Structure-Strength Correlations in Five Species of Seaweed

A Major Qualifying Project Report Submitted to the Faculty of the WORCESTER POLYTECHNIC INSTITUTE

In partial fulfillment of the requirements for the Degree of Bachelor of Science in Mechanical Engineering Submitted by: ______Rachel Swanson ______Rachel Sudol

Date: 05/01/2018

Keywords: 1. Seaweed 2. Biopolymers 3. Strength 4. Mechanical Properties 5. Cellulose

Approved by: ______Prof. Satya Shivkumar, Major Advisor Abstract

Seaweeds are plentiful and simple to cultivate in most coastal areas of the world. Current industrial uses of seaweed utilize chemically extracted polysaccharides and hydrocolloids found in seaweeds, not entire seaweed fibers. This study investigates the structure and strength of seaweeds found in New England and CA. The samples were analyzed by microscopy, TGA, and tensile testing. Typical breaking forces were between 3 N and 30 N for red and brown seaweed, respectively. Strength for these samples ranged from 0.7 to 29 MPa. The properties of seaweed are determined by the fractions of polysaccharides, cellulose and inorganics. In addition, cellular orientation increases the strength. The organized structure in seaweed and its high strength may enable its use in new biopolymer products.

Acknowledgements The authors of this MQP would like to thank Professor Pamela J. Weathers for her aid in helping identify various species of seaweed. We would also like to thank Professor JoAnn L. Whitefleet- Smith for her aid in microscopy as well as Professor Tiffany Butler who helped us with testing our samples. We thank Daryl Johnson for teaching us the TGA machine in the Gateway lab. Finally, we would like to thank Professor Satya Shivkumar for his guidance throughout this process as our advisor. This project would not be complete without him.

Table of Contents

1. List of Figures 5 2. Introduction 6 3. Literature Review 8 3.1 Seaweed Structure 8 3.1.1 Morphology 8 3.1.2 Composition 9 3.1.3 Effects of Ecology on Structure 11 3.2 Material Properties and Uses of Seaweed 12 3.2.1 Brown Seaweed 12 3.2.2 Green Seaweed 13 3.2.3 Red Seaweed 13 3.2.4 Carrageenan and Agar Films 13 3.3 Seaweed Biomaterials 14 4. Objectives 15 5. Methods 16 6. Modeling of Tensile Properties 20 7. Results 24 8. Conclusions 27 9. References 29 10. Journal Article 31 11. Appendix 47

1. List of Figures

Figure 1: Morphology of Seaweed 8

Table 1: Various Components of Seaweed 10

Figure 2: Cellulose Degradation 10

Figure 3: Tensile Strength versus Percent Composition of Cellulose 11

Figure 4: Impact of Temperature on Cell Wall Thickness 12

Figure 5: Seaweed Pressing Method 17

Figure 6: Sample Loaded in Instron Tensile Testing Machine 18

Figure 7: Netzsch TGA Machine and TGA Tested Sample 19

Figure 8: SOLIDWORKS Description of Seaweed Structure: Small Fibers 20

Figure 9: SOLIDWORKS Description of Seaweed Structure: Medium Fibers 21

Figure 10: SOLIDWORKS Description of Seaweed Structure: Large Fibers 21

Figure 11: Small Fiber Tensile Test Simulation 22

Figure 12: Medium Fiber Tensile Test Simulation 22

Figure 13: Large Fiber Tensile Test Simulation 23

Figure 14: Representative Force-Displacement for Each Species of Seaweed 25

2. Introduction

Seaweed can grow in most coastal areas of the world and needs very little input and care during its growth cycle. This makes it a cheap and widely accessible base for many different applications. It has recently gained attention for its potential to be used to manufacture materials, specifically plastics. These plastics are generally made using solely the polysaccharides in the seaweed, neglecting the mechanical strength that could be gained by using the entire fiber structure of the seaweed plant.

Red seaweed, in the phylum Rhodophyta, grows typically in tropical environments in the intertidal and subtidal zones (40 meters to 250 meters deep) at a depth typically deeper than where green and brown seaweed grow due to red seaweed’s ability to absorb blue light [1]. The color of red seaweed comes from the pigments phycoerythrin and phycocyanin. An important subgroup of red seaweed is the coralline algae which releases calcium carbonate onto the surfaces of their cells. Over 6,500 species of red seaweeds are known, the majority of which are marine species [1].

Brown seaweed, in the phylum Phaeophyta, includes mostly marine species and grows in the tidal zones of colder oceans in the world. It is the largest category of seaweed and is usually brown or yellow-brown in color [2]. This brown color is a result of the xanthophyll pigment, fucoxanthin, which masks the chlorophyll pigments. Brown seaweed typically has a branched and filamentous thallus, kelp being the best known example. Brown seaweed is the only type of algae that have internal tissue differentiation into conducting tissue. There are approximately 1800 known species of brown seaweed [1].

The fiber structure of red seaweed is generally filamentous or made of chains of cells attached by end walls and is usually branched. Cortification (husk) or ‘pit plugs’ can occur. ‘Pit plugs’ are granular protein masses that fill holes created during cell division [2]. Brown seaweed are generally filamentous with branches. Many have air bladders which aid in flotation. Some species can be grown meters in height, forming marine kelp forests [1].

The important polysaccharides in red seaweed are floridoside, cellulose, agar, and carrageenan. Floridoside serves as a food reserve and is made up of alpha-d-glucose and amyopectin. Cellulose consists of Beta-1,4 glucose molecules that form microfibrils which make up the cell wall. Agar is a structural polysaccharide that contains D- and L- galactose molecules which makes up part of the matrix that encases the cellulose in the cell wall. Carrageenan is made of only d-galactose units and also surrounds cellulose in the cell wall [3]. The important polysaccharides in brown seaweed are lamarin, alginic acid, and cellulose. Lamarin consists of 20-25 glucose units and beta-1,3 linked glucean which serve as food reserves. Alginic acid is a beta-1,4 linked d-manunonic acid and alpha-1,4 linked l-guluronic acid that forms alginate when the alginic acid chains form around calcium ions to make a stiff gel. Cellulose functions the same way as in the red algae [3]. Trace amounts of iron (Fe), zinc (Zn), Manganese (Mn), and copper (Cu) have been found in both red and brown seaweed [3].

Currently, seaweed is used in a variety of applications. The main use is as a food product, with two million tons of seaweed processed into one type of food or another [2]. The second major use is mainly for the extraction of the phycocolloids alginate, agar and carrageenan [2]. A typical technique that has been used to utilize seaweed and other biopolymers is to grind the material and incorporate it into commodity and engineering thermoplastics [4]. A composite material was made by adding particles of Posidonia oceanica, a red seaweed, with an average particle size of 250 µm with a biobased high density polyethylene. However, seaweed is hydrophilic and most plastics are generally hydrophobic, which leads to improper mixing and thus poor mechanical properties.

Materials have also been made from seaweed using only the polysaccharides that can be extracted from it [4]. The polysaccharides are extracted from seaweed and processed in order to produce a plastic resin in combination with traditional thermoplastics [5]. Wong, et al. [6] developed such a plastic in the form of a film, by adding sago starch, agar and glycerol to a solution and drying it overnight at 50°C. These processes do not exploit the fibers of seaweed, which could be used to increase strength of these plastics. A methodology that utilizes the structural feature of seaweed without the need for a matrix of traditional plastics would be highly beneficial both for sustainability, recycling, and material properties. In order to develop such structures, it is imperative to understand the structure-property relationships of different seaweeds. It is the purpose of this contribution to obtain information in this regard.

