Development of Biomass-Based Plastic Resins from Natural Algal Polysaccharides

Chapter I INTRODUCTION

A. Background of the Study Plastics are carbon-based polymers that are usually derived from petroleum. With the discovery of plastics, life became much more convenient because they are used to create a wide variety of useful materials. But discarded plastics are considered to be a big cause of pollution because they are so resilient that it will take many centuries for these plastics to completely degrade while other types of plastics will last forever. Because of that, plastics make our environment a much less attractive place (Atienza, 2009). Getting rid of plastics is extremely difficult. Burning these plastics gives off harmful chemicals that could cause traumatic stress to the environment. Recycling these plastics is also difficult because there are many forms of plastics and each form has to be recycled by a different process (Woodford, 2008). For this reason, greener solutions have to be made like the development of biomass-based plastics to somehow lessen the impact of the improper use and disposal of petrochemical plastic products. Biomass-based plastics or bioplastics are forms of plastics that are derived from renewable biomass resources like vegetable oil and cornstarch rather than the conventional plastics that are made from petroleum (Sweeney, 2008). These bioplastics allow an intelligent, cascading usage of natural resources with less waste and less emissions of greenhouse gases (Bioplastics24, 2007). Seaweeds are best known for the natural polysaccharides that can be extracted from them. Some of these polysaccharides are agar, alginate and carrageenan. These

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seaweed extracts are widely used now in various disciplines of science like food technology and microbiology but so far, there were no scientific approaches yet in the use of these polysaccharides as polymers for plastic products (Montaño, 2010). Since natural algal polysaccharides are renewable carbon-containing polymers, this research aimed to effectively and efficiently synthesize biomass-based plastic resins from agar, alginate and carrageenan. B. Statement of the Problems This study geared with the development of biomass-based plastic resins from natural algal polysaccharides using steaming process. It aimed to create good, environment-friendly, inexpensive and toxic-free bioplastic resins by utilizing agar, alginate and carrageenan as novel polymers for plastic products. It also sought to test the biodegradability and the general chemical resistance of the bioplastic resin to provide scientific bases for their possible applications in the industry. Specifically, it aspired to answer the following questions: 1. Are the bioplastic resins capable of biodegrading when immersed in loam soil and exposed in open air for 42 days? 2. Are the bioplastic resins capable of resisting the effect of the following chemical solutions for an hour? a. distilled water b. hydrochloric acid c. sodium hydroxide d. sodium chloride

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C. Statement of the Hypotheses Ho1: The bioplastic resins are not capable of biodegrading when immersed in loam soil and exposed in the open air for 42 days. Ha1: The bioplastic resins are capable of biodegrading when immersed in loam soil and exposed in the open air for 42 days. Ho2: The bioplastic resins are not capable of resisting the effect of the following chemical solutions for an hour. a. distilled water b. hydrochloric acid c. sodium hydroxide d. sodium chloride Ha2: The bioplastic resins are capable of resisting the effect of the following chemical solutions for an hour. a. distilled water b. hydrochloric acid c. sodium hydroxide d. sodium chloride A. Significance of the Study Official figures show that worldwide, about a million tons of petroleum-based plastics per year are being produced and used and about seven million barrels of oil per day are being consumed to make these plastics (Sweeney, 2008). Now, imagine that number dropping to zero! With the help of bioplastics, one day, that might be a reality.

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With this research, the conventional petroleum-based plastics will soon be replaced by bioplastics made from sustainable resources like natural algal polysaccharides. An advantage of this is that, they will not fill up the landfills because they are largely biodegradable and just for months, disposed bioplastics are completely gone unlike petroleum-based plastics which takes about many centuries. Since another use of seaweeds has been discovered again, the seaweed industry, specifically in the Philippines, might rise at an increased rate. Plastic factories soon will tap into vats of natural algal polysaccharides instead of petroleum. The seaweed reserve will not be endangered since these seaweeds grow at a fast rate so depleted stocks can be replaced too (Montaño, 2010). This research will also be significant to the whole scientific community since it would provide added information about how to make a good, environment-friendly, inexpensive and toxic-free bioplastic resins from natural algal polysaccharides. This research can also serve as a springboard for future researchers who want to develop safe and cost-effective bioplastics resins. B. Scope and Limitations The main purpose of this study was to synthesize and develop novel biomassbased plastic resins from natural algal polysaccharides and phycocolloids using steaming process in such ways that the resulting products are cost-effective, highly efficient, environment-friendly and toxic-free. It sought to determine the biodegradability of the bioplastic resins in loam soil and open air within a time frame of 42 days and the general chemical resistance of the bioplastic resins in plain water and in acid, base and salt solutions for an hour.

