The role of chitin-rich skeletal organic matrix on the crystallization of calcium carbonate in the crustose coralline alga Leptophytum foecundum

The organic matrix (OM) contained in marine calcifiers has a key role in the regulation of crystal deposition, such as crystalline structure, initiation of mineralization, inhibition, and biological/environmental control. However, the functional properties of the chitin-rich skeletal organic matrix on the biological aspect of crystallization in crustose coralline algae have not yet been investigated. Hence, the characterization of organic matrices in the biomineralization process of this species was studied to understand the functions of these key components for structural formation and mineralization of calcium carbonate crystals. We purified skeletal organic matrix proteins from this species and explored how these components are involved in the mineralization of calcium carbonate crystals and environmental control. Intriguingly, the analytical investigation of the skeletal OM revealed the presence of chitin in the crustose coralline alga Leptophytum foecundum. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the OM revealed a high molecular mass protein as 300-kDa. Analysis of glycosylation activity exposed two strong glycoproteins as 300-kDa and 240-kDa. Our study of the biominerals of live collected specimens found that in addition to Mg-calcite up to 30% aragonite were present in the skeleton. Our experiment demonstrated that the chitin-rich skeletal OM of coralline algae plays a key role in the biocalcification process by enabling the formation of Mg-calcite. In addition, this OM did not inhibit the formation of aragonite suggesting there is an as yet unidentified process in the living coralline that prevents the formation of aragonite in the living skeletal cell walls.

Leptophytum foecundum has been placed in the same genus as the widespread Arctic/Subarctic Leptophytum leave. However, as Adey et al 2015 2 have shown with DNA analysis, it belongs in a separate genus. At this time, that change has not been made. L.
foecundum is known to occur from the western Canadian Arctic across the Arctic Ocean and adjacent North Atlantic to Novoya Zemlya in Russia. It extends the furthest south in the Gulf of Maine in the western North Atlantic (Athanasiadis and Adey, 2006) 3 .
Typically, L. foecundum is a thin crust, approximately one-half hypothallium and one-half perithallium, with distinctive, raised and rimmed conceptacles. However, as shown by Athanasiadis and Adey 2006 3 , L. foecundum, especially in the form of? rhodoliths and on pebble bottoms, tends to repeatedly overgrow itself, not only earlier-formed crusts but also the postmature conceptacles on those crusts. In so doing, it can form crusts that are one cm in thickness or greater. However, unlike the Clathromorphum crusts, which are typically formed by continuously growing perithallium, thick L. foecundum crusts are repeated hypothallium and perithallium units, sometimes with entrained foreign inclusions and spaces. Care must be taken to link anatomy with chemistry in defining bulk chemistry in this species.

Preparation of skeletons
To ensure contamination-free of samples of other associated tissues (e.g., fungus, bacteria etc.) or other skeletal minerals (e.g., shells, corals etc.), the preparation of skeletons of the algal body was performed very carefully. We used both upper and lower levels (lower level normally stays attach to rock or other organisms in the sea) to confirm the skeletal mineral. We used a series of mechanical and chemical treatments. (1) Experimental skeletons were made small pieces and all pieces were washed with 95% ethanol (five times) and distilled water (ten times) while stirring; (2) The cleaned skeletons were dried and examined using a microscope to determine whether they were completely free of unwanted tissues and other contaminants. We also confirmed via Scanning electron microscope (SEM) that all skeletons are completely contamination-free; (3) The selected skeletons were finely ground; (4) Skeletal powder was stirred with 1 M NaOH for 2 h; (5) To ensure again that the skeletons were tissue free (especially fungus and bacteria), they were stirred vigorously in a 10% sodium hypochlorite (NaOCl) bleaching solution for 1 h to remove fleshy tissues and debris. The treated samples were washed with distilled water until all chemicals were removed. Finally, the samples were washed with MilliQ water five times. All steps in the preparation of skeletons were conducted at room temperature, and the materials obtained were stored at 4°C until further analysis.