3. Literature Review

3.1 Seaweed Structure

Seaweed structure encompass many different components of the seaweed organism. The relevant components to this study, morphology, composition and effect of ecology on structure, are discussed below.

3.1.1 Morphology

Seaweed is made up of the thallus, stipe, floats and holdfast. The thallus, seen in Figure 1, is the large leaf-like structure of the organism. The stipe functions as the stem, connecting thallus and holdfast. The holdfast is the roots of the plant, anchoring it to a substrata. The floats are air filled structures that help the plant to be buoyant. The thallus and stipe are consistent across all types of seaweed. Some species of seaweed do not have floats, or are free-floating, so do not have holdfasts [7].

Figure 1: Morphology of Seaweed [7]

Seaweeds tend to grow in filaments, which are chains of cells attached by cell walls. These chains are rarely linear, mostly branching at different points throughout growth. Red seaweeds exhibit “pit plugs”, granular protein masses that fill holes left in cell division and filament branching. Red seaweeds often have cortication, in which some cells form a husk or bark around the rest [8].

Seaweeds sometimes show parenchymatous construction, meaning they are made up of differentiated cells. This type of cell is only seen in brown seaweeds. Further, brown and green seaweeds display plasmodesmata, which are structures that link adjacent cells and suggest some degree of cell-to-cell communication [8].

3.1.2 Composition

Polysaccharides A study in the British Phycological Journal found the main food reserve for green seaweed to be starch. The main polysaccharides in all the green algae tested were found to be polydisperse heteropolysaccharides, a strong acid. Polysaccharide proportions in green seaweed vary from genus to genus. The function of each polysaccharide can be food storage, structural support, or to prevent destruction from tides. The structural polysaccharides of green seaweed synthesize xylans and mannans. The xylans are the main structural component in some species and give a strong fibrous structure, making up the main skeletal component of the seaweed. In other species, mannans are the main crystalline skeletal component [3]. One food storage molecule of red seaweed is floridean starch, which is part of the reason the seaweed appears red. Another polysaccharide in red seaweed is water soluble xylan, which suggests it is mainly used for food storage as well. Quantitatively, the main polysaccharide of red seaweed are galactans which entirely consist of galactose, commonly known as carrageenan and agar. As in green seaweed, proportions of polysaccharides vary among genuses. The functions of these polysaccharides are similar to those of green seaweed [3]. In brown seaweed, the food reserve polysaccharides are mannitol, alcohol sugar, and glucans. The main polysaccharide of brown seaweed is alginic acid. Again, composition varies species to species. Alginate occurs in the inner part of the cells as well as the cell walls. Young, healthy brown seaweed has high proportions of alginate. The main function of polysaccharides in brown algae are to prevent destruction caused by waves and tides [3].

Cellulose Cellulose, a polysaccharide, is the most abundant organic substance on earth. It is composed of a chain of beta 1-4 linked glucose molecules. Because it is cheap and accessible, seaweed has been considered as a potential source for cellulose [9]. A study conducted tested cellulose content of twelve species of seaweed of various families found off the coast of India. The study concluded that green and brown seaweeds tested had high cellulose and alpha cellulose content, whereas the red seaweeds tested had low cellulose content. The highest cellulose content was found in Gelidiella acerosa, a brown algae, at 13.65%. The species with the lowest cellulose content was Kappaphycus alvarezii, a red seaweed, at 2% [9].

Table 1: Various Components of Seaweed [3]

Thermogravimetric analysis was conducted on cellulose to determine the effects of water content on thermal behavior and glass phase transition of cellulose [10]. The cellulose was heated to 500 ℃ at various heating rates to determine accurately where the cellulose starts to decompose. Water loss was determine to occur from 0-120 ℃ (about 7% of total mass). Cellulose decomposition was determined to occur in two stages. The first between 220 and 300 ℃ and the second between 300 and 475 ℃, with 62% and 28% of mass loss occurring at these regions respectively. Cellulose powder was concluded to be chemically stable to about 180 ℃ [9].

Figure 2: Thermal degradation behavior of cellulose indicating various temperature regimes. [10]

Cellulose content, due to its microfibrillar structure, is responsible for the tensile strength found in tree roots [11]. Roots were tested from fives species of trees from different parts of

France. Mean root strength was found to be 28.4 MPa however large variation occurred between species. Two species of tree were dosed with additional cellulose content (60% and 69% cellulose content). A linear relationship was determined between cellulose content and tensile strength but variability still remained high [11].

Figure 3: Tensile Strength versus Percent Composition of Cellulose [11]

3.1.3 Effects of Ecology on Structure

A study was performed at the Institute of Experimental Plant Biology examining the effects of low temperature on Winter Oilseed Rape leaf growth and structure of cell wall. The study found that thickening of cell wall occurred in leaves that were grown in the colder temperature. The cell wall thickness increased three fold over the course of the study [12].

Figure 4: Impact of Temperature on Cell Wall Thickness [12]

3.2 Material Properties and Uses of Seaweed

3.2.1 Brown Seaweed Kelp forests are one of the most productive marine communities and are cultivated for a variety of purposes, such as food production and biofuel. The structural properties of two related species of Laminaria kelp were determined in a study [13]. Previous studies conducted found that aging of seaweed influences the mechanical properties. This study focused on morphological characteristics at a given site as a function of the season. The two species of kelp, L. digitata and L. hyperborea, were sampled in winter as well as summer but only minor differences were noted between the species [13]. The species were collected by divers off the coast of Norway. The blade width, blade ratio (ratio of blade width and blade length), stipe length, and density were determined for each sample. The blade top and bottom, stipe top, middle, and bottom were all tested. The density of L. digitata was found to be 1067 kg/m3 and the density of L. hyperborea was 1086 kg/m3 [13]. Ratio of the blade as well as thickness were found to be too varied and as a result inconclusive. For the stipe of the plants, the Young’s Moduli ranged from 13.2 ± 8.6 MPa for L. digitata and 28.2 ± 10.5 MPa for L. hyperborea. No variation of density occurred for the different seasons. Kelps tested from the winter were found to have better strength [13]. Nereocystis luetkeana, another species of giant kelp, had a mean

12 breaking stress of 3.64 MN/m2. The fibers were also found to be arranged in crossed right and left helices [14]. Lowell et al tested the effects of herbivore damage on seaweed and the mechanical properties of Ascophyllum, a genus of brown seaweed. Samples were tested from a random stretch of shore in Nova Scotia in February. Samples were taken from the stipe of the plant. The undamaged Ascophyllum samples tested ranged from 4.5 to 6 MPa [15].

3.2.2 Green Seaweed Anderson et al tested several species of green seaweed from Panama to determine how forces from water movement affect overall biomechanical properties [16]. Breaking force for each species ranged from 6.6N to 22.3N and strength ranged from 1 MN/m2 to 7.9 MN/m2. The study also found that calcified algae were significantly stronger than non-calcified algae [16]. Another species of green seaweed, Udotea flabellum, which grows anchored in the sand, was found to have a breaking force of 19.2 ± 1.10 N [17].

3.2.3 Red Seaweed Shaughnessy et al studied two closely related species of red seaweed from British Columbia, Mazzaella splendens and Mazzaella linearis. M. Splendens has a short thin stipe with wide blades. M. linearis has a long thick stipe with narrow blades. Junction (stipe-holdfast) breaking force for the two species ranged from 10 N to 17 N. The cross sectional area of the species at the junction was 1.0 mm2 and 4.0 mm2 for M. splendens and M. linearis, respectively [18]. M. splendis had two reported values of 6.5 MPa and 9 MPa for tensile strength corresponding with two different geographical locations. The tensile strength of M. linearis was reported at 4.5 MPa [18]. Demes et al tested thirteen species of red seaweed found in Washington, USA. Breaking strength ranged from 0.074 MPa (Polyneura) to 0.08 MPa (Mazzaella). High variation was seen among species [19].

