Mechanical Properties of the Skeleton of Acropora Cervicornis

University of Central Florida STARS

Honors Undergraduate Theses UCF Theses and Dissertations

2018

Bridget Masa University of Central Florida

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MECHANCIAL PROPERTIES OF THE SKELETON OF ACROPORA CERVICORNIS

BRIDGET A. MASA

A thesis submitted in partial fulfillment of the requirements for the Honors in the Major Program in Mechanical Engineering in the College of Engineering and Computer Science and in the Burnett Honors College at the University of Central Florida Orlando, Florida

Spring Term, 2018

Thesis Chair: Nina Orlovskaya, PhD.

Abstract

This research explores the instantaneous mechanical behavior of the skeleton of the critically endangered staghorn coral Acropora cervicornis. Both bleached and sanded skeletons were used in this experiment. The Raman spectroscopy test showed that there was no significant change in the Raman shift between the three branches tested. The shifts were nearly identical to Raman shifts of calcium carbonate. Vickers hardness test found that 1 Bleached had the average hardness of

3.44 GPa with a standard deviation of 0.12 GPa. The sanded sample also had a similar value of

3.54 GPa with a standard deviation of 0.13 GPa. Samples from 2 Bleached had a hardness value that was significantly lower at only 2.68 GPa with a standard deviation of 0.37 GPa. The axial compressive stress test determined that the average strength for the bleached samples was 18.98

MPa and for the sanded, 29.16 MPa. This information can be used to assist in the restoration of this species.

Dedication

In dedication to my hard working parents Alan and Silvia Masa. For the many opportunities they have given me along with their constant support and words of encouragement Thank you.

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Acknowledgments

I would like to thank the following people who helped me over the course of conducting my research and writing this thesis: Dr. Nina Orlovskaya, my thesis chair and mentor, for graciously taking me on as a part of her team and for her kindness and constant guidance throughout this research. Dr. David Gilliam of Nova Southeastern University, for providing me with these beautiful corals. Dr. John Fauth, for being a member of my thesis committee and for entrusting me with this research that he had so much excitement for. Dr. Ghatu Subhash at the University of Florida, for allowing me access to his lab and indentation machine. Dr. Romain Guame, for allowing me to use his diamond saw and for sparking my interest and understanding of materials during his Structures and Properties of Materials class. Dr. Ranajay Ghosh, for being a member of my thesis committee and for helping me better understand the structure of the coral I was working with. Lastly, a special thanks to my friend and mentor Alejandro Carrasco-Pena. I can’t thank you enough for the help you’ve given me throughout this entire process from start to finish. Thank you for your constant guidance, patience, willingness to answer questions, and for doing all of it with a smile. You truly made this experience a joy. This research was supported in part by MRI NSF project “133775” Development of a Multi-Scale Thermal-Mechanical-Spectroscopic System for in-Situ Materials Characterization, Research and Training.

Table of Contents

1. Introduction ...... 1 2. Literature Review ...... 3 2.1 Microorganisms in Corals ...... 3 2.2 Structure of Coral Skeletons ...... 4 2.2.1 Chemical Composition ...... 5 2.2.2 Crystal and Microstructure ...... 5 2.3 Mechanical Behavior of Coral Skeletons ...... 9 2.3.1 Vickers Hardness ...... 9 2.3.2 Compressive Strength ...... 9 2.4 Raman Spectroscopy Analysis ...... 12 3. Goals ...... 13 4. Materials and Methods ...... 14 4.1 Raman Spectroscopy Analysis ...... 15 4.2 Micro-images ...... 15 4.3 Vickers Micro Hardness Test ...... 17 4.4 Compression Tests ...... 17 5. Results ...... 19 5.1 Raman Spectroscopy Analysis ...... 19 5.2 Vickers Hardness ...... 19 5.3 Compression Tests ...... 20 6. Conclusion ...... 23 Appendix A ...... 24 Appendix B ...... 43 References ...... 45

List of Tables

Table 1: Summary of Previous Literature Results ...... 9 Table 2: Hardness Results of Polished Samples ...... 19 Table 3: Bleached Samples: Max Peak, Mentionable Peaks, and Mean Max Peak Stress ...... 21 Table 4: Sanded Samples: Max Peak, Mentionable Peaks, and Mean Max Peak Stress Results . 21 Table 5: Dimensions of Polished Coral Samples ...... 25 Table 6: Dimensions of 1 Bleached Coral Samples ...... 25 Table 7: Dimensions of 2 Bleached Coral Samples ...... 25 Table 8: Dimensions of 3 Sanded Coral Samples ...... 25 Table 9: Vickers Hardness of 1 Bleached and Polished Sample ...... 32 Table 10: Vickers Hardness of 2 Bleached and Polished Sample ...... 34 Table 11: Vickers Hardness of 3 Sanded Sample ...... 36

