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The presence and localization of organic matrix associated with the aragonite phase in the fibers of blue coral Heliopora coerulea skeletons were stud...
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In Situ Raman Spectral Mapping Study on the Microscale Fibers in Blue Coral (Heliopora coerulea) Skeletons Fenfen Zhang,*,† Weiying Cai,‡ Jichun Zhu,‡ Zhenrong Sun,‡ and Jing Zhang† †

State Key Laboratory of Estuarine and Coastal Research, East China Normal University, 3663 North Zhongshan Road, 200062 Shanghai, P.R. China ‡ State Key Laboratory of Precision Spectroscopy, East China Normal University, 3663 North Zhongshan Road, 200062 Shanghai, P.R. China ABSTRACT: The presence and localization of organic matrix associated with the aragonite phase in the fibers of blue coral Heliopora coerulea skeletons were studied by in situ microRaman mapping spectra, with a spatial resolution of ∼0.3 μm. Spatial variations in the amounts and chemical compositions of the fibers were imaged. The results showed that the amide I and the α-helix of amide III were perpendicular to the c-axis of fibers’ growth, whereas the β-turns/sheet of amide III was in the parallel conformation. Visible S S and C S bonds were consistent with the XANES results, which indicated the existence of organic sulfur in coral skeletons. Regular cyclic changes between aragonite and organic matrix refined a stepping growth mode of the fibers’ biomineralization. An inorganic PO4 bond was detected and exhibited the same concentration variation trends as the v4 aragonite bands. Instead of providing an ocean P proxy on the subseasonal to centennial scale by LA-ICPMS, the possibility was raised of producing high resolution surface ocean phosphorus records on daily environmental variation via P/Ca variation cycles determined from Raman mapping data.

he fine-scale structure and composition of fibers in coral skeletons are of great interest to sedimentological and paleoenvironmental reconstruction scientists,1 because these skeletons might provide chemical proxies for highly detailed ocean environmental changes on both short and long time scales.2 The stable isotopes,3,4 trace elements,5 proxy records of nutrients,6 8 and anthropogenic pollutions2 in coral skeletons have been well studied. Numerous works on coral skeleton P/Ca proxy for seawater phosphate by LA-ICPMS (laser ablation-inductively coupled plasma mass spectrometry) have been published,6,8,9 as P is a key biolimiting nutrient for marine life and a tracer for water masses. Moreover, increased P runoff and pollution exhibit deleterious effects on reef health.10 Therefore, a reliable scheme for the coral skeleton biomineralization process and P incorporation mechanism would lay the groundwork for the reconstruction of ambient ocean water nutrient P.8 However, until now, the skeleton biomineralization or P incorporation mode still remains controversial. From the viewpoint of most geologists, a fiber is “a single orthorhombic crystal of aragonite” and serves as a sort of environmental archive, which was proposed as a purely mineralogical mode of fiber growth.11 However, this well-accepted mineralogical interpretation has been contradicted by biologically controlled mode on fiber growth.12 On the basis of staining techniques, Johnston has claimed that “attempts to implicate these organic materials in the process that control and direct mineralization have largely been made in ignorance of this material’s spatial distribution and microarchitecture within the skeleton”.12 Gautret further pointed out the organo-mineral composition of coral fibers and that mineralizing matrixes persist entrapped within crystal-like fibers.13 FT-IR,14,15 HPLC,16 electrophoresis,15 in situ acridine orange staining,17,18 and Raman19,20 data have further