3.2.4 Carrageenan and Agar Films The strength of carrageenan film extracted from Kappaphycus alvarezii and Eucheuma spinosum, both red seaweeds, was determined through tensile testing of the material [19]. Two types of carrageenan were produced, k and l type. The k type carrageenan produced higher tensile strength, 3.875 ± 0.2 kN/mm2, compared to the l type which had a strength of 0.945 ± 0.10 kN/mm2. The strength difference in types is due to chemical makeup [20]. Mechanical properties of agar films in a humid climate has also been determined [21]. Agar was extracted from Gelidium robustum in Mexico to create films. Between 45 and 60 days, the films became brittle, and after 90 days the samples were too brittle to perform tensile tests on. At day 0, tensile strength was approximately 3.25 MPa but by day 60, tensile strength was only about 1.5 MPa. It was determined that the absorption of solar energy caused the degradation [20]. In a separate study, agar was determined to thermally degrade from 250-400 ℃ [22].

3.3 Seaweed Biomaterials

There are currently two main ways seaweed is used to create biomaterials. The first utilizes extracted polysaccharides to manufacture items that are purely biopolymer. The second incorporates ground seaweed into other polymers, biobased and conventional. There are many published examples of the first method. One of these examples used Posidonia Oceanica washed up on beaches in the Mediterranean combined with polyethylene (PE) made from sugarcane [23]. In this study, the seaweed was ground to an average particle size of 250 micrometers. It was then combined with PE at various concentrations through mechanical mixing. The study found increasing the proportion of seaweed particles increased the tensile strength of the samples and decreased elongation [23]. The drawback to this method is the poor interaction of seaweed filler and thermoplastic matrix in composite materials [24]. Seaweed, along with other lignocellulosic materials, are hydrophilic while thermoplastics are hydrophobic. This results in composites with poor bonding and poor mechanical properties. Different methods are being explored as routes for the utilization of seaweed for materials without this drawback. The second method mentioned above is one way the drawback of seaweed filler can be negated. One study that manufactured materials in this way prepared a solution of agar, a polysaccharide extracted from seaweed [25]. This solution was allowed to dry, producing a film of agar. These films have a relatively high tensile strength and have potential to be used as packaging, among other uses. However, neither of these methods preserves the whole seaweed filaments. The tensile strength of the seaweed structures themselves are not utilized in either of these methods. The structure of seaweed has potential to be developed into materials that are biodegradable and strong, as well as not having the negative interactions between the seaweed and thermoplastic.

4. Objectives

 To understand the structure in different types and genus of seaweed,  To use tensile testing to determine the mechanical properties of various species of seaweed,  To establish structure-property correlations in different seaweeds,  To examine the potential for seaweed fibers to be used in combination with various polymers.

5. Methods Five species of seaweed (Brown and red) were collected for this project, four from the New England coast (Rye, NH, Revere, MA, Falmouth, MA, and Newport, RI) and one from Southern California (Ventura, CA). All samples were taken from salt water sources in these regions. The water temperature was recorded at each collection site. Samples were preferred when collected alive, identified as seaweed attached to rocks or rooted in the ground. However, samples were collected obtained from shore if they appeared fresh (no signs of whitening or drying out). Samples were placed in airtight containers after inspection and completely submerged in seawater from the collection location. The containers were moved to a chilled cooler for transportation before they were stored in a sterile refrigerator at 5 °C. The samples were not stored for more than 48 hours untouched.

The genus of each species was identified through background research and consultation of Professor Pamela Weathers of the WPI Biology Department.

Within the 48 hours, samples were pressed. The process for pressing samples is as follows. Samples were removed from the air tight container and rinsed in cold water to remove any sand or other particles. The sample was then patted dry and placed on a piece of cardstock. Branches were spread as much as possible. A sheet of parchment paper was placed over the sample followed by a sheet of blotting paper. Samples were layered between pieces of foam board with no more than three samples between each board pair. Rubber bands were used to hold the stack together. The stack was then placed under a 20 pound weight for 48 hours. After the completion of pressing, samples were removed and kept in an airtight container with a drying agent (drierite) and stored in a sterile fridge. Pressed samples were not kept longer than a month.

A) B) C)

D) E) Figure 5: Seaweed Pressing Method: A) Seaweed is washed and pat dry, B) Seaweed is arranged on cardstock, C) Seaweed is covered with parchment paper, D) Seaweed is covered with newspaper, and E) Seaweed is covered with a paper towel.

All test were completed within two weeks of sample collection.

Tensile testing was performed using an Instron tensile testing machine. Test samples were prepared by selecting a section of a pressed sample that was uniform (no branches, air pockets or deformities). Epoxy was used to secure the test sample segment between two pieces of cardboard in order to fit into the machine. The test sample was allowed to dry overnight. Before any test was performed, the length, width and thickness of each sample was recorded. The part of the seaweed the test sample was removed from was recorded visually. Test samples were then secured in the machine clamps with a load range of 1000N, data collection interval of 10ms and clamp speed of 0.000166 m/s. The test samples were loaded until failure. At least three successful (broken during testing and no glue failure) tensile tests were completed for each species of seaweed.

Figure 6: Sample Loaded in Instron Tensile Testing Machine

The force-displacement data retrieved from the Instron machine was used to calculate the Stress, Strain, and Elastic Modulus for each sample using a program in Matlab. The equations are below.

v = clamp velocity, l0 = original length, t = time

Strain = (lf –l0)/l0 = [l0 + (l0*v*t)]/l0 Stress = Cross sectional area * Load = Width * Thickness * Load Modulus = Max Strain/Max Stress

Plots for each Stress-Strain data set were generated in Matlab.

Samples were observed under a microscope to determine fiber width, fiber orientation and cell orientation.

Thermogravimetric Analysis (TGA) was completed on four of the five genera collected. The test were conducted using a Netzsch TGA machine. 10 mg samples were removed from any part of the seaweed to be tested. The Netzsch crucible was burned out prior to testing to remove any remaining material from previous tests. The sample was heated at a rate of 5 °C per minute to a final temperature of 800 °C.

Figure 7: Netzsch TGA Machine and TGA Tested Sample

An introductory computational analysis was conducted to model the principal structural features in seaweed. The goal was to delineate the microscopic features that can influence strength. Additional details are provided in Section 6.

6. Modeling of Tensile Properties In order to understand the impact of the fiber structure on the stress behavior of seaweed, simulations were conducted using SOLIDWORKS and ANSYS. In these simulations, the fiber diameter was varied, in order to illustrate how the fiber diameter influences the stress distribution during a tensile test.

First, three descriptions were created in SOLIDWORKS. The fiber diameter varies from .2615 mm to .5219 mm and the number of fibers vary from 9 to 18. These descriptions most closely represent the Membranoptera genus, due to the diameter and the circular perimeter of the description. The descriptions do not exactly represent any of the genera tested in this project, in order to solely understand the behavior of the fibers during tensile loading. The descriptions can be seen in Figures 8, 9, and 10.

Figure 8: SOLIDWORKS Description of Seaweed Structure: Small Fibers

Figure 9: SOLIDWORKS Description of Seaweed Structure: Medium Fibers

Figure 10: SOLIDWORKS Description of Seaweed Structure: Large Fibers

Second, a simulation of the tensile tests was done using the above models loaded into ANSYS. The “Static Mechanics” mode was used. The model was fixed on one end and a displacement of 0.0001667 m/s was applied on the other end. This simulation was done for each of the three models created. The simulations can be seen in Figures 11, 12 and 13.