List of Figures

Figure 1: Acropora cervicornis ...... 1 Figure 2: Diseased Acropora cervicornis ...... 2 Figure 3: Structure of coral tissue ...... 3 Figure 4: Bleached coral skeletons ...... 4 Figure 5: Morphological skeleton features common to all stony corals ...... 6 Figure 6: Growth process ...... 7 Figure 7: Working model of coral biomineralization ...... 8 Figure 8: Sketch of growth of the coral axial corallite ...... 8 Figure 9: Physical stresses in a coral branch...... 10 Figure 10: Fracture of test cores as a function of skeletal structure ...... 11 Figure 11: Coral branches used in experiment ...... 14 Figure 12: Top view microimages of the polished samples ...... 16 Figure 13: Coral sample undergoing indentation ...... 17 Figure 14: Coral undergoing compression test ...... 18 Figure 15: Normal Raman spectra of calcium carbonate ...... 19 Figure 16: Fractured coral skeleton samples ...... 20 Figure 17a: Profile and Cross-sectional pictures of 1 Bleached samples ...... 26 Figure 18: Profile and Cross-sectional pictures of 2 Bleached samples ...... 27 Figure 19: Profile and Cross-sectional pictures of 3 Sanded samples ...... 28 Figure 20: First point of Raman taken on coral 1 Bleached ...... 29 Figure 21: Second point of Raman taken on coral 1 Bleached ...... 29 Figure 22: Third point of Raman taken on coral 1 Bleached ...... 29 Figure 23: First point of Raman taken on coral 2 Bleached ...... 30 Figure 24: Second point of Raman taken on coral 2 Bleached ...... 30 Figure 25: Third point of Raman taken on coral 2 Bleached ...... 30 Figure 26: First point of Raman taken on coral 3 Sanded ...... 31 Figure 27: Second point of Raman taken on coral 3 Sanded ...... 31 Figure 28: Third point of Raman taken on coral Sanded ...... 31

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Figure 29: Indentations of 1 Bleached ...... 33 Figure 30: Indentations of 2 Bleached ...... 35 Figure 31: Indentations of Sanded ...... 37

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1. Introduction

Figure 1: Acropora cervicornis [1]

Corals are found throughout the world and are an important part of the oceans eco-system.

Fish and many other creatures rely heavily on these corals for their habitats. Unfortunately, many coral reefs have become unhealthy and are dying out due to various factors such as a rising change in ocean temperature and increased ocean acidity. This has a negative impact on the strength and structural integrity of the corals skeleton and affects the health of the microorganism residing in the skeleton. Other factors include storm intensity and frequency, which cause turbulent waters that damage and dislodge the corals [2], disease and harmful types of macroalgae [3], as well as many other unknown factors.

Many research experiments have been conducted involving the restorations of reefs through various methods. However, there is little information regarding the mechanical properties of these coral skeletons which is the structural part responsible for maintaining stability and

rigidity. Due to constant pressure imposed from ocean waves, corals are subjected to cyclic fatigue which affects their survival and lifespan. Therefore, it is important to study both instantaneous and time dependent mechanical properties of coral skeletons to be able to predict, both experimentally and theoretically, their behavior under several types of loadings they experience in the ocean.

Figure 2: Diseased Acropora cervicornis [4]

Knowledge of the mechanical properties of corals can improve the ways humans aid in the recovery and restoration of these threatened and endangered species. For example, coral rehabilitation experiments can be better equipped in improving the production and maintenance techniques of ocean based coral nurseries by selecting synthetic materials to be used as a framework for damaged coral reefs. The type of coral skeleton used to conduct this research is the critically endangered staghorn coral, Acropora cervicornis. This species of corals, as well as many others, have faced a drastic decline in population due to the previously mentioned factors. By studying its mechanical properties, including the hardness and compressive strength of its skeleton, one can better understand its mechanical stability.

2. Literature Review

2.1 Microorganisms in Corals

Corals share a mutualistic interaction with the algae genus Symbiodinium (also referred to as zooxanthellae), as well as many other types of microorganisms. The microorganisms are members of the three kingdoms: Bacteria, Archaea, and Eucarya. Symbiodinium benefits corals by transferring photosynthetically fixed carbon to coral polyps. Several types of bacteria are housed in the surface mucus layer, coral tissue (including the gastrodermal cavity), and the calcium carbonate (CaCO3) skeleton (Fig. 3) [5].

