe can cover many majorcells arranged in the growth rings of diameters around 120–300 µm,separated by thin layers of ~15 µm thick, clearly of different

composition. The distribution of the elements detected in the electronmicroprobe measurements is very characteristic (Fig. 3). It can beobserved that significant concentrations of C, Na, O, N, Mg and Br werepresent within the interior of the wide cells. To a lesser extent, Sr (notshown here) and S are located in the interior of cells. I, Ca, K and Zn(two latter not shown) occur in the narrow interlayer (gluing) zones.The results revealed that Ca, observed mainly in the gluing zone, wasanticorrelated with Mg and Sr, observed in the interiors. Similarly, Iwas anticorrelated with Cl and Br. In other materials, those groups ofelements are as a rule positively correlated. It can be postulated that asignificant separation process occurs during the formation of thelayers of the skeleton.

Mappings made by the use of PIXE (Fig. 4) confirmed the uniquelocation of I in the narrow gluing zones between the rings. Moreover,calcium, silicon, iron, chromium and potassiumwere located togetherin the thin linear structures (~35 µmwide), presumably in or close tothe gluing zones. The results obtained revealed that these zonesseemed to be wider than the area covered by the I (~15 µm) and werenot strictly the same zones as those occupied by this element. Ba (notshown here) and partially K were the only elements strictly correlatedwith the I. Al and Cl were much more dispersed than the elementsmentioned. The gluing zone seemed to be depleted of S — the effectwas clearer than in EPMA measurements.

To show the separation of the elements between different zones(interior of the cells, gluing zone and the skeleton core region),relevant data were grouped and shown in Table 2. The concentrationratios were presented and the ratios were normalized against thelevels in the interiors of cells. It is clearly observed that I, Ca and Zn areconcentrated in the gluing zones. For Ca and Zn, even more striking istheir great concentration in the core region.

4. Raman results

Raman mappings were spanned over the width of the cells, tocover the interior together with the gluing zones. Many oscillationswere detected in the spectra. We selected those which could be at-tributed to the interesting us chemical groups. The results which arepresented in Fig. 5 are cited only on a basis of one oscillation but theywere confirmed by at least another one, not shown here. In Fig. 5B, theC-I symmetric stretching vibration of ring at 190 cm−1 is shown; infact it illustrates the location of thyroxine and it is obvious that itcorresponds to our previous findings about the location of I. Thethyroxine occurs mainly in the gluing zone. The width of iodinatedzone is in a range of 9-12 µm and well corresponds to value of 15 µm,determined by EPMA and similar value from PIXE. In Fig. 5C, thepresence of amide I (1656 cm−1) is revealed in the same locations.The same zone is completely depleted of the amino acids invol-ving sulphur (methionine and cystein – S–S stretching deforma-tion 500 cm−1). This result is rather in accordance with our previousunclear results concerning S from EPMA and PIXE measurements(Figs. 3 and 4) and in contradiction to the results by Goldberg [15],suggesting that S-bearing aminoacids can be extracted from the gluingcement. The C–Br oscillations (650 cm−1) are deficient in the gluingzone, while very abundant close to this zone. It is in accordancewith our data from EPMA (Fig. 3), showing the absence of Br in gluingzone and sudden enrichment in the interior of cells, especially close tothe gluing zone. It is quite clear from Raman studies, that chitin(1108 cm−1) is nearly absent in gluing zone.

The careful treatment of Raman spectra allowed revealing of thedetails of the subtle structure of the cell interior. It was impossible toextract similar information from EPMA or PIXE measurements. Fig. 6shows the chitin fibrils, 4 µmwide, inside interior, parallel each otherand oriented along the longer axis of the cell (perpendicular to thedirection of the annual growth). The chitin fibrils are roughly in thesame locations as the sulphur-bearing aminoacids and brominatedcompounds. At the same time, the fibrils are deiodinated and depleted

Fig. 6. Fragmentof the abovemapping, 40×50µm, showingA) four chitinfibrils; D) S-richand E) Br-rich compounds follow chitin fibrils; B) I-rich and C) amide I containing com-pounds avoid the chitin fibrils.



of compounds with amide I groups; both of the latter are much in thesame matrix, surrounding the chitin fibrils.