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confirmed the presence of organic compounds. Unfortunately, in spite of its’ great potential for illustrating the fiber biomineralization process, the biologically controlled mode has not exerted much practical influence on the field of earth sciences.18 Recently, several micrometric or even nanometric level analytical methods, including SEM (scanning electron microscope),21 AFM (atomic force microscopy),22 NanoSIMS (secondary ion mass spectrometer),23 and XANES (X-ray absorption near edge structure spectroscopy),15 have uncovered micromorphological patterns of skeletal components. Particular attention should be paid to the XANES studies,15,24,25 which showed biochemical maps that allow the localization of sulfated polysaccharides in micrometer coral fibers. These results provide evidence for the step-bystep cyclic growth process of the mineral phase within the fibers. As an alternative to the “step-by-step” mode, a layered mode has been proposed by Stolarski.21 Each growth layer acted as the actual Environment Recording Unit.24 However, all these methods detect the morphology, mineral phase, or organic matrix separately. If another nondestructive microanalytical method, such as microRaman mapping, is able to simultaneously obtain information from both the mineral component and the organic matrix, it would provide a complete picture of the variation in the major heterogeneous constituents with high spatial resolution.26,27 Thus, microRaman mapping studies on coral skeletons may give clues to advance our understanding of the fiber biominerlization process and bring out some semiquantitative chemical proxies. Received: July 8, 2011 Accepted: September 1, 2011 Published: September 01, 2011 7870

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Analytical Chemistry In the present work, differences in the chemical compositions and structures of fibers in blue coral Heliopora coerulea skeleton were studied by in situ microRaman mapping. The Heliopora coerulea is unique among the Alcyonaria, for its’ trabecular skeleton is made of fibrous aragonite which has a characteristic permanent blue pigmentation of iron salts.28,29 It is also described as a “living fossil” and was once widely distributed in tropical and subtropical areas.28,30 The prominent bands in the Raman spectra of the blue coral skeletons, such as the v4 aragonite and amide I bands, were quite sensitive to the fibers’ growth orientation. The amide III had two different modes; the α-helix mode was perpendicular to the direction of the fibers’ growth along the c-axis, and the β-turns/ sheet one exhibited a parallel conformation. C S and S S signals were detected, which was consistent with XANES mapping results of organic sulfur in fibrous form.15,31 The mineral and organic matrix varied regularly, which evidenced the previous cyclic secretion of organic or mineral components in layered biomineralization mode.21,23 This result illustrated that the crystallization of the fibers growth may be driven by the organic matrix.24 In addition, these regular organo-mineral cycles and microbandings in blue coral fibers were possibly coupled to an extrinsic diurnal (e.g., temperature, light, tides) or semidiurnal (e.g., tides) cycles, which may also be ascribed to daily cycles of carbonate secretion.1 From a practical standpoint, a direct geochemical P proxy for the reconstruction of surface ocean P concentrations would be very useful. Here, visible PO4 stretching bond in the spectra indicated that phosphorus was possibly present as substituted β-tricalcium phosphate (β-TCP) substitution32 or as Fe2(PO4)33 inside the fibers.33,34 The alternation of phosphorus was mapped, and the P/Ca ratio was calculated. Instead of subseasonal to centennial scale P records from LA-ICPMS results,8 this study presents the possibility of producing high resolution P proxy records based on daily environmental variation, which may be especially important in the interpretation of extreme P related ocean events. The results demonstrate that Raman spectroscopic mapping might become a powerful tool in the study of coral skeleton biominerialization and in paleoenvironmental reconstruction.

’ MATERIALS AND METHODS Blue Coral Samples. The blue coral Heliopora coerulea, one of the zooxanthellate corals, was recently collected from the shallow waters of the South China Sea. For the investigation, the coral cores were sliced into 1 mm thick slabs along the growth axis using a water-cooled diamond saw. The slabs were then sectioned into ∼5 mm lengths for SEM and Raman analysis. All slabs were polished and etched for approximately 2 min in dilute hydrochloric acid solution. Additionally, nonacid etched and acid etched coral samples were ground to nanometer-scale powders for XRD detection. Although the XRD signal intensity of the acid etched sample was a bit less than that of the nonacid etched sample, both powder XRD patterns were coincident with the peak positions of the standard powder sample of aragonite (JCPDS Card No. 050453 Quality: 1 (aragonite, syn)), which indicated that the dilute acid etching process does not change aragonite crystallites in blue corals (XRD data not shown). To avoid the random errors, all samples and measurements were prepared or tested in the duplicates. SEM Analysis. SEM observations were conducted using a Field Emission SEM JSM-6700F housed at the Shanghai Institute of Ceramics, Chinese Academy of Sciences. The polished