Figure 3: Structure of coral tissue. Bacteria resides in the surface mucus layer, coral tissue and the calcium carbonate (CaCO3) skeleton [5]

The porous coral skeletons house bacteria that satisfy approximately 50% of a coral’s total nitrogen needs. Loss of these endosymbiotic algae and/or its photosynthetic pigments results in

the corals losing their colors in a disease known as coral bleaching (Fig. 4). Bleaching usually occurs during extremely cold or warm ocean temperatures which affect the microorganisms within the coral. When corals are exposed to environmental stresses, such as climate change or water pollution, the microbial population and the coral’s polyps can either adapt to this new condition or succumb to coral disease [5].

Figure 4: Bleached coral skeletons in the Great Barrier Reef near Port Douglas, Australia [6]

2.2 Structure of Coral Skeletons

The skeletal structure of corals greatly influences their ability to withstand hydrodynamic forces within their habitats. Skeleton strength influences a variety of factors such as branch size, growth form, and the ability to thrive in environments suitable to that specific species of coral [7].

The Acropora type of coral grows by a branching out method. This is beneficial because the extension of its branches up and outwards allows the coral to receive more space and light than other low-growing coral species [8]. Unfortunately, this growth method also makes branched

colonies prone to greater stress per unit of hydraulic force because their branches become subjected to additional bending stresses [7]. This method of growth also makes the coral prone to breakage.

Since A. cervicornis is often found in shallow waters it must withstand the stresses caused by the force of breaking waves. However, a moderate amount of breakage is beneficial in assisting the corals in asexual fragmentation, their primary way of reproduction. Some of the fragments that have broken off survive by reattaching to the substrate. This allows the corals’ colonies to reduce stress, enable survivorship, and begin the growth of new colonies [2].

2.2.1 Chemical Composition

A general chemical composition of the coral skeleton of A. palmata was determined to have the principal elements oxygen, calcium, and carbon; the minority elements sodium, sulfur and magnesium; and trace elements (less than 0.1%) [8]. The skeleton is aragonite, which is a naturally occurring form of calcium carbonate (CaCO3) [5].

2.2.2 Crystal and Microstructure

Figure 5 shows scanning electron microscopes (SEM) images of the stony coral Stylophora pistillata. The walls of coral skeletons are made up of corallites (Fig. 5A). A single corallite, along with additional elements of the skeleton including the columella, the speta, and the theca, are shown in Fig. 5B. By using polarized light microscopy (PLM) and electron backscatter diffraction

(EBSD) higher resolution images are taken of the acicular aragonite crystals extending outwards from the center of calcification (COC) as shown in Fig. 5, C and D [9].

Figure 5: Morphological skeleton features common to all stony corals (A) Combination of SEM images showing the intact surface of a skeletal branch. (B and C) PLM micrographs of a single corallite and a trabecula, respectively. (D) ESBD inverse pole figure orientation map of trabecula. [9]

The process of coral growth is biomediated, meaning the skeletal organic matrix secreted by the coral plays the key role in this process. Amorphous calcium carbonite (ACC) nanoparticles are deposited in microenvironments that are enriched in skeletal organic matrix (SOM). Skeleton proteins are then interwoven with the calcium carbonate coating the aragonite crystals. As these

ACC nanoparticles are formed and move away from the center of calcification, they eventually lose their magnesium and become acicular aragonite crystals which then form the skeletal fibers

(Fig. 6) [9].

Figure 6: Growth process [9]

The first step of coral biomineralization is the animal cells secreting the SOM. The second step involves the deposition of magnesium-rich ACC nanoparticles. The first and second step are able to take place simultaneously. The third step is the growth of acicular aragonite crystals by attachment of amorphous precursor nanoparticles. The final step is the formation of skeletal fibers

(Fig. 7) [9].

Figure 7: Working model of coral biomineralization [9]

As shown in Fig. 8, A. cervicornis grows from the tip by the continuous addition of needle like crystals on aragonite crystals. These groups form the fasciculi which make up the porous primary skeleton. From the tip, the porosity and permeability of the skeleton decreases, while its density increases. The addition of aragonite to the fasciculi and the increase of calcium carbonate in the channels of the coral skeleton causes the density to increase [10].

Figure 8: Sketch of growth of the coral axial corallite [10]

2.3 Mechanical Behavior of Coral Skeletons

Table 1: Summary of Previous Literature Results

Study Coral Compressive Tensile Elastic Vickers Species Strength Strength modulus microhardness (MPa) (MPa) (GPa) (GPa) Chamberlain Acropora 21-80.8 8.7-38 (1978) [7] palmata Vosburgh Acropora 62-89.8 10.3-40.9 24.8-82.4 (1982) [11] reticulata Alvarez Acropora 48 (direction of 3.31 (2002) [8] palmata polyp growth) 23 perpendicular Baldock Acropora 57 ±32 8.84-12.72 55-77 (2014) [2] intermedia

2.3.1 Vickers Hardness

Vickers Hardness can be calculated using the equation:

θ 2퐹푠푖푛 퐹 Equation 1: 퐻푉 = 2 = 1.854 푑2 푑2

Where: F = load (gf), d = mean diagonal of the indentation (mm), θ = angle between opposite faces of the diamond = 136º

The mean value of Vickers microhardness of A. palmata was found using a load of 50 gf applied for 15s [7].