5. Discussion

The results of our investigations have shown precisely themicrostructure of the black corals. It was confirmed that the matrixis generally composed of organic matter, mainly chitin and as knownfrom other contributions — proteins. Although, accordingly to thepreviously publishedworks, the chitin constituted only between 6 and18% (on average −15%) in the black corals, this compound organizedthe whole structure of the coral cell interiors. Both the diffraction andFTIR experiments indicated that the chitin was the main organiccompound, with some less important changes in the relevant spec-tra suggesting that other components may be present as well. Thediffraction experiments were carried out at the microscopic levelaimed at selected locations. Despite the limitations mentioned earlierwith regard to the rather large beam diameter (~15 µm) incomparison with the width of the gluing zones (15 µm), theexperiments showed relatively small but clear differences betweenthe phases in the locations of gluing zones and cell interiors. The chitinwas detected in both locations. Although the three main peaks in thestandard of theα−chitinmatchedwell the threemajor features in ourdiffractograms of the internal and boundary locations in the blackcoral, one could observe the additional reflexes/bands in both lattercases. An additional peak was observed in a position of 7° of 2θ anglesin the diffractogram of the black coral cell interior. This is due to thepresence of an unidentified organic component in the interior zone,with the interplanar distance of 1.26 nm. In the region between 13 and26°, the peaks in the synchrotron-based diffractograms were widelyscattered, which can be partially attributed to the limitation in theangular resolution of the synchrotron-based device in comparisonwith the conventional table-top instrument used in the analysis ofchitin. However, peak broadening can indicate that chitin in blackcoral has a lower crystallinity than standard chitin. Nevertheless, boththe peak in the 7°, 2θ position and some features of the spectrum inthe 13–26° range do not result fully from the chitin spectrum. It isbelieved that these should be attributed to the presence of someorganic constituents (proteins?) in the matrix. The analyses also showthat the chitin component in the black coral is characterized by thepresence of great amount of small crystallites; this is demonstrated bythe full rings and not spots observed in the diffraction patterns.Intensity ratios between [110] and [020] reflexes in the wall andinterior locations of the sample were shown to differ, indicating diffe-rent orientation of crystallites in both locations. Here, the orientationof the crystallites in the interior is much closer to that characteristicfor the crystalline samples of the standard α-chitin [32].

MicroRaman measurements showed more precisely the arrange-ment of the organic matter. The distribution of I-containing compounds(thyroxine) was the most characteristic among all the results. It wasmainly observed in the gluing zone. In the interior, the presence of thesame compounds was detected in the interfibrillar space, between thechitin fibrils. Chitin fibrils were ~4 µmwide and parallel each other andparallel to the longer axis of the cells, i.e. perpendicular to the radialdirection. Fibrils were enriched in compounds with Br and S. Moreover,chitin fibrils were nearly absent in the gluing zone, the result which isonly partially in agreement with the micro-XRD results. Such distribu-tion of the organic compoundswithin the cell interiorwas shown for thefirst time. Moreover, the regular distribution of the chitin fibrils helps tounderstand, that their location introduces the order inside the cellinterior. It looks as if the chitin fibrils stretched along the main axesof the cells were embedded in the solid solutions of other organicsubstances.

Additional information about the matrix could be extracted fromthe organic matter analysis. Importantly, subtle differences betweenour data and those by Goldberg in the C, H, and N analyses were

observed. This difference is believed to result from the presence ofdifferent sets of amino acids, with the greater amounts of the nitrogenin our samples (it corresponds to greater amount of small-chain aminoacids) and the greater amount of hydrogen observed in Goldberg'scorals. In addition, from the comparison of the iodine content betweenthese samples, a decrease in the concentration of thyroxine by anorder of magnitude in the samples used for this study can be inferred.