Preparation of soluble and insoluble organic matrices
The mechanically and chemically cleaned alga (ca. 10 g) were decalcified in 0.5 M EDTA-4Na (pH 7.8) overnight. The decalcifying solution was centrifuged (Eppendorf-5430) at 4,000 rpm (15 minutes), and the soluble organic matrix (SOM) in the supernatant was filtered and collected for further purification. The insoluble organic matrix (IOM) in the precipitate was subsequently lyophilized and washed with distilled water (5 times). The solution was dialyzed prior to lyophilization to remove EDTA against 5x1 L of distilled water for 64h while water was changed five times using dialysis tubing. Additional details on sample preparation can be found in Rahman et al 4,5 . To purify SOM, filtrated samples were passed through two Sep-Pak C18 cartridges connected in tandem (Waters Associates, Milford, MA) to remove the EDTA completely and separate the soluble macromolecules, followed by passing through 10% acetonitrile (2 mL/three times). Finally, the absorbed macromolecules were eluted in 50% acetonitrile (2 mL/three times), frozen in a deep freezer and lyophilized. Deacetylation with NaOH (see preparation of skeletons above) and decalcification of the calcified skeletons following the above-mentioned procedures are perfect to obtain high content of soluble chitin from the skeletal samples of coralline algae.

Analysis of organic matrix proteins (OMP)
The Precision Plus SDS-PAGE standard (Bio-Rad) was used as a protein marker for electrophoresis analysis. An eluate (derived from 5 g of the algal skeleton) was run on 12% polyacrylamide gel with several replications. The purification of the organic matrix (OM) and total OMP were done following the methods of Rahman et al., (2011) 4 . Periodic acid-Schiff (PAS) staining was used to identify glycoproteins, and the two-high abundant chitin associated glycoproteins were identified to be the only OMPs occluded in the algal skeletons of L. foecundum.

Preparation of the solution for in vitro crystallization
To investigate the effect of matrix proteins on the CaCO3 formation, in vitro crystallization experiments was carried out using a solution containing the components typically present in seawater. Two crystallization solutions were prepared, one that induces calcite (a calcitic crystallization solution) and one that induces aragonite (an aragonitic crystallization solution).
The calcitic solution consisted of a supersaturated solution of Ca (HCO3)2 that was prepared by purging a stirred aqueous suspension of CaCO3 with carbon dioxide. Calcium carbonate was dissolved in CO2-aerated water, and excess precipitates were removed by filtration using filter

Raman Spectroscopy and Microscopy
Two kinds of samples were analyzed using Raman spectroscopy, skeletal powder, and original skeletons. Prior to conduct Raman analysis, we ensured that both powder and skeletons were contamination free by other associated tissues or other skeletal minerals as described in the "Preparation of skeletons section" above. The samples were analyzed using Thermo Fisher Scientific DXRxi Raman Imaging Microscope configures with 455 nm or 532 nm laser and a full range grating providing 5 cm -1 spectral resolution. The samples were fixed on a standard microscope glass slide using double-sided adhesive tape. To analyze by Raman spectroscopy with the original skeletons, several small pieces with a smooth surface were selected from the middle part of the skeletons. A different type of Raman spectroscopy was also used to investigate the samples. In this case, all measurements were obtained in a NTEGRA Spectra system (NT-MDT, Zelenograd/Moscow, Russia) coupled with a Solar TII Spectrometer. This system is equipped with an upright confocal laser microscope, a photomultiplier tube (PMT) and a Raman spectrograph coupled with a high-resolution CCD camera (1600 x 200 pixels). PMT imaging was used to find the areas of interest for Raman spectra acquisition. Raman spectra were obtained at room temperature and at ambient pressure using a 532 nm wavelength laser with a maximal power of 8.7 mV. Backscattered light was collected through a 100x objective, 0.7 numerical aperture (NA) and redirected to the CCD camera (cooled at -70 ºC). The spectra were obtained with a spectral resolution of 1.6 cm −1 for a 600 lines/mm grating.

In Situ AFM
The finely polished samples were used for the Atomic Force Microscope (AFM) analysis.
The topography of the surface was mapped using an AFM in phase imaging mode. Phase images can be generated as a consequence of variations in material properties such as friction. AFM observations were conducted with an Atomic Force Microscopy NT-MDT Ntegra Spectra at room temperature.

X-Ray Diffraction (XRD)
The same finely ground skeletal powder samples as for FTIR were used for this analysis.
The polymorphism of crystals in the skeletons was determined by an X-ray diffractometer