Figure 11: Small Fiber Tensile Test Simulation (Stress Values not to scale)

Figure 12: Medium Fiber Tensile Test Simulation (Stress Values not to scale)

Figure 13: Large Fiber Tensile Test Simulation (Stress Values not to scale)

The ANSYS simulations showed nodes of low stress on the high points of the modeled fibers. These nodes were very close to the fixed end of the model. As the number of fibers increased in the models, the number of low stress nodes also increased. This indicates that a larger number of smaller diameter fibers contributes to a higher tensile strength due to the increased distribution of stress. This finding is reflected in the experimental data, with the genus with the smallest fiber diameter, Ascophyllum, having the highest measured tensile strength.

7. Results

All the major results are presented in section 10. A summary is given in the following paragraphs.

Fiber diameter ranged from 0.01 mm (Egregia) to 0.1 mm (Petalonia).

The tensile testing data collected showed variance throughout morphological location and genus. Out of approximately 20 successful tensile tests per specimen, groups were created of samples that were taken from the same general morphological location (at least three successful samples per group). Membranoptera had the lowest breaking strength at 2 N and Ascophyllum produced the largest breaking strength at 30 N. In general, the breaking force of the brown seaweed tested ranged from 3 - 30 N depending on the morphological location of the sample. However, the breaking force of the red seaweed ranged from 1- 3N and was highly consistent due to small morphological variation between test samples.

The data collected from the tensile tests were used to determine elastic modulus, specific modulus, tensile strength, and specific strength for each species of seaweed at different morphological locations. The largest elastic moduli as well as the largest tensile strengths were seen generally at the stipe of the plant close to the root. Ascophyllum had the largest modulus, 0.43 GPa, from samples selected less than 40 mm from the root of the plant. However, the smallest elastic modulus, 0.007 GPa, was also from Ascophyllum but samples were taken more than 100 mm away from the root of the plant. The elastic modulus ranged from 0.03 to 0.07 GPa for Fucus. Similarly to the Ascophyllum, the modulus was highest for the Fucus at the stipe of the plant closest to the root. The Petalonia tested was a large piece of thallus so no root or stipe could be identified. Samples taken from the center of the thallus had a modulus of 0.67 GPa and samples taken from the edge of the thallus produced a modulus of 0.04 GPa. Egregia, which had an identifiable stipe, produced a modulus of 0.18 GPa at said stipe and a modulus of only 0.04 GPa at the thalli. Membranoptera had the second highest modulus of 0.38 GPa for all samples tested. Specific modulus ranged from 7.5*104 m2/s2 (Fucus) to 62*104 m2/s2 (Petalonia) close to the root of the plant.

The largest tensile strength was 29 MPa (Ascophyllum) also located at the stipe closest to the root of the sample. The lowest strength at a similar location on a separate species, Fucus, was 2.4 MPa. Strength also appeared to correlate to morphological location. For examples, Egregia at the stipe had a strength of 4.8 MPa but only a strength of 0.69 MPa at the thallus or leaves. The stipe near the roots had a higher strength, except in Petalonia for which no root was identified. Strength at the center of the thallus of Petalonia was 5.5 MPa and 0.74 MPa at the edge of the thallus. Specific strength ranged from 2.4*104 m2/s2 (Ascophyllum) to 0.26*104 m2/s2 (Fucus) at the stipe.

Figure 14: Representative Force-Displacement Data for Each Species of Seaweed

All the results of this MQP are presented as a Journal Paper as indicated below:

R. Swanson, R. Sudol and S. Shivkumar, "Structural Features and Mechanical Properties of Five Species of Seaweed" The Journal of Experimental Marine Biology and Ecology, 2018. Submitted for publication. The paper is attached in section 10.

8. Conclusions Plastic waste is a pressing environmental problem, interfering with the quality of life of humans, plants and animals. Biodegradable materials have the potential to reduce the impact of plastic waste. Seaweed is one plant that has gained attention recently for its potential to be a base stock for materials, due to its abundance and the ease of cultivation. Current biomaterials involving seaweed consist of ground seaweed powder in thermoplastics. This results in polymers that have a slightly higher tensile strength than pure polysaccharides, but the hydrophilic nature of the seaweed and the hydrophobic nature of thermoplastics results in poor mechanical properties.

The goal of this project was to determine the potential of seaweed to be used in material applications by utilizing the mechanical properties of the fibers found in seaweed. Structural features of seaweed and how they contribute to mechanical properties were explored to better understand the possible impact of utilizing seaweed fibers.

Fiber structure was investigated by identifying fiber diameter, fiber orientation, as well as cell orientation. Fiber diameter ranged from 0.01 to 0.1 mm. The red species of seaweed tested, Membranoptera, had a highly organized cell structure compared to the brown species of seaweed tested which were identified to have a more branched cellular structure. This highly, organized structure may be responsible for Membranoptera having the second highest tensile strength, even though it had a significantly smaller cross sectional area.

Breaking force ranged from 3 to 35N (Membranoptera and Petalonia, respectively) and tensile strength ranged from 2.5 to 29 MPa (Fucus and Ascophyllum, respectively). Different morphological locations on the same species were found to produce different strengths. The stipe, which is the densest part of the seaweed most likely as a result of a higher cellulose content, generally produced the largest tensile strength.

It was determined that although cellulose is only 2-10% of the total composition of seaweed, it is the main contributing factor to strength. Hence, seaweed with a higher cellulose content is likely to have higher strength. This conclusion was confirmed by both thermogravimetric analysis as well as literature.

Two similar species of kelp were tested, Egregia and Petalonia. The species from Southern California, Egregia, produced a lower strength compared to the species from New England, Petalonia. The TGA results were used to confirm that this difference between species is a result a lower cellulose content caused by a thinner cell wall needed to survive in the warmer climate of Southern California.

The percentage of inorganics in the seaweeds tested ranged from 35% (Fucus) to 55% (Egregia) determined from the thermogravimetric analysis. The amount of inorganic material was found to correlate with the mechanical strength. Generally, a higher percentage of inorganics resulted in a higher strength.

The presented data in this study aims to help further and develop new applications of various types of seaweed widely available through a better understanding of the material properties of red and brown seaweed. Seaweed with a higher cellulose content and density produced a higher material strength. Red seaweed, with a more organized cellular structure, also produced a higher strength than the majority of brown species tested. The results of this study also indicate that seaweed in colder climates produce a higher tensile strength. This data suggests that high cellulose and fibrous seaweed with organized cell structures show the most potential for future applications.

9. References

[1] C. L. Hurd et al, Seaweed Ecology and Physiology. Cambridge University Press, 2014. [2] Anonymous Handbook of Marine Macroalgae : Biotechnology and Applied Phycology. 2011Available: http://ebookcentral.proquest.com/lib/wpi/detail.action?docID=818581 . [3] E. Percival, "The polysaccharides of green, red and brown seaweeds: Their basic structure, biosynthesis and function," British Phycological Journal, vol. 14, (2), pp. 103-117, 1979. [4] J. F. Balart et al, "Manufacturing and properties of biobased thermoplastic composites from poly (lactic acid) and hazelnut shell wastes," Polymer Composites, vol. 39, (3), pp. 848-857, 2018; 2016. [5] K. P. Rajan et al, "Blends of thermoplastic polyurethane (TPU) and polydimethyl siloxane rubber (PDMS), part-I: assessment of compatibility from torque rheometry and mechanical properties," Journal of Polymer Research, vol. 19, (5), pp. 1-13, 2012. [6] S. HII et al, "AGAR FROM MALAYSIAN RED SEAWEED AS POTENTIAL MATERIAL FOR SYNTHESIS OF BIOPLASTIC FILM," Journal of Engineering Science and Technology, vol. 11, pp. 1-15, 2016.