2.3.2 Compressive Strength

In compression tests, corals seem to be strongest along the grain of the structure. As shown in Fig. 9, the force of water often acts perpendicular to the branch. Corals overcome this with

design features that allows maximum strain in the direction of these stresses. The polyp orientation is also closer to 60°, which allows for the coral to better withstand the principle stresses [10].

Figure 9: Physical stresses in a coral branch. The lines in the drawing indicate the grains of a coral branch. Arrows indicate the direction of the forces due to water, and the resulting tensile and compressive stresses acting on the coral [10].

The growth direction of polyps are good indicators of the fractures that develop from applied stress (Fig. 10). Corals with polyp growth parallel to the axis of the core tend to develop planar extension fractures that are roughly parallel to the stress. As the angle between the polyp growth direction and stress direction increases, fracture resistance also increases [7]. The growth direction of the A. cervicornis polyps is like diagram A of Fig. 10. Therefore, it can be assumed that an axial compressive load will cause it to fracture down the middle of the specimen.

Porosity (defined as pore volume/total skeletal volume) also plays a factor in coral skeletal strength. This strength varies inversely with skeletal porosity. Although coral skeletons do not deal with strain energy as well as other skeletal materials, they compensate by having a relatively high

strength even with less material per unit volume. Massive colonies are less porous and can withstand mechanical stresses better than colonies of branching corals [7].

Figure 10: Fracture of test cores as a function of skeletal structure. Fracture shown by heavy line in sketches at top. Actual appearance of fractured cores shown at bottom. A- Montastrea annularis, growth parallel to stress. B- Montastrea annularis, growth oblique to stress. C- Ancropora palmata, [7]

Mechanical tests on A. palmata consisted of applying a load range of 200-400 kg at a strain rate of 0.02mm/min. This resulted in a compressive strength of 48 MPa in the direction of polyp growth and 23 MPa perpendicular to it [8].

In another study, samples were cut to a diameter to length ratio of 1:3 and a static compressive load of 2 mm/min was applied at both ends until failure. The compressive strength was calculated from the equation:

4푁 Equation 2: 휎 = 푐 휋퐷2

This led to a mean average compressive strength of 57 MPa with a standard deviation of 32 MPa.

This high standard deviation was from two samples with a compressive strength of under 30 MPa

[2].

2.4 Raman Spectroscopy Analysis

In 1929, Sir Chandrasekhr Venkata Raman started the beginning of Raman Spectroscopy with the use of his telescope. Over time, it gradually developed into an extremely useful way to analyze material [12]. Raman spectroscopy analyzes a material’s chemistry by using scattered light. As the light (i.e. laser) passes through a sample, it causes the molecules to vibrate in a way specific to that particular material. An extremely small amount of this scattered light shifts in energy from the laser frequency due to electromagnetic waves and vibrations from the molecules.

This shifted light is then plotted in accordance to its intensity levels and frequency and results in a

Raman spectrum of that material [13].

3. Goals

The goal of this research is to explore the mechanical instantaneous behavior of the skeleton of Acropora cervicornis by determining its Vickers hardness and compressive strength.

Raman spectroscopy will also be observed to find the Raman spectrum associated with the coral.

This information can aid in making of synthetic materials that can be used as a framework for damaged coral reefs.

4. Materials and Methods

This research was conducted at the University of Central Florida using dried skeletons of

Acropora cervicornis. The three skeletal branches used were provided by Nova Southeastern

University’s permitted offshore nursery (Appendix B). Two skeletons were cleaned using bleach

(sodium hypochlorite, NaCIO), while the other skeleton was cleaned in-site by microorganisms.

The three pieces of coral used to conduct these experiments are shown in Fig. 11. Throughout this paper they will be referred to as 1 Bleached, 2 Bleached and 3 Sanded. The corals were cut using a diamond blade to an approximate length to diameter ratio of 2:1.

22 cm

Figure 11: Coral branches used in experiment: 1 Bleached, 2 Bleached, 3 Sanded (left to right)

The diameter of each cut sample was measured from their top surface (i.e. in the direction of the growth of the polyps). The height of each sample was measured as well. As shown in Tables

5-8, the diameters of the compressed sampled ranged between 7.37 to 14.12 mm and the heights ranged between 16.86 to 28.73 mm.