Although the matrix of black corals was essentially composed oforganic materials and supported on the structure of chitin, it was alsoobserved that substantial amounts of chemical elementswere present.The presence of I and, to less extent Br, was anticipated, since blackcorals selectively collect both of these elements from seawater. Theelements were later absorbed in different locations within the coral.The position of I is quite specific. It is located selectively in the gluingzones, what is clearly observed on the relevant mappings. Hence,assumptions can be made that I is an important component of thegluing substance found within this zone. We did not make the in-vestigationswhat kind of the chemical compound included the iodine,but the presence of the thyroxine, confirmed for the black corals inother studies, was certain. Similarly, calcium was observed in muchhigher concentrations in gluing zone. One can join this fact with theinformation by Nielson [18] and Goldberg [15] that the carboxyaminoacids and lipids also concentrate in the gluing zone. It can be a reasonfor forming the coordination bounds with Ca. Similarly, Zn is enrichedin the same region. Due to the known role of Ca and Zn as the co-enzymeatoms, their increased presence in the gluing zone can indicatesignificant enzymatic activity of this zone. K is also overrepresented inthe gluing zone. Similarly, several heavy elements such as Fe and Cr,registered by PIXE, were present in the same zone. Their role can besimilar as that of Ca and Zn. Thus, the studies on the different kinds ofthe enzymatic activity inside gluing zones seem to be quite essential inthe future. The clear deficit of the chitin (Raman measurements) andsulfur-bearing amino acids (Raman and electron microprobe) puts indoubt the role of the gluing zone as themechanical delimitation of thecell. Our findings emphasize rather the enzymatic activity of this zonethan its mechanical significance. Perhaps, the well known elasticity ofthe black coral rods can result from the small rigidity and ability ofstretching of the gluing zone, found in our contribution.

PIXE measurements clearly showed the elemental differencesbetween the gluing and skeletal zones and more complex structure ofthe gluing zone than it was revealed by the electron microprobe. OnlyBa and K are associated strictly with I (a zone ~15 µm thick). Ca, Fe, Crand Si only slightly overlap with the I zone. The whole gluing zone isover twice as wide than I-bearing layer. The complex structure of thegluing zone and the role of particular elements demand additionalstudies.

6. Conclusions

A number of characterization methods showed that black coralscollected from three different regions morphologically contain dis-tinctive multilayer hybrid structures. The matrix is composed ofα−chitin, which constitutes approximately 15%, supplemented by aset of proteins and carbohydrates. The orientations of the chitincrystallites were different in different zones; in the interior, the chitinfibrils were 4 µm wide and oriented along the long axes of the cells.Morphological observations showed that the structure consisted of acore, then a number of structural cells bonded with intermediategluing layers.

The elemental analysis of gluing layer showed high I, K, Zn, and Caconcentrations. These elements are probably contacted with thecarboxyamino acids and lipids which also concentrate in the gluingzones, according to independent studies. The core contains highconcentrations of Zn and Ca among inorganic constituents. Thematerial of gluing zone seems to be themost interesting among all theconstituents of the black corals — it involves highly concentrated



iodine inside the organic zone, material potentially very interestingfor pharmaceutical applications.

The chemical complexity of the intermediate gluing zone betweenthe skeletal layers suggests that further studies are required to betterunderstand this natural unique composite structure and to enablebiomimetic synthesis for a range of synthetic materials.

Acknowledgements

Authors are deeply grateful to Prof. W.M. Goldberg and Mr M. Pal,whose generous donation of the samples of black corals made thiswork possible. The third kind of samples was taken from the collectionof one of the authors (B.B-N.).