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Figure 1. Scanning electron micrographs obtained from the blue coral skeleton.

and acid etched slabs were coated with a thin layer of gold to reduce charging during the SEM imaging. Raman Microspectroscopy and Mapping Measurements. Raman spectra were collected using a Raman spectrometer (Jobin Yvon, type T64000) equipped with an integrated Olympus BX81 microscope. Spectra of the coral were collected using an ultralong working distance microscope objective lens that provided a magnification of 100. Raman spectra were excited by 514.5 nm light from a water-cooled argon/krypton ion laser. The Rayleigh radiation was blocked by a holographic notch filter, and the backscattered Raman light was dispersed by an 1800 grooves mm 1 holographic grating to a liquid nitrogen cooled CCD chip that consisted of an array of 1024  256 pixels. The laser power at the sample was ca. 3 mW during the collection of the Raman spectra of the blue coral skeleton. The blue coral slab was placed on the stage of the microscope, and the transverse cross section was oriented perpendicular to the incident laser beam from a 100 (NA = 0.9, Nikon) microscope objective lens. With this lens, the instrument was confocal and probed a depth of approximately 1 μm. The part near the core of the blue coral skeleton was identified and positioned using a motorized XY stage and an optical camera. Raman images were acquired in 15  35 μm2. The images were created using a 0.3 μm step size. The mapping analysis was concentrated on the region from 600 to 1800 cm 1 in the study.26 The background fluorescences of all the samples were subtracted from the calculated baseline computation, and a polynomial filtering was performed.

’ RESULTS SEM Micrographs of Blue Coral Skeletons. The main microstructures of the etched blue coral slabs are presented in high magnification SEM photomicrographs (Figure 1). It consisted of large bundles of tapered fibers. A layered surface morphology composed of regular incrementally grown fibers was observed, whereas the concentric growth lines were well visible after etching. The length of the individual banding was approximately 4 to 7 μm. Similar textural features have been observed in other coral species.35 Raman Spectra of Blue Coral Skeletons. Corresponding Raman Spectrum of Blue Coral Skeletons. Figure 2A shows a typical Raman spectrum of the fibers section. The prominent bands were labeled. The v4 aragonite stretching vibration at 1084 cm 1 was the strongest marker for the fiber minerals. The v4 aragonite 7871

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Figure 3. Raman spectral deconvolution of amide III band.

Figure 2. (A) Typical Raman spectrum of the blue coral skeleton; (B) the highlights of the 450 to 780 cm 1 bands in (A).

vibration was also observed at 702 cm 1. The broad bands showed in the high-frequency region were attributed to amide III band region (1200 1380 cm 1), the C H bending mode (1451 cm 1), the N H bending mode (1484 cm 1), and amide I band region (1570 1710 cm 1).36 39 These broad organic peaks were mainly from chitin or other proteins. Organic sulfate S S and C S vibrations at 510, 650, and 667 cm 1, which mostly originated from S-proteins,40,41 were also visible (Figure 2B). The PO4 deformation and stretching bonds were detected at 974 and also at 623, 1140, and 1178 cm 1,42 which indicated β-tricalcium phosphate (β-TCP) substitution32 or Fe2(PO4)33 inside the fibers because of the large amounts of calcium or iron slats existed in blue coral skeletons. The Raman spectrum from the calcification center was similar to that of the fiber section and was only distinguished by a bit high ratio of organic matrix/v4 aragonite. A band-fitting approach with Gaussian and Lorentzian line shapes was employed to identify the presence and changes in the relative contribution of orientational effects, on the basis of the Raman spectra of secondary structure of amide III backbone converted to α-helix or β-turns/sheet conformation.38,39,43 Figure 3 showed after deconvolutions of the Raman amide III bands. 1292 cm 1 was attributed to CH;44 1267 cm 1 and 1310 cm 1 were assigned as α-helix and β-turns/sheet of amide III.39 Typical Raman Spectral Mapping Zone on Fibers. Figure 4a e shows the calculated contrast images (generated by the Labspec 5.0 software pixel by pixel, the intensity scores were calculated for relative band areas, and the scores are displayed by different color codes) of v4 aragonite, P, amide I, and the α-helix and β-turns/ sheet of amide III, respectively. A layered structure was observed.