4.1 Raman Spectroscopy Analysis

To perform the Raman Spectroscopy, a Renishaw® inVia Raman microscope (Renishaw,

Gloucestershire, the UK) was used. This system contains a 514.5 nm line of Ar+ ion laser with a maximum power of 25 mW. When testing the samples, 50% of this power was used. The system also contained a spectrograph fitted with holographic notch filters and an optical microscope. A

100x lens was used for the magnification with a focus shift of 100-2000 cm-1 when taking the

Raman spectrum [14].

4.2 Micro-images

The corals samples 1 Bleached, 2 Bleached, and 3 Sanded were examined under a microscope. Images were taken at a magnification of 50x as shown in Fig. 12. The top row in

Figures 17-19 also shows this image with the use of a camera and the naked eye. From these images the symmetrical structure of coral skeletons can be seen.

1 Bleached 2 Bleached 3 Sanded

600µm

Figure 12: Top view microimages of the polished samples: 1 Bleached, 2 Bleached, and 3 Sanded (left to right)

4.3 Vickers Micro Hardness Test

One sample from each branch was taken and prepared for hardness testing. Samples 1

Bleached, 2 Bleached, and 3 Sanded were polished by hand on SiC sand paper with grits of P1000,

P2000, and P3000, to achieve a mirrored finish suitable for hardness measurements. The height and diameter were recorded for each sample (Table 5). To find the indentation marks with ease, the circumferences of the corals were wrapped in scotch tapes and marked with an arrow.

Indentations for Vickers hardness tests were done using a Tukon 2100B (Wilson Instruments,

Illinois, USA). Ten indentations were made on each sample with a load of 50 g applied for 15 s.

Figure 13: Coral sample undergoing indentation

4.4 Compression Tests

Uniaxial compression tests were performed on the samples using a universal testing machine (Criterion® 43, MTS, Minnesota, USA). A compressive axial load of 0.003 mm/s was

applied until noticeable failure. Using the main compressive axial load at failure, compressive strength was calculated using the equation:

푃 Equation 3: 휎 = 푐 퐴

휋푑2 where σc is the compressive strength, P is the load, A is the area of a cylinder 퐴 = , and d is the 4 diameter of the sample.

Figure 14: Coral undergoing compression test

5. Results

5.1 Raman Spectroscopy Analysis

There was no significant change in the Raman shift among the three samples (Figures 20-

28). Their chemical composition was almost identical to Raman shifts studies done on materials composed of calcium carbonate (Fig. 15). This result was expected because coral skeletons are composed of calcium carbonate [14] in the form of aragonite, which produces needle-like crystals.

1087 Calcium Carbonate

713 283

Intensity

-1 Wavenumber/ cm

Figure 15: Normal Raman spectra of calcium carbonate (calcite, chalk) [15]

5.2 Vickers Hardness

Table 2: Hardness Results of Polished Samples 1 Bleached Mean Hardness Standard Deviation Units 350.56 11.95 Hv 3.44 0.12 GPa 2 Bleached Mean Hardness Standard Deviation 273.11 38.08 Hv 2.68 0.37 GPa 3 Sanded Mean Hardness Standard Deviation 361.28 13.40 Hv 3.54 0.13 GPa

Samples from 1 Bleached had a mean hardness of 3.44 ± 0.12 GPa. Samples from 2

Bleached had a hardness value significantly lower at only 2.68 ± 0.37 GPa. The samples from 3

Sanded also had a similar value to 1 Bleached at 3.54 ± 0.13 GPa.

5.3 Compression Tests

Examples of the compression fractures are shown in Fig. 16. All samples compressed fractured in this way. The results of the compression tests can be found in Appendix A, Plots 1-9.

The trend reveled throughout these plots show that as the load increases minor breakage occurred.

Despite these fractures, the coral structure held and continued to support the load. Stress continued to rise until it reached a maximum stress peak where a major fracture occurred. After this fracture, the stress level decreased. Tables 3 and 4 show significant numbers associated with each sample tested.

Figure 16: Fractured coral skeleton samples 1.d Bleached (left) and 2.c Bleached (right)

Table 3: Bleached Samples: Max Peak, Mentionable Peaks, and Mean Max Peak Stress

Bleached Samples 1.c 1.d 2.c 2.d Max Failure Peak Stress (MPa) 15.55 12.05 27.2 21.12 10.31 8.56 7.48 10.67 Mentionable Peaks (MPa) 4.65 Mean Max Peak Stress (MPa) 18.98

Table 4: Sanded Samples: Max Peak, Mentionable Peaks, and Mean Max Peak Stress Results

Sanded Samples 3.a 3.b 3.c 3.d 3.e Main Failure Peak Stress (MPa) 37.67 21.38 28.36 31.69 26.71 19.7 18.21 16.15 23.04 15.05 Mentionable Peaks (MPa) 13.33 20.94 13.32 18.57 Mean Max Peak Stress (MPa) 29.16

6. Discussion

The Raman spectroscopy portion of this study fell in accordance to the Raman spectra of similar materials also composed of calcium carbonate.