[7] Catalina Island Marine Institute. (2018). Tag Archives: Algae. [Online] Available at: http://cimioutdoored.org/tag/algae/ [Accessed 25 Apr. 2018].

[8] A. H. Buschmann, "Hurd, C. L., Harrison, P. J., Bischof, K. & Lobban, C. S. 2014. Seaweed Ecology and Physiology. Cambridge University Press, Cambridge, UK, 551 pp," Journal of Phycology, vol. 52, (2), pp. 315-316, 2016. [9] A. K. Siddhanta et al, "Profiling of cellulose content in Indian seaweed species," Bioresour. Technol., vol. 100, (24), pp. 6669-6673, 2009. [10] L. Szcześniak, A. Rachocki and J. Tritt-Goc, "Glass transition temperature and thermal decomposition of cellulose powder," Cellulose, vol. 15, (3), pp. 445-451, 2008. [11] M. Genet et al, "The influence of cellulose content on tensile strength in tree roots," Plant Soil, vol. 278, (1/2), pp. 1-9, 2005. [12] M. STEFANOWSKA et al, "Low Temperature Affects Pattern of Leaf Growth and Structure of Cell Walls in Winter Oilseed Rape (Brassica napus L., var. oleifera L.)," Annals of Botany, vol. 84, (3), pp. 313-319, 1999. [13] P. Y. Henry, "Variability and similarities in the structural properties of two related Laminaria kelp species," Estuarine Coastal and Shelf Science, vol. 200, pp. 395-405, 2018. [14] M. A. R. Koehl and S. A. Wainwright, "Mechanical Adaptations of a Giant Kelp," Limnol. Oceanogr. vol. 22, (6), pp. 1067-1071, 1977. [15] R. B. Lowell, J. H. Markham and K. H. Mann, "Herbivore-Like Damage Induces Increased Strength and Toughness in a Seaweed," Proceedings of the Royal Society of London.Series B: Biological Sciences, vol. 243, (1306), pp. 31-38, 1991.

[16] K. Anderson et al, "Biomechanical properties and holdfast morphology of coenocytic algae (Halimedales, Chlorophyta) in Bocas del Toro, Panama," J. Exp. Mar. Biol. Ecol., vol. 328, (2), pp. 155-167, 2006. [17] L. Colladovdesi, R.E. Dewreede and K.L.D. Mil, "Biomechanical properties of Udotea (Halimedales, Chlorophyta) in a Mexican reef lagoon," Phycologia, vol. 37, pp. 443-449, 1998. [18] G. W. Saunders, "Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications," Philosophical Transactions of the Royal Society B: Biological Sciences, vol. 360, (1462), pp. 1879-1888, 2005. [19] K. W. Demes et al, "VARIATION IN ANATOMICAL AND MATERIAL PROPERTIES EXPLAINS DIFFERENCES IN HYDRODYNAMIC PERFORMANCES OF FOLIOSE RED MACROALGAE (RHODOPHYTA)," J. Phycol., vol. 47, (6), pp. 1360-1367, 2011. [20] A. V. Briones et al, "Tensile and Tear Strength of Carrageenan Film from Philippine Eucheuma Species," Marine Biotechnology, vol. 6, (2), pp. 148-151, 2004. [21] Y. Freile-Pelegrín et al, "Degradation of agar films in a humid tropical climate: Thermal, mechanical, morphological and structural changes," Polym. Degrad. Stab. vol. 92, (2), pp. 244- 252, 2007. [22] E. Raphael et al, "Agar-based films for application as polymer electrolytes," Electrochim. Acta, vol. 55, (4), pp. 1455-1459, 2010. [23] B. Ferrero et al, "Development of natural fiber‐reinforced plastics (NFRP) based on biobased polyethylene and waste fibers from Posidonia oceanica seaweed," Polymer Composites, vol. 36, (8), pp. 1378-1385, 2015. [24] M. M. Hassan, M. Mueller and M. H. Wagners, "Exploratory study on seaweed as novel filler in polypropylene composite," J Appl Polym Sci, vol. 109, (2), pp. 1242-1247, 2008. [25] P. Kanmani and J. Rhim, "Antimicrobial and physical-mechanical properties of agar-based films incorporated with grapefruit seed extract," Carbohydr. Polym. vol. 102, pp. 708-716, 2014.

10. Journal Article

The journal article, submitted to The Journal of Experimental Marine Biology and Ecology, can be seen on the following page.

Structure-Mechanical Property Correlations in Five Species of Seaweeds

R. Swanson, R. Sudol and S. Shivkumar*

Department of Mechanical Engineering Worcester Polytechnic Institute 100 Institute Rd. Worcester, MA 01609

Abstract

Seaweeds are plentiful and simple to cultivate in most coastal areas of the world. Current industrial uses of seaweed utilize chemically extracted polysaccharides and hydrocolloids found in seaweeds, not the entire seaweed fibers. The present study investigates various material properties and structural features of seaweed found in New England and Southern California. Samples from five species of seaweed were collected and dried. The dried samples were analyzed using microscopy and thermogravimetric analysis. The mechanical properties of the different seaweeds were measured. Typical breaking forces were between 3 N and 30 N for red and brown seaweed, respectively. The elastic modulus and strength were estimated to be on the order of 50-100 GPa and 10-20 MPa, respectively. Seaweeds that contain more inorganics generally exhibit a higher strength. Filamentous seaweed with organized cells were found to have a higher modulus and strength. The presence of cellulose and polysaccharides, along with an organized cell structure, may provide an opportunity to utilize the whole seaweed to develop new biopolymer products, with comparable properties to existing polymers.

Corresponding Author: *S. Shivkumar ([email protected])

Keywords: seaweed, biopolymers, mechanical properties

1. Introduction Seaweeds are marine, benthic algae that are multicellular and macrothallic. These algae are separated into three main phyla, Chlorophyta, Phaeophyta and Rhodophyta, which are classified by the predominant pigment found in the cells. The common terminology for these types are green, brown, and red seaweeds, respectively. Seaweeds are morphologically highly varied, some having a stipe (stem) and thallus (blade) [Pereira, Neto, 2015]. Others have microfilaments that can be branched or unbranched, or parenchymatous thalli [Pereira, Neto, 2015]. A variety of species have a holdfast that attaches the body of the seaweed to hard substrata. Several grow on the surface of other marine flora and others grow unattached [Pereira, Neto, 2015]. Seaweeds must be submerged in salt water for a least a portion of the day to survive and, because they are sessile organisms, are highly sensitive to the conditions and composition of the environment in which they grow [Pereira, Neto, 2015].

Each of the types of seaweed outlined above have different structural morphologies and cellular growth patterns. Brown seaweed typically has a branched, filamentous thallus, at the macro level, which is most apparent in species of kelp [Anderson et al, 2006]. This is the only algae type that has internal tissue differentiation into conducting tissue. Red seaweed is generally filamentous or made of chains of cells attached by end walls and is usually branched. Cortification (husk) or ‘pit plugs’, granular protein masses that fill holes between branches that are created during cell division, can occur in red seaweed [Koehl, Wainwright, 1977]. Mechanical properties of different seaweeds also depend on the composition of cell wall skeleton and matrix [Fowler, Quatrano, 1977]. Cell walls of seaweed are composites, with a matrix of polysaccharides and fibers, called microfibrils, of cellulose. The cell wall is mostly polysaccharides, which contribute to the overall rigidity of the plant. In brown algae, this polysaccharide is alginate and, in red, it is carrageenan. The microfibril skeleton that makes up the rest of the cell wall is typically composed of cellulose in seaweeds [Northcote, 1972].