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The research was performed at the residence house of the researcher at #3 Daisy St, Baptista Village, Calao East, Santiago City, Isabela and at the Science Laboratory of the Philippine Science High School-Cagayan Valley Campus, Masoc, Bayombong, Nueva Vizcaya from April 8, 2011 to November 11, 2011. The data that were gathered from the various tests were analyzed and interpreted using Two-Way Repeated Measures Analysis of Variance and Bonferroni Posttests via GraphPad Prism version 5.01 for Windows. The research was not further extended in the mass production of the bioplastic resins for public use in the industry but this might also be possible. C. Definition of Terms Agar. A phycocolloid of repeating galactose units that is primarily extracted from the cell walls of red algae. It was used as one of the polymers to make the bioplastic resins. Alginate. A phycocolloid of repeating mannuronic and guluronic acids that is primarily extracted from the cell walls of brown algae. It was used as one of the polymers to make the bioplastic resins. Biodegradability. A special property of plastic which tells whether the plastic is capable of biodegrading when immersed in substrates that promote biodegradation like loam soil and open air. It was one of the properties of the bioplastic resins that were determined. Bioplastic. A special form of plastic that is made from renewable biomass resources like corn starch and vegetable oil. It was the main experimental unit tested in this research.

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Bonferroni Posttests. A statistical tool used to compare every pair of means. It was one of the main statistical tools used in the analysis of the data gathered in the biodegradability test. Carrageenan. A phycocolloid of repeating units of sulfated galactose that is primarily extracted from red algae. It was used as one of the polymers to make the bioplastic resins. Distilled Water. A colorless polar solvent having the formula H2O. It was one of the chemicals used in the general chemical resistance test as a control. General Chemical Resistance. A property of plastic which tells the set of chemicals the plastic can resist from deterioration and to the chemicals that deteriorates the fundamental properties of the plastics. It was one of the properties of the bioplastic resins that were determined. Glycerol. A viscous organic compound having the formula of C3H8O3 which is primarily used as a non-toxic plasticizer in the bioplastic industry. It was used as the main plasticizer added to the bioplastic resins. Hydrochloric Acid. A colorless strong acid having the formula of HCl. It was one of the chemicals used in the general chemical resistance test. Loam Soil. A substrate that promote biodegradation due to its high content of organic matter and moisture on which microorganisms may feed and flourish on. It was used as one of the substrates used in the biodegradability test. Natural Algal Polysaccharides. Polymeric sugars that are mainly extracted from red and brown algae which exhibit starch-like properties and the capacity to form sturdy

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gels; also known as phycocolloids. These were the polymers used in making the bioplastic resins. Open Air. A substrate that promotes biodegradation due to its high content of oxygen and air-borne microorganisms and materials like bacteria, spores, dust, etc. It was used as one of the substrates used in the biodegradability test as a control. Percent Weight Loss. A quantity that tells the percentage lost in the masses of the bioplastic resins after being exposed to substrates that promote biodegradation and this due to the effect of aerobic respiration and the enzymatic actions of microorganisms. It was one of the parameters used in the biodegradability test and was computed using the formula: percent weight loss = [(Wi - Wf) / Wi] * 100% Plasticizer. A chemical that is used to give additional elasticity, flexibility and strength to materials like plastics. This was used to provide plastic-like properties to the bioplastic resins. Sodium Chloride. A white crystalline salt having the formula of NaCl. The aqueous form of the chemical was one of the chemicals used in the general chemical resistance test. Sodium Hydroxide. A colorless strong base having the formula of NaOH. It was one of the chemicals used in the general chemical resistance test. Two-Way Repeated Measures Analysis of Variance. A statistical tool used to determine how a response of paired or matched subjects is affected by two factors. It was one of the main statistical tools used in the analysis of the data gathered in the biodegradability test.