Figure 4f j shows along growth axis c, the corresponding intensities of v4 aragonite, P, amide I, and the α-helix and β-turns/ sheet of amide III, respectively. Aragonite and the organic matrix were permanently existed in the fibers, although their concentrations in different parts changed. Interestingly, variations in the almost successive cyclic trends of the aragonite, P, amide I, and the α-helix and β-turns/sheet of amide III are dipicted at approximately 4 7 μm. This result indicates that, when the concentration (intensity) of aragonite, P, and β-turns/sheet of amide III reached the maxima, the concentrations of amide I and the α-helix of amide III decreased to their lowest observed values. However, the total amide III band and all bonds in the other direction (y-axis) did not show this cyclic relationship. On the basis of the intensity of aragonite and P, the P/Ca variability was assessed (Figure 5), and the daily layer was determined from the aragonite variation cycles.

’ DISCUSSION On the basis of the results of Johnston,12 Stolarski,21,35 and Cuif,25 fiber skeletogenesis was used to develop a reliable model of the growth process through fine-scale structure observation combined with the organic matrix or trace element spatial distribution data. From a practical standpoint, the results presented here may refine the biologically controlled growth mode of fiber and contribute to a proposed new high resolution approach to the environmental daily records in coral skeletons based on P/Ca assessments.24 Heterogeneous Mineralization Processes between Mineral and Organic Matrix in the Mapping Zone and Daily Banding in the Fibers. The coexistence of mineral and organic

matrix has been widely studied in the past. Organic sulfate (S S, C S) signals were detected in the Raman spectra, which was consistent with XANES results from Cuif et al.15,31 Cuif and Sorauf have pointed out that the spatial arrangement of the organic compounds controlled the microstructural organization of the mineral phase.45 However, the inability to effectively link the microstructure to mineral and organic data has remained a major obstacle to understanding the fiber biomineralization pathway. In the Raman spectra, the organic signals were mostly from chitin and other S-proteins. Both chitin and S-proteins are reactive in the calcification process46 and serve as a meshwork to control the crystal size and shape in the fibers.21 The backbone of the organic matrix also contributes to the orientation effects. The amide I (CdO) bond was perpendicular to the c-axis of fiber growth; on the other hand, the v4 aragonite band was parallel to the axis of 7872

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Figure 4. Raman mapping on v4 aragonite (a), P (b), amide I (c), amide III, α-helix (d), and amide III, β-turns/sheet (e); c- axis parallel to growth orientation; corresponding intensity of aragonite (f), P (g), amide I (h), amide III, α-helix (i), and amide III, β-turns/sheet (j).

the fiber, and they presented the opposite trends. The secondary structure of amide III (C N) also showed orientation

sensitivity: the α-helical band of amide III represented a similar orientation pattern with amide I, whereas the β-turns/sheet band 7873

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Figure 5. P/Ca ratio assessed by Raman mapping of fibers, and daily layers were determined from the aragonite variation cycles.