Currently, seaweed is used in a variety of applications. The main use is as a food product, with two million tons of seaweed processed into one type of food or another [Jenson, 1993]. The second major use is mainly for the extraction of the phycocolloids alginate, agar and carrageenan [Jenson, 1993]. A typical technique that has been used to utilize seaweed and other biopolymers is to grind the material and incorporate it into commodity and engineering thermoplastics. Balart, et al. made a composite material by adding particles of Posidonia oceanica, a red seaweed, with an average particle size of 250 µm to a biobased high density polyethylene. However, seaweed is hydrophilic and most thermoplastics are hydrophobic, which leads to improper mixing and thus poor mechanical properties.

Materials have also been made from seaweed using only extracted polysaccharides. The polysaccharides are extracted from seaweed and processed in order to produce a plastic resin in combination with traditional thermoplastics [Rajam, et al., 2012]. Wong, et al. [Wong, et al.,

2016] developed such a plastic in the form of a film, by adding sago starch, agar and glycerol to a solution and drying it overnight at 50°C. These processes do not exploit the fibers of seaweed, which could be used to increase strength of these plastics. A methodology that utilizes the structural feature of seaweed without the need for a matrix of traditional plastics would be highly beneficial both for sustainability, recycling, and material properties. In order to develop such structures, it is imperative to understand the structure-property relationships of different seaweeds and it is the purpose of this contribution to obtain information in this regard.

2. Materials and Methods

Five species of seaweeds were collected from a variety of locations in New England (Rye, New Hampshire, Hampton, New Hampshire, Revere, Massachusetts and Newport, Rhode Island) and one location in Southern California (Ventura, California) (Table 1). All collection locations were at a saltwater, oceanic source. During the period of the study, collection dates ranged from early fall to early winter. The water temperatures in New England vary from 5℃ to 22℃ from late summer to early winter. The water temperature in Southern California ranged from 14℃ to 20℃ from late summer to early winter [Global Sea Temperature, 2018].

Genus Collection Location Water Temperature (°C)

Petalonia Revere, MA 5℃ to 22℃

Egregia Ventura, CA 14℃ to 20℃

Ascophyllum Falmouth, MA 5℃ to 22℃

Fucus Rye, NH 5℃ to 22℃

Membranoptera Newport, RI 5℃ to 22℃ Table 1: Genus of seaweed collected, location of seaweed collected, season collected, water temperature of where seaweed was collected

Of the five species collected, four were brown seaweed, including the species from California. The taxonomy of the seaweed specimens were determined as the five following genera: Petalonia, Egregia, Ascophyllum, Membranoptera, and Fucus. Petalonia, Egregia, and Ascophyllum samples were found on the shore after high tide. Membranoptera and Fucus were collected alive either from sand or rocks.

Samples were preferred when collected alive, determined by if the plant was attached to rocks or rooted in ground. They were also collected from the shore if they were fresh and had no signs of whitening or drying out, usually after high tide. Seaweed specimens were stored in airtight containers with the seawater they were found in completely covering them. The containers were

34 stored in a cooler during transportation and until they could be properly refrigerated. The samples were stored, refrigerated and untouched, for a maximum of two days before they were pressed and dried.

The samples were cleaned in cold water and pressed for 48 hours to prepare for further testing. The samples were then stored in an airtight container containing a desiccant and stored at 5⁰C. Pressed samples were kept up to one month before testing.

The structure in the seaweed samples were examined using a Jiusion 40 -1000X Digital Microscope Camera in order to determine fiber size and orientation of each genus. Tensile tests were conducted using an Instron tensile testing machine, with a load range of 1000 N and an accuracy of 5 N. Test samples around 3 cm in length were obtained from a uniform (no branches, air pockets or other deformities as much as possible) section of the pressed sample. The samples were tensile tested at a displacement rate of 0.166 mm/s. The samples were loaded until failure. Force-deformation data were collected at an interval of 10 ms. At least five successful (i.e. fracture occurred in the central portions of the sample and not due to delamination of the sample at the grips) were completed for each genus of seaweed.

Thermogravimetric (TGA) testing was conducted using a Netzsch thermobalance. Approximately 10 mg of the seaweed sample was loaded into a Netzsch crucible. The crucible was burned out prior to the loading of each sample. The initial mass of the crucible and sample were recorded using an electronic scale. Each sample was heated to 800℃ at a rate of 5℃ per minute.

3. Results and Discussion

The fiber width and orientation varied with each species of seaweed collected. The fiber width for the different seaweeds was between 0.01 (Egregia) and 0.1 mm (Petalonia) (Table 2). The fibers in Petalonia were oriented randomly to the longitudinal axis and the direction of growth of the seaweed. Petalonia was the only genus to exhibit random orientation of fibers; all other genera exhibited a high degree of longitudinal fiber orientation (Table 2). The highest degree of orientation was observed in red seaweed. In brown seaweeds, the fibers are not as oriented, but there was an intricate network of cells observed [Anderson et al, 2006], whereas red seaweed grows in chains, with cells attached by end walls [Koehl, Wainwright, 1977]. The orientation of cells can have a significant impact on mechanical properties, as will be shown in the following sections.

Genus Sample A Location on D Fiber Density E SM TS SS (mm2) Sample (mm) (mm) Orientation (kg/m3) (GPa) (m2/s2*104) (MPa) (m2/s2*104) Ascophyllum 0.037 parallel 1200[1]

I 2.9 <40 from root 0.43 35 29 ± 7.3 2.4

II 1.4 40 - 100 0.08 6.6 3.6 ± 0.28 0.30

III 2.8 >100 0.007 0.58 1.7 ± 0.54 0.14

Fucus n/a parallel 929[2]

I 3.6 60 - 150 from 0.07 7.5 2.4 ± 0.39 0.26 root II 3.3 <60 0.07 7.5 3.4 ± 0.09 0.37

III 2.3 >150 0.03 3.2 1.4 ± 0.23 0.15

Petalonia 0.09 random 1086[3]

I 1.4 30 - 50 from edge 0.10 9.2 4.3 ± 0.44 0.40

II 1.2 30 - 50 0.67 62 5.5 ± 0.26 0.51

III 1.5 10 - 30 0.13 12 1.8 ± 0.21 0.17

IV 4.6 0 - 10 0.04 3.7 0.74 ± 0.07 0.31 Egregia 0.009 parallel 1067[3]

I 3.3 0 from stem 0.18 17 4.8 ± 1.8 0.44

II 3.2 >0 0.04 3.7 0.69 ± 0.06 0.01 Membranoptera 0.027 parallel 1200[1]

I 0.32 0 - 60 from root 0.38 32 5.6 ± 0.24 0.47

Table 2: Genus of seaweed, tested location on seaweed, cross sectional area of representative sample, representative diameter of fiber width, fiber orientation of seaweed in regards to longitudinal axis, density, elastic modulus, specific modulus, average tensile strength, and specific tensile strength. [1] (Rajan, Al-Ghamdi, Ramesh, & Nando, 2012). [2] (Hurd, Harrison, Bischof, & Lobban, 2014). [3] (Henry, 2018).