The hardness calculated from samples 1 Bleached and 3 Sanded fell in accordance to the study done by Alverez (2002), who found the mean value of A. palmata to be 3.31 GPa (329.48 ±

3.44 HV). Sample 2 Bleached had a lower value of 2.68 ± 0.37 GPa. The reason for this seemingly skewed value is most likely that the top and bottom surfaces were not completely horizontal to the surface. This error likely occurred because the samples were polished by hand. This would affect the indentations, which need to be performed on a flat horizontal surface for accurate results.

Direction of fractures from the compression tests were to be expected after observing the results of Chamberlin’s (1978) findings of the relationship between the direction of the growth of polyps to the axis of the core. In A. cervicornis, the parallel growth of the polyps causes fractures to develop that are roughly parallel to the applied stress. The reason the coral structure was able to support the load despite fractures may have been due to its porous skeleton, which prevented the fractures from spreading throughout the skeleton and jeopardizing its skeletal structure. The mean axial compressive strength of the bleached samples was 18.98 MPa while the sanded samples were

29.62 MPa. Samples from 1 Bleached are lower than the other samples at 15.55 MPa and 12.05

MPa. A possible reason for this may be because these samples are from younger regions of the branch. This would cause them to be less dense than the other samples and would impact their ability to support the given load.

7. Conclusion

Bleached (chemically cleaned) and Sanded (biologically cleaned) coral skeletons of

Acropora cervicornis were used to study their mechanical properties in this experiment. The microstructure of A. cervicornis shows their complex skeletal organization. In future studies it would be beneficial to use a Focus Ion Beam that can produce a 3D image of the coral skeletons to observe and study their structures.

Raman spectroscopy measurements of the coral skeletons verified that they are composed of calcium carbonate (CaCO3). The mechanical properties from Vickers hardness and axial compression tests were also studied. Vickers hardness tests shows that the coral skeletons of the chemically cleaned corals had no significant difference when compared to the coral skeletons cleaned by microorganisms. After making 10 indentations at a 50 g load, Vickers hardness values found that 1 Bleached samples had the mean hardness of 3.44 ± 0.12 GPa. The sanded sample also had a similar value of 3.54 ± 0.13 GPa. However, samples from 2 Bleached had a hardness value that was significantly lower at only 2.68 ± 0.37 GPa. This lower value was most likely due to a sloped surface which affected the accuracy of the indentations and measurements. Mean axial compressive stress tests determined that the maximum mean strength for the bleached samples was

18.98 MPa, compared to the 29.16 MPa value of the sanded sample. One can see from the stress vs. time deformation plots, the coral skeletons exhibited “gracious” failure during compressive loadings. Due to the corals polyp growth direction, when compressed axially, the fracture happened parallel to the load.

Appendix A

Table 5: Dimensions of Polished Coral Samples

Coral Height (mm) Diameter (mm) 1 Bleached 22.28 13.30 2 Bleached 22.36 12.64 3 Sanded 13.73 8.56

Table 6: Dimensions of 1 Bleached Coral Samples

Coral Height (mm) Diameter (mm) 1B.a (Not used) 27.73 11.22 1B.b (Not used) 27.13 8.17 1B.c 24.69 12.52 1B.d 22.10 13.11

Table 7: Dimensions of 2 Bleached Coral Samples

Coral Height (mm) Diameter (mm) 2B.a (Not used) 25.59 8.16 2B.b (Not used) 23.23 7.87 2B.c 31.58 14.12 2B.d 28.73 9.85

Table 8: Dimensions of 3 Sanded Coral Samples

Coral Height (mm) Diameter (mm) 3S.a 21.69 7.97 3S.b 16.86 9.30 3S.c 20.75 9.43 3S.d 26.76 7.37 3S.e 20.22 9.16 3S.f (Not used) 20.53 9.89 3S.g (Not used) 22.02 7.94

Figure 17a: Profile and Cross-sectional pictures of 1 Bleached samples

Figure 18: Profile and Cross-sectional pictures of 2 Bleached samples

Figure 19: Profile and Cross-sectional pictures of 3 Sanded samples

1st Point 1 Bleached 120000 1082 100000 80000 60000 150 701

Intensity 40000 203 20000 0 100 300 500 700 900 1100 1300 1500 1700 1900 Raman shift/cm-1

Figure 20: First point of Raman taken on coral 1 Bleached [14]

2nd Point 1 Bleached 100000 1082 80000

60000 701 40000 150

Intensity 203 20000 0 100 300 500 700 900 1100 1300 1500 1700 1900 Raman Shift/cm-1