showed a pattern similar to that of v4 aragonite, as shown in Figure 4d,e. When both regions of amide III were integrated, no orientational pattern was evident any more. This observation fitted the fiber orientation theory proposed by Wagermaier et al:47 the CdO (amide I) vibration was perpendicular to the fiber axis, whereas the amide III band consisted primarily of C N vibration modes. The amide III had two different modes, one parallel to the mineral c-axis and the other parallel to CdO axis. Here, the α-helical band of amide III was parallel to CdO axis, and the β-turns/sheet band was parallel to the mineral c-axis. Therefore, the total amide III did not show any orientation effect. As a result, the calculated and mapped v4 aragonite and amide I bands could be directly used to estimate the orientations of the fibers. Skeletal fibers are considered to be matrix-mediated biominerals built by the stepping growth mode. From the SEM results of the Heliopora coerula (blue coral) skeleton, every growth increment range was around 3 7 μm.48 Each growth layer obtained from SEM (4 7 μm) and Raman (around 4 7 μm) data is in the range of the daily scales. The Raman mapping data on the growth rate of approximately 6 μm per day provided microstructural evidence of the successive deposition of the layers by fibers. On the basis of microRaman mapping, successive regular cyclic alternations between mineral and organic fibers at heterogeneous mineralization patterns were imaged. A plausible explanation for the observed phenomenon is that zooxanthellate corals generally exhibit distinct differences in day night calcification growth rates and in physiological cycles on distribution of organic matrixes. These growth rates and physiological cycles are similar to the mineral and organic matrix mineralized in other biomineralized materials;36,37 thus, the fibers displayed the cyclic and rhythmic changes corresponding to daily banding. Possibility of Daily Phosphorus Records in Blue Coral Fibers. Phosphorus is an essential nutrient for marine organisms, and its availability will affect marine biota and thus link to global climate.7 The P/Ca ratio from LA-ICPMS results for several coral skeletons would be a reliable proxy record of the seasonal phosphorus in ambient seawater.6,8 Such a direct proxy could

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assist in the reconstruction of long-term variations in ocean P content. However, whether the P is bound in the inorganic lattice, in organic phases, or in ferromanganese coatings is still a controversial matter.7 Because calcium and iron salts existed in blue coral skeletons, P was most probably introcrystallined with calcium as a β-tricalcium phosphate (β-TCP) substitution32 or with iron as inorganic Fe2(PO4)33 phase according to the Raman spectra.33,34 Raman spectroscopy failed to detect organic or other forms of P. Although the P incorporation mechanism remains unclear,7 the LA-ICPMS results showed that almost 60% of the P was in the organic phase.9 LaVigne et al. have hypothesized that this organic skeleton P most probably served as a background level of P and was set the coral’s calibration y-intercept, whereas inorganic skeleton P varied consistently with changes in PO4 sw (calibration slope).8 If so, the P/Ca variation was in direct response to ambient seawater P because only the inorganic P signal was detected in the Raman maps. Because of cyclicity in the biominearalization process, each growth layer acts as an “Environment Recording Unit”.24 The possibility of elaborating reliable P records at the daily scale is now proposed. This is especially important in interpreting the coastal eutrophication or costal P runoff and pollution incidents. Further assessment and comparation work is required.

’ CONCLUSIONS MicroRaman mapping allowed the simultaneous analysis of aragonite and the organic matrix in coral skeletons. Cyclic repartitions between mineral and organic matrix indicated daily increments in fibers. The orientation sensitivity of aragonite and amide I almost exactly matched the skeletal growth steps. The mapping results refined a biochemically driven mode in the fiber biomineralization process. The distribution of phosphorus was also mapped. Because each growth layer acts as an “Environment Recording Unit”, the possibility of producing phosphorus records on daily environmental variation was proposed. Further improvements by other methods need to be established in the future. ’ AUTHOR INFORMATION Corresponding Author

*Tel: +86 21 6223 2572. Fax: +86 21 6254 6441. E-mail: ffzhang@ sklec.ecnu.edu.cn.

’ ACKNOWLEDGMENT We thank Dr. Huang Hui (South China Sea Institute of Oceanology, Chinese Academy of Sciences) for identifying blue coral species. This work was supported by project grants from the Ministry of Science and Technology of China (No. 2007DFB20380), the funds for Creative Research Groups of National Natural Science Foundation of China (No. 40721004), and Major Program of National Natural Science Foundation of China (No. 40830850). ’ REFERENCES (1) Gill, I. P.; Dickson, J. A. D.; Hubbard, D. K. J. Sediment. Res. 2006, 76, 683–688. (2) Meibom, A.; Cuif, J. P.; Houlbreque, F.; Mostefaoui, S.; Dauphin, Y.; Meibom, K. L.; Dunbar, R. Geochim. Cosmochim. Acta 2008, 72, 1555–1569. 7874

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