References

[1] http://www.waquarium.org/, March 6, 2007.[2] D.M. Opresco, Zootaxa 852 (2005) 1.[3] M. Yoklavich, M. Love, J. Mar. Educ. 21 (2005) 27.[4] R.W. Grigg, Proc. 3rd Int. Coral Reef Symp. 2 (1977) 609.[5] http://www.arkive.org/species/GES/invertebrates_marine/, 2008.[6] R.W. Grigg (1976) Sea Grant Tech Rept UNIHISEGRANT-TR-77-03, pp 48.[7] K.R. Grange, N.Z. J. Mar. Freshw. Res. 19 (1985) 467.[8] K.R. Grange, R.J. Singleton, N.Z. J. Zool. 15 (1988) 55.[9] W.M. Goldberg, Mar. Biol. 49 (1978) 669.[10] C. Ellis Jr., R.J. Chandross, R.S. Bear, Comp. Biochem. Physiol. 66B (1980) 163.[11] C.H. Brown, Structural materials in animals, J Wiley and Sons, New York, 1975,

p. 448.[12] A.C. Neville, D.A.D. Parry, J. Woodhead-Galloway, J. Cell Sci. 21 (1976) 73.

[13] K. Kim, W.M. Goldberg, G.T. Taylor, Biol. Bull. 182 (1992) 195.[14] W.M. Goldberg, Hydrobiologia 216/217 (1991) 403.[15] W.M. Goldberg, T.L. Hopkins, S.M. Holl, J. Schaefer, K.J. Kramer, T.D. Morgan, K. Kim,

Comp. Biochem. Physiol. 107B (4) (1994) 633.[16] J. Roche, M. Fontaine, J. Leloup, in: M. Florkin, H.S. Mason (Eds.), Halides,

Comparative Biochemistry, vol. 4, Academic Press, New York, 1963, p. 493.[17] W.M. Goldberg, Mar. Biol. 35 (1976) 253.[18] A.J. Nielson, W.P. Griffith, J. Histochem. Cytochem. 26 (1978) 138.[19] S.M. Holl, J. Schaefer, W.M. Goldberg, K.J. Kramer, T.D. Morgan, T.L. Hopkins, Arch.

Biochem. Biophys. 292 (1992) 107.[20] B. Kaczorowska, A. Hacura, T. Kupka, R. Wrzalik, E. Talik, G. Pasterny, A.

Matuszewska, Anal. Bioanal. Chem. 377 (2003) 1032.[21] M.N.V.R. Kumar, React. Funct. Polym. 46 (2000) 1.[22] S. Lebed, M. Cholewa, Z. Cioch, B. Cleff, P. Golonka, D.N. Jamieson, G.J.F. Legge, S.

Łazarski, A. Potempa, C. Sarnecki, Z. Stachura, Nucl. Instrum. Methods B 158 (1999)44.

[23] P. Chevallier, P. Dhez, F. Legrand, A. Erko, Y. Agafonov, L.A. Panchenko, A. Yakshin,J. Trace Microprobe Tech. 14 (1996) 517.

[24] P. Dillman, P. Populus, P. Chevallier, P. Fluzin, G. Beranger, A. Firsov, J. TraceMicroprobe Tech. 15 (1997) 251.

[25] A.P. Hammersley, K. Brown, W. Burmeister, L. Claustre, A. Gonzalez, S. Mcsweeney,E. Mitchell, J.-P. Moy, S.O. Svensson, A.W. Thompson, J. Synchrotron Radiat. 4(1997) 67.

[26] M. Ermrich, F. Hahn, E.R. Wölfel, Textures Microstruct. 29 (1997) 89.[27] D. Genty, Y. Quinif, J. Sediment. Res. 66 (1996) 275.[28] D. Carlström, J. Biophys. Biochem. Cytol. 3 (1957) 669.[29] Y. Kato, J. Kaminaga, R. Matsuo, A. Isogai, Carbohydr. Polym. 58 (2004) 42.[30] M. Zhang, A. Haga, H. Sekiguchi, S. Hirano, Int. J. Biol. Macromol. 27 (2000) 99.[31] F. Manoli, S. Koutsopoulos, E. Dalas, J. Cryst. Growth 182 (1997) 116.[32] R. Minke, J. Blackwell, J. Mol. Biol. 120 (1978) 167.[33] G. Schwartzman, D.M. Opresko, Bull. Mar. Sci. 50 (2) (1992) 352.


Morphology and the chemical make-up of the inorganic components of black corals

Documents

Transcript of Morphology and the chemical make-up of the inorganic components of black corals