The force-deformation curves for the various seaweed are shown in Figures 1 through 5. Two genera of kelp, a type of brown seaweed, were tested in this study, Petalonia and Egregia. These specimens had breaking forces ranging from 3 to 26 N (Figure 1) and 3 to 31 N (Figure 2), respectively. Ascophyllum samples produced breaking forces from 5 to 28 N (Figure 3). Fucus samples had breaking forces of 6 to 38 N (Figure 4). Membranoptera produced breaking forces from 1 to 3 N (Figure 5), the lowest of all genera tested. This low breaking force may partially be attributed to the small cross-sectional area of this genus. While the cross section area of the samples of seaweed tested was between 1 to 5 mm2 for most genera, it was 0.32 mm2 for Membranoptera (Table 2).

Previous studies on the material properties of seaweed have focused on the structure of seaweeds to determine the effects wave force and currents have on the morphology [Koehl, Wainwright, 1977]. Several previous studies have tried to examine the effects of ocean waves and currents on

36 the structural morphology and tensile behavior of seaweeds. The thalli of kelp tend to be very large and flat and are impacted by currents much more than the other species due to this morphology. The other species tested are smaller and tend to have thalli that are rounded, and so do not interact with ocean currents as much. The higher breaking strength of kelps may be an adaptation to the larger forces experienced by the thalli. An examination of Table 2 demonstrates a similar finding in this study, and indicates that there are differences in material properties among different genera of seaweed, as well as at different morphological locations on the same seaweed.

The elastic modulus of the seaweed specimens varied from 0.5 to 25 GPa depending on the genus and the location of the sample (Table 2). These values compare well with other biological tissues. The moduli for plant cell walls, starch, bone and cellulose crystals are on the order of 1, 3, 20 and 130 GPa respectively (Meyers et al, 2008). The measured tensile strengths ranged from 0.5 to 30 MPa (Table 2). Petalonia produced a mean breaking strength of 5.46 ± 0.26 MPa at the center of the thallus, and Egregia had a mean strength of 4.83 ± 1.77 MPa at a location on the stem. In this case, the seaweed found in the colder water temperature, Petalonia, had the highest breaking strength (Table 2). Stefanowska et al conducted a study on temperature effect on the structure of rapeseed found that there was a significant increase in cell wall thickness of plants in colder temperatures [Stefanowska et al, 1999]. This data is consistent with another investigation which found that a species of giant kelp, Nereocystis zuetkeana, had a average breaking strength of 3.64 ± 2.2 MPa [M. A. R. Koehl & Wainwright, 1977]. Both samples tested in the present study were also found free floating, consistent with N. zuetkeana described above. Another separate study tested two species of kelp, Laminaria digitata and Laminaria hyperborea, from the coast of Norway that produced breaking strengths of 8.44 ± 3.49 MPa and 10.9 ± 2.69 MPa [Henry, 2018]. These data are similar but slightly higher compared to the data collected from Petalonia and Egregia in the current study. L. digitata and L. hyperborea were collected by divers, resulting in fresher samples which may explain the larger breaking strengths observed than those of the present study [Henry, 2018]. The higher strengths also may be due to the colder water temperatures of Norway.

Figure 1: Variation of force with displacement during tensile testing of Petalonia. The insert shows an expanded force-displacement curve, drawn for clarity. The photographs of the seaweed and the location of the tensile sample are also shown. The labels I, II, III and IV coincide with the designations in Table 2.

Figure 2: Variation of force with displacement during tensile testing of Egregia. The insert shows an expanded force-displacement curve, drawn for clarity. The photographs of the seaweed and the location of the tensile sample are also shown. The labels I and II coincide with the designations in Table 2. Ascophyllum samples tested in this study were obtained at a distance of 40 mm to 100 mm from the holdfast of the plant and had an average breaking strength typically of 3.59 ± 0.28 MPa (Table 2). A previous study, Lowell et al, tested the strength and toughness of Ascophyllum nodosum from Nova Scotia, Canada, before and after it was damaged by an herbivore. The study

38 reported strengths of the undamaged specimens to be between 4.5 MPa and 6 MPa [Lowell et al, 1991]. Their samples were obtained at least 50 mm from the base of the plant and no more than 100 mm away. Fucus samples tested in this study had a mean breaking strength of 3.41 ± .09 at the root, similar to that of Ascophyllum. Fucus has a similar morphology to that of Ascophyllum, and is also a brown seaweed. These similarities make a comparison between Fucus and Ascophyllum possible. There are few other recorded tests on the tensile properties of Fucus.

Figure 3: Variation of force with displacement during tensile testing of Ascophyllum. The insert shows an expanded force-displacement curve, drawn for clarity. The photographs of the seaweed and the location of the tensile sample are also shown. The labels I, II, and III coincide with the designations in Table 2.

Figure 4: Variation of force with displacement during tensile testing of Fucus. The insert shows an expanded force-displacement curve, drawn for clarity. The photographs of the seaweed and the location of the tensile sample are also shown. The labels I, II, and III coincide with the designations in Table 2. 39

The genus of red seaweed in this study, Membranoptera, produced a breaking strength of 5.62 ± 0.24 MPa (Table 2). Similar results were obtained on the breaking strength of the junction between the stipe and holdfast of two species of red seaweed, Mazzaella splendens and Mazzaella linearis [Saunders, 2005]. The majority of samples were collected in Vancouver, Canada, but some were collected in warmer climates such as Australia. The strength of M. splendens had two reported values, of approximately 6.5 MPa and 9 MPa, corresponding with the two geographic collection areas. The lower strength specimen was collected from the warmer climate. The reported value for M. linearis is approximately 4.5 MPa [Saunders, 2005]. It was assumed that the strength of the main part of the stipe would be higher, because the fracture occurs at junctions between the stipe and thallus of seaweed. Similar results were observed in the present study. Demes et al tested thirteen species of red seaweed and reported broad results across species found in Washington, USA [Demes et al, 2005]. In this case, breaking strength ranged from 0.074 MPa in Polyneura to 0.8 MPa in Mazzaella [Demes et al, 2005]. The red seaweed tested in the present study was closest to the results from Saunders [Saunders, 2005]. However, neither study included Membranoptera used in the present investigation.

Figure 5: Variation of force with displacement during tensile testing of Membranoptera.

The insert shows an expanded force-displacement curve, drawn for clarity. The

photographs of the seaweed and the location of the tensile sample are also

shown. The label I coincides with the designations in Table 2.

All the seaweeds tested had significant differences in breaking strength depending on the morphological location of the seaweed tested (Table 2). In different samples, all tests conducted at the same morphological location produced a similar breaking strength. Figure 5 shows Membranoptera all tested from the same location had an average breaking strength of 5.62 ± 0.24 MPa (Membranoptera I). For most species examined, the test samples closest to the root of

40 the plant produced the highest breaking strength. In Table 2, it can be seen that samples of Ascophyllum less than 40 mm from the root had a breaking strength of 29.00 ± 7.31 MPa (Ascophyllum I), and samples tested more than 100 mm had a breaking strength of 1.65 ± 0.54 MPa (Ascophyllum III). Similar results were observed with Egregia (Egregia I) and Fucus (Fucus I). The tests on Petalonia were conducted 0-50 mm from the edge of the thallus (Table 2). Because the collected specimens had washed up on the shore, no root was present and sample locations were recorded in relation to the edge of the thallus. These samples in the center of the thallus produced the highest breaking strength compared to samples taken towards the edge of the seaweed: 4.30 ± 0.44 MPa (Petalonia I) and 5.46 ± 0.26 MPa (Petalonia II) respectively. This variation is most likely the result of damage, such as small tears, on the edge of the kelp due to waves and current [Koehl, Wainwright, 1977]. The stipe also tended to be the morphological location with the highest tensile strength, as seen in Egregia (Egregia I). This result may be due to a higher density or thicker cell walls in the stipe than in other morphological locations of seaweed.