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Chapter II REVIEW OF RELATED LITERATURE AND STUDIES

A. Natural Algal Polysaccharides A.1.Description Phycocolloids are the natural polysaccharides that are found from both brown and red algae. These are gelatinous starch-like chemicals that are capable to form viscous gels. They are extracted from the cell walls of certain seaweeds and mainly used for their colloidal properties (Montaño, 2010). Up to present research, only agar, alginate and carrageenan are the phycocolloids that contain economic and commercial significance. They are essential since these polysaccharides exhibit high molecular weights, high viscosity, excellent gelling, stabilizing, thickening and emulsifying properties. They can also act as an agent for the maintenance of moisture. They are all soluble in water which makes them very versatile materials (Peck, 2010). A.2.Major Classes of Phycocolloids A.2.i. Agar Agar is a cell wall constituent of red algae. It is a natural polymer made from repeating units of galactose. It is an odorless, slightly transparent and sugar-reactive substance which takes form of a gel. Unlike gelatin which is a protein-based gel derived from animals, agar is a polysaccharide extracted mainly from red seaweeds (Montaño, 2010).

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Figure. 2.1. The Chemical Structure of an Agar Polymer Agar is the most potent gel-forming agent among the three. It is mainly used as a medium for microorganisms to grown since a wide range of organisms cannot digest agar. It is also used as a binder for medical tablets and capsules. It is also mixed in beers and wines to add clarity. Since it can withstand high temperatures reaching up to 70 degrees Celsius, it has been utilized now in canned meat products. Nowadays, agar gels are already used to analyze proteins and DNA and to immobilize cells (Peck, 2010). A.2.ii. Alginate Alginate is a cell wall constituent of brown algae. It is a natural polymer from repeating units of mannuronic and guluronic acid. It is an odorless, slightly transparent and viscous gum which takes the form of a liquid gel. It has a strong hydrophilic nature which makes alginate capable of absorbing water much greater than its own weight. It is also a thermally stable seaweed gum (Montaño, 2010).

Figure. 2.2. The Chemical Structure of an Alginate Polymer

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Alginate is considered to be the most common stabilizer, emulsifier and thickener used in the manufacture of dairy products, cosmetics and personal care merchandise like soaps, shampoos and conditioners. Aside from it is a binding agent in medical tablets, it also add a protective covering to it. It is also used for the fabrication of medical dressings that are easier to remove causing less pain and disruption to the wound (Peck, 2010). A.2.iii. Carrageenan Carrageenan is a cell wall constituent of red algae. It is a natural polymer made from repeating units of sulfated galactose. It is an odorless and slightly transparent substance which takes the form of a semi-solid gel. Carrageenan can retain a strong negative charge over the normal pH change (Montaño, 2010).

Figure. 2.3. The Chemical Structure of a Carrageenan Monomer Carrageenan is widely use in the manufacture of air freshener gels. It is also applied in pastries and confectionaries to add more creaminess. It is also used to improve and balance the behavior of gums and even to control crystal growth. It can also be used to test the reactivity of milk. But most of all, they are usually utilized as stabilizers for emulsions (Peck, 2010).