Figure 21: Second point of Raman taken on coral 1 Bleached [14]

3rd Point 1 Bleached 20000 1082

15000

10000 699

Intensity 150 5000 203

0 100 300 500 700 900 1100 1300 1500 1700 1900 Raman Shift/cm-1

Figure 22: Third point of Raman taken on coral 1 Bleached [14]

1st Point 2 Bleached 50000 1082 45000 40000 35000 30000 25000 20000 700

Intensity 149 15000 203 10000 5000 0 100 300 500 700 900 1100 1300 1500 1700 1900 Raman Shift/cm-1

Figure 23: First point of Raman taken on coral 2 Bleached [14]

2nd Point 2 Bleached 50000 1082 40000 30000 20000 149 700 Intensity 202 10000 0 100 300 500 700 900 1100 1300 1500 1700 1900 Raman Shift/cm-1

Figure 24: Second point of Raman taken on coral 2 Bleached [14]

3rd Point 2 Bleached 70000 1081 60000 50000 40000 30000 149 700 Intensity 20000 202 10000 0 100 300 500 700 900 1100 1300 1500 1700 1900 Raman Shift/cm-1

Figure 25: Third point of Raman taken on coral 2 Bleached [14]

1st Point Sanded 60000 1081 50000 40000 30000 700 Intensity 20000 149 10000 203 0 100 300 500 700 900 1100 1300 1500 1700 1900 Raman Shift/cm-1

Figure 26: First point of Raman taken on coral 3Sanded [14]

2nd Point Sanded 60000 1081 50000 40000 30000

Intensity 20000 700 149 10000 202 0 100 300 500 700 900 1100 1300 1500 1700 1900 Raman Shift/cm-1

Figure 27: Second point of Raman taken on coral 3 Sanded [14]

3rd Point Sanded 60000 1081 50000 40000 30000

Intensity 20000 149 700 10000 203 0 100 300 500 700 900 1100 1300 1500 1700 1900 Raman Shift/cm-1

Figure 28: Third point of Raman taken on coral 3 Sanded [14]

Table 9: Vickers Hardness of 1 Bleached and Polished Sample

1 2 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 16.21 16.68 50 17.01 16.41 50 Hardness (Hv) 342.8 Hardness (Hv) 332.0 Hardness (GPa) 3.36 Hardness (GPa) 3.26 3 4 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 16.07 15.81 50 16.7 16.45 50 Hardness (Hv) 364.8 Hardness (Hv) 337.4 Hardness (GPa) 3.58 Hardness (GPa) 3.31 5 6 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 16.58 16.26 50 16.18 15.93 50 Hardness (Hv) 343.8 Hardness (Hv) 359.6 Hardness (GPa) 3.37 Hardness (GPa) 3.53 7 8 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 16.18 15.8 50 16.04 15.99 50 Hardness (Hv) 362.6 Hardness (Hv) 361.4 Hardness (GPa) 3.56 Hardness (GPa) 3.54 9 10 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 16.66 16.4 50 15.83 16.18 50 Hardness (Hv) 339.3 Hardness (Hv) 361.9 Hardness (GPa) 3.33 Hardness (GPa) 3.55

1 Bleached Indentation 1 Indentation 2

Indentation 3 Indentation 4

Indentation 5 Indentation 6

Indentation 7 Indentation 8

Indentation 9 Indentation 10

Figure 29: Indentations of 1 Bleached

Table 10: Vickers Hardness of 2 Bleached and Polished Sample

1 2 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 17.2 17.37 50 17.1 17.58 50 Hardness (Hv) 310.3 Hardness (Hv) 308.3 Hardness (GPa) 3.04 Hardness (GPa) 3.02 3 4 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 17.93 17.21 50 17.83 16.31 50 Hardness (Hv) 300.3 Hardness (Hv) 318.1 Hardness (GPa) 2.94 Hardness (GPa) 3.12 5 6 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 16.69 18.6 50 18.08 20.88 50 Hardness (Hv) 297.7 Hardness (Hv) 244.3 Hardness (GPa) 2.92 Hardness (GPa) 2.40 7 8 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 17.67 19.14 50 20.53 22.57 50 Hardness (Hv) 273.7 Hardness (Hv) 199.6 Hardness (GPa) 2.68 Hardness (GPa) 1.96 9 10 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 19.39 19.79 50 20.56 18.97 50 Hardness (Hv) 241.6 Hardness (Hv) 237.3 Hardness (GPa) 2.37 Hardness (GPa) 2.33