Geographic location differences can best be observed between samples of the same genus taken from different areas. This study did not examine the variation in properties for the same genus found in different locations, but these comparisons can be made with data of similar genera from other studies. The data from this study of a kelp genus collected in New England produced tensile strength data of 3.59 ± 0.28 MPa (Table 2) while a study from Nova Scotia produced tensile strengths of 4.5 MPa and 6 MPa [Lowell et al, 1991]. Water temperature in Nova Scotia typically ranges from 2.5 ℃ to 14.2 ℃ between early autumn and early winter compared to 5 ℃ to 22 ℃ in New England, which may be the cause of a higher breaking strength compared to kelp specimens tested in this study (Figure 1 and Figure 2) [Global Sea Temperatures, 2018].

Figure 6: TGA curves at a heating rate of 5 oC/min. The legend shows the TGA curves of Fucus, Petalonia, Egregia, and Membranoptera seaweed. Four degradation regions can be observed in the TGA data from the present investigation (Figure 6). The first region is from 0°C to 120°C in which all samples exhibit water loss. A study by Jang et al, tested the thermal properties of Laminaria japonica, a brown seaweed, and Enteromorpha crinite, a green seaweed [Jang et al, 2013]. Their data indicates a mass loss at approximately 100 °C due to water vaporization, consistent with the findings of this study. The next degradation region occurs from 120 to 250 °C (Figure 6). Jang et al indicates that decomposition of carbohydrates occurred in this region, which may account for the mass loss observed in this temperature range [Jang et al, 2013]. The third region of degradation occurs from 250 to 400 °C. This region was reported to be in the range in which cellulose degradation may occur [Jang et al, 2013]. Egregia, the species collected from Southern California, experienced the least mass loss (~23 %) of the brown species tested (Figure 6). This specimen was collected from a warm climate (14 ℃ to 20⁰C) and thus may contain thinner cell walls due there is less environmental stress and consequently less cellulose loss. Membranoptera experienced the smallest overall percent mass loss (~14 %). Siddhanta et al found that red seaweed generally had significantly lower levels of cellulose compared to green and brown seaweed (Siddhanta et al., 2009). This low cellulose content of red seaweed may again be the reason that Membranoptera in the current study experienced the smallest mass loss in the third degradation region. The final region of mass loss occurs between 400 to 700 °C where all samples exhibit a total mass loss between 50% and 65% after exposure to 700 °C. This is consistent with the results from a previous study, which reports a mass loss between 50 and 55 %

[Jang et al, 2013]. Fucus had the highest mass loss (65%), indicating that it contained a lowest fraction of inorganics. There are many different inorganic compounds that may be present which may contain macroelements, such as Ca, Mg and P (up to 5%) and much lower concentrations of Fe, Cu, Mn and Zn (< 1%) [Misurcova, Ladislava, 2011]. These elements may make up a portion of this leftover inorganic material. Petalonia, and Egregia exhibit a mass loss of 62% and 50% respectively (Figure 6). This difference in mass loss may be attributed to the difference in geographic collection locations and the growth conditions. The fraction of inorganics may also contribute to the tensile strength as can be seen from Table 2. For example, Fucus with the lowest inorganic fraction of about 35%, also produced the lowest breaking strength. However, as indicated in the following paragraphs, the tensile strength may be affected by many other factors, including growth conditions, cell wall composition, thickness and structural orientation and no direct correlation between the fraction of inorganics and tensile strength can be established.

The degradation behavior of the genera tested in the study indicate that the composition of the cells can affect tensile strength. The TGA profile of Membranoptera shows a significant degradation area, about 15%, around 200°C (Figure 6). This degradation may be associated with the degradation of polysaccharides found in the cell wall [Anastasakis et al, 2011]. The anioinic polysaccharide alginic acid exhibits a similar degradation behavior, with a significant mass loss around 200°C [Anastasakis et al, 2011]. However, the majority of structural polysaccharides in red seaweed are agar and carrageenan, which demonstrate significant degradation around 350°C and 300°C respectively [Raphael et al, 2010][Martins et al, 2012] [Freile-Pelegrín et al, 2007]. The carbohydrate loss of Membranoptera continues for a total mass loss of 30%, a finding consistent for the other genera tested in this study. This degradation behavior suggests a composition of 30% of different polysaccharides (agar, carrageenan and alginate) in the species tested and the tensile strength of these polysaccharides is similar to that of those samples. Azamar, et al. reports a tensile strength of 3.2 MPa of a 1.5% concentration agar film. A 5% concentration carrageenan film has a tensile strength of 3.9 MPa [Briones et al, 2004]. Hence, the amount of polysaccharide may have an effect on the tensile strength.

The second major degradation area seen in the TGA tests which occurs around 300°C may correlate to the degradation of cellulose (Figure 6). Szcześniak et al and Genet et al have reported similar degradation behavior for cellulose powder [Szcześniak, Rachocki, & Tritt- Goc, 2008, Genet et al, 2005]. Cellulose has a highly organized structure and thus contributes to strength. For example, tree roots with 90% cellulose content have a tensile strength of around 36 MPa [Szcześniak, Tritt-Goc, 2008]. The samples in this study lose 5 to 10% of their mass at 300°C (Figure 6), suggesting that cellulose accounts for 5 to 10% of the specimen mass. These results are consistent with the data of Siddhanta et al that brown seaweeds typically contain 2 to 10% cellulose. The stipe area in the seaweed is likely to have a cellulose content greater than 10% (Siddhanta et al., 2009). The high crystallinity of cellulose may contribute to the tensile strength as well, and may account for the relatively high tensile strength of the seaweed samples in Table 2 (3 to 29 MPa) that are generally above that of pure polysaccharides (~ 3 MPa) [Briones et al, 2004]. 43

Note that the stipe areas (Table 2) generally had the highest strength. Thus, the two components of cell walls, polysaccharides and cellulose, can exercise significant control of the strength properties [Quatrano, et al., 1988]. Although the effect of cellulose on the strength is greater than that of polysaccharides, the overall amount of polysaccharides can be as much as 30%, while that of cellulose is ~ 2 to 10%. Hence, the polysaccharides can also have a major impact on the strength.

4. Conclusions

Seaweed fibers can have different composition and cell wall arrangement depending on location. The highest levels of fiber orientation were observed in the genus of red seaweed, Membranoptera, while Petalonia, a brown seaweed contains a relatively random network of large fibers. Depending on the type of seaweed and the testing location, the breaking force was measured to be between 3 and 30 N, which corresponds to a tensile strength between 0.5 and 30 MPa. The elastic modulus of various types of seaweed is typically on the order of 0.5 to 25 GPa and compares well with other biological structures. It was observed that seaweed from colder water temperatures generally show a higher breaking force among similar species. The structural components of seaweed include polysaccharides, cellulose and inorganics. The types and fractions of these component can significantly contribute to the strength properties. Polysaccharides make up a larger portion (30%), with cellulose ranging from 2 to 10% and inorganics between 35% and 55%. Morphologic locations of seaweed with higher densities (stipe) may contain a higher cellulose content and therefore exhibit relatively high breaking strength compared to other locations on the same species. Brown seaweed from a warmer climate had less cellulose content compared to similar species from cold climates, likely due to a thinner cell wall required to survive in warmer temperatures. Seaweed fibers, with organized structure and high strength compared with extracted polysaccharides, have a significant potential to be a candidate for bulk biopolymer applications. The genera of seaweeds with high cellulose content, such as Ascophyllum, may be better suited for applications requiring moderate to high strength.

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11. Appendix

All data files that are used in the MQP are attached as a zip file. Folders are labeled with analysis type (TGA, Force-time, and Microscopy) and genus. Within those folders, individual data files are titled by sample number and date tested.