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B. Biomass-based Plastics B.1. Description Biomass-based plastics or simply bioplastics are forms of plastics that are made from renewable biomass sources like starch, polylactide acid and poly-3-hydroxybutyrate (Sweeney, 2008). These plastics are based on the concept of the natural cycle of matter and they promote an intelligent, cascading handling of natural resources with less waste and less emissions of greenhouse gases (Bioplastics24, 2007). Most bioplastics are being engineered to be biodegradable meaning that they will aerobically biodegrade into substances that blend harmlessly with the soil (Woodford, 2008). B.2. Applications Many bioplastics look virtually indistinguishable from conventional oil-based plastics but they lack the performance and ease of processing of traditional petrochemical plastics (Woodford, 2008). But despite of their unusual characteristics, these bioplastics are now gaining popularity in the market. So far, the most common purpose of these materials is for packaging which includes plastic covers and plastic bags. They are also used as organic waste bags wherein they can be composted together with the green waste. Blister foils and trays for fruits and vegetables are also being manufactured now. Other bioplastics are now utilized as mobile phone casings, carpet fibers, car interiors and even in pipe applications (Absolute Astronomy, 2010.). B.3. Environmental Impact Bioplastics are believed to be the solution to the big problem about improper plastic waste disposal (Valdez, 2010). This generation of green plastics promotes less reliance on fossil fuels as a carbon source and also drastically reduces the amount of

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hazardous wastes caused by oil-derived plastics (Absolute Astronomy, 2010). When bioplastics are composted, they decompose back into carbon dioxide, water and biomass and emit fewer amounts of greenhouse gases (World Centric, 2010). Bioplastics are easier to recycle than petrochemical plastics because less energy is required to recycle them and most can be reprocessed thermoplastically (Valdez, 2010). The advantages of using bioplastics are innumerable but most of all, bioplastics encourage a greener and better living on the planet. C. Methods of Evaluating the Effectiveness and Quality of Plastic Resins C.1. Biodegradability According to the American Society for Testing Materials (2010), for a plastic to be defined as biodegradable, it should meet the following specifications: 1. the material has to reach 60% biodegradation within 180 days;

2. the material has to disintegrate into very small pieces; 3. the residue has to contain certain specified limits of heavy metals and other contaminants. Biodegradability is usually measured by the amount of carbon dioxide released by the plastic as it aerobically biodegrades over time. Standard procedures involve putting the plastic into laboratory composting conditions, trapping the released carbon dioxide into a solution and titrating the solution with an acid in order to measure the amount of carbon dioxide produced over time. This measure of carbon dioxide is used to determine the percentage of carbon that has been converted by the aerobically respiring microbes (Stevens, 2010).

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Biodegradability can also be measured in terms of percent weight loss. The procedures are much similar with the standardized tests of ASTM and ISO, the only difference is that, instead of measuring the amount of carbon dioxide released over time, the dry weights itself of the plastics are being used as one of the descriptors of biodegradation. If there are differences in the dry weights of the plastics over time, this is a sure sign of microbial biodegradation due to the enzymatic actions of microorganisms (Biyo & Temelo, 2008). The rate of biodegradation is highly dependent upon the composition, thickness and surface area of the plastic. Laboratory composting conditions such as moisture and temperature can also affect the rate of biodegradation of the material (World Centric, 2010). C.2. General Chemical Resistance Various chemicals have different reactions to different types of plastics. Polystyrene, for example, is resistant to salt solutions and non-oxidizing acids but not to aromatic compounds because they tend to form cracks. Some plastics like polypropylene lose their resistance to inorganic salt solutions when the temperature exceeds to 60 degrees Celsius. Other plastics like polyethylene decomposes when they are exposed to halogens and polycarbonate expands when placed in a solution of benzene. This process of degradation is called corrosion (Pella, 2010). Corrosion is defined as the process wherein the material is exposed to chemicals or environmental factors that causes a significant change in the material’s chemical structure resulting in the deterioration of the fundamental and essential properties of the material (Tatum & Harris, 2010).

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Basic corrosion tests are highly visual. Common methods of corrosion monitoring tests involve dropping a small amount of chemical such as an acid, a base or salt solution into the surface of the specimen. Some usual indications of corrosion include thinning, swelling and cracking. Discoloration and the formation of holes are also signs of corrosion. Also remember that the thickness of the material, concentration of the chemical and the temperature will greatly affect the results of the test (Division of Alabama Specialty Products, Inc., 2011).

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Chapter III METHODOLOGY

A. Research Design In this study, the following biomass-based plastic resins from different combinations of natural algal polysaccharides were utilized as the main experimental units: 1. 3 g agar + 5 mL glycerol + 30 mL distilled water 2. 3 g alginate + 5 mL glycerol + 30 mL distilled water 3. 3 g carrageenan + 5 mL glycerol + 30 mL distilled water 4. 1.5 g agar + 1.5 g alginate + 5 mL glycerol + 30 mL distilled water 5. 1.5 g agar + 1.5 g carrageenan + 5 mL glycerol + 30 mL distilled water 6. 1.5 g alginate + 1.5 g carrageenan + 5 mL glycerol + 30 mL distilled water 7. 1 g agar + 1 g alginate + 1 g carrageenan + 5 mL glycerol + 30 mL distilled water The bioplastic resins underwent two tests in which their effectivity and quality were determined. The biodegradability and general chemical resistance tests were the test methods performed. Each test method used Completely Randomized Design (CRD) as a system in the assigning of the experimental units in their respective treatments and in the observation and gathering of the results. To maintain the accuracy and efficiency of data, there were three replicates used in each treatment of each test method. For the biodegradability test, the percent weight loss and the observed signs and forms of biodegradation were the parameters used to evaluate whether the bioplastic resins are capable of biodegrading under controlled laboratory conditions. This test method involved the exposure of the bioplastics resins in loam soil and open air for 42 days and the

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measurement of the dry weights of the bioplastic resins every seven days. Figure 3.1 shows the paradigm of the biodegradability test which was used to assign the different experimental units in their respective treatments. Table 3.1 also shows the paradigm of the application of Multiple Time Series Design with Randomization in the observation of results in the biodegradability test. Type of Substrate

Bioplastic Resin Mixture

Ag Al Ca Ag + Al Ag + Ca Al + Ca Ag + Al + Ca

E1 E1 E1 E1 E1 E1 E1

Loam Soil E2 E2 E2 E2 E2 E2 E2

E3 E3 E3 E3 E3 E3 E3

C1 C1 C1 C1 C1 C1 C1

Open Air C2 C2 C2 C2 C2 C2 C2

C3 C3 C3 C3 C3 C3 C3

Figure. 3.1. Paradigm of the Biodegradability Test Legend: Ag – Agar Al – Alginate Ca – Carrageenan

E – Experimental Group C – Control Group

Table 3.1. Multiple Time Series Design with Randomization for the Biodegradability Test R X1 O1 O2 O3 O4 O5 O6 R X2 O1 O2 O3 O4 O5 O6 R X3 O1 O2 O3 O4 O5 O6 R X4 O1 O2 O3 O4 O5 O6 R X5 O1 O2 O3 O4 O5 O6 R X6 O1 O2 O3 O4 O5 O6 R X7 O1 O2 O3 O4 O5 O6 Legend: R – Randomization X – Application of treatments O1 – 1st observation on the 7th day O2 – 2nd observation on the 14th day

O3 – 3rd observation on the 21st day O4 – 4th observation on the 28th day O5 – 5th observation on the 35th day O6 – 6th observation on the 42nd day

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For the general chemical resistance test, the effect of the different chemicals to the bioplastic resins was the only parameter used to evaluate whether the bioplastics are able to resist the corrosive properties of the chemicals when they are exposed to them. This test method involved the dropping of the chemicals onto the surface of the bioplastic resins and the observation of the effect of the chemicals to the bioplastics resins. Figure 3.2 shows the paradigm of the general chemical resistance test which was used to assign the different experimental units in their respective treatments. . Table 3.2 also shows the paradigm of the application of Multiple Time Series Design with Randomization in the observation of results in the biodegradability test. 2 M HCl

Bioplastic Resin Type

Ag Al Ca Ag + Al Ag + Ca Al + Ca Ag + Al + Ca

Type of Chemical 2 M NaOH 2 M NaCl

Distilled H2O

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Figure. 3.2. Paradigm of the General Chemical Resistance Test Legend: Ag – Agar Al – Alginate Ca – Carrageenan HCl – Hydrochloric Acid NaOH – Sodium Hydroxide

NaCl – Sodium Chloride H2O – Water E – Experimental Group C – Control Group

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Table 3.2. Multiple Time Series Design with Randomization for the General Chemical Resistance Test R X1 