2 Bleached Indentation 1 Indentation 2

Indentation 3 Indentation 4

Indentation 5 Indentation 6

Indentation 7 Indentation 8

Indentation 9 Indentation 10

Figure 30: Indentations of 2 Bleached

Table 11: Vickers Hardness of 3 Sanded Sample

1 2 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 16.4 15.9 50 16.93 16.08 50 Hardness (Hv) 355.4 Hardness (Hv) 340.3 Hardness (GPa) 3.49 Hardness (GPa) 3.34 3 4 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 15.68 15.52 50 16.59 15.78 50 Hardness (Hv) 380.9 Hardness (Hv) 353.9 Hardness (GPa) 3.74 Hardness (GPa) 3.47 5 6 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 16.02 15.98 50 16.36 15.73 50 Hardness (Hv) 362.1 Hardness (Hv) 360.1 Hardness (GPa) 3.55 Hardness (GPa) 3.53 7 8 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 16.74 16.06 50 15.31 15.74 50 Hardness (Hv) 344.7 Hardness (Hv) 384.6 Hardness (GPa) 3.38 Hardness (GPa) 3.77 9 10 d1(μm) d2(μm) kgf d1(μm) d2(μm) kgf 15.79 16.21 50 16.13 15.58 50 Hardness (Hv) 362.1 Hardness (Hv) 368.8 Hardness (GPa) 3.55 Hardness (GPa) 3.62

3 Sanded Indentation 1 Indentation 2

Indentation 3 Indentation 4

Indentation 5 Indentation 6

Indentation 7 Indentation 8

Indentation 9 Indentation 10

Figure 31: Indentations of Sanded

Plot 1: Compression Results of Sample 1.c Bleached

1.c Bleached 18 100.95,15.55, 16 14 12 10 8 75.55, 10.31

6 Stress (MPa) Stress 4 2 0 -2 0 50 100 150 200 250 300 350 Time (s)

Plot 2: Compression Results of Sample 1.d Bleached

1.d Bleached 14 79.42, 12.05 12 59.32, 8.56 10

8 40.32, 4.65 6

4 Stress (MPa) Stress 2

0 0 50 100 150 200 250 -2 Time (s)

Plot 3: Compression Results of Sample 2.c Bleached

2.c Bleached 375.91, 27.20 30

15 123.61, 7.48

10 Stress (MPa) Stress 5

0 0 50 100 150 200 250 300 350 400 450 -5 Time (s)

Plot 4: Compression Results of Sample 2.d Bleached

2.d Bleached 25 155.22, 21.12 53.52, 10.67 20

Stress (MPa) Stress 5

0 0 100 200 300 400 500 600 700 -5 Time (s)

Plot 5: Compression Results of Sample 3.a Sanded

3.a Sanded 83.37, 37.67 40 35 30 25 65.22, 19.70 20 15

Stress (MPa) Stress 10 5 0 0 50 100 150 200 250 300 -5 Time (s)

Plot 6: Compression Results of Sample 3.b Sanded

3.b Sanded 25 152.81,18.21 218.01, 21.38

20 52.04, 13.33

Stress (MPa) Stress 5

0 0 100 200 300 400 500 600 -5 Time (s)

Plot 7: Compression Results of Sample 3.c Sanded

3.c Sanded 35 107.92, 28.36 30

25 63.72, 16.15 20

10 Stress (MPa) Stress 5

0 0 50 100 150 200 -5 Time (s)

Plot 8: Compression Results of Sample 3.d Sanded

3.d Sanded 35 227.11, 23.04 297.82,31.69 30 193.82, 20.94 25 160.42, 18.57 20

Stress (MPa) Stress 10

0 0 50 100 150 200 250 300 350 400 Time (s)

Plot 9: Compression Results of Sample 3.e Sanded

3.e Sanded 30 205.21, 26.71 25

20 121.11, 15.05

15 99.11, 13.32

10 Stress (MPa) Stress 5

0 0 50 100 150 200 250 300 -5 Time (s)

Appendix B

25 August 2017

Dr. Nina Orlovskaya

Associate professor

College of Engineering and Computer Science University of Central Florida 12760 Pegasus Blvd.

P.O. Box 162450

Orlando, FL • 32816-2450

Dear Dr. Orlovskaya

This letter confirms temporary transfer of 30 skeletons of the stony coral Acropora cervicornis from my possession to yours. Acropora cervicornis is listed as threatened under the Endangered Species Act. These specimens came from our (Nova Southeastern University) permitted offshore A. cervicornis nursery. Per 50 CFR 223.208, transport of legally-obtained specimens is permitted as long as it is not in the pursuit of commerce. The purpose of this temporary transfer is education and research.

A copy of this letter should remain with the specimens at all time. If any questions please contact me at the information provided below.

Sincerely,

David Gilliam, PhD Nova Southeastern University Halmos College of Natural Sciences and Oceanography 8000 North Ocean Drive; Dania Beach FL 33004 Office: 954 262-3634 [email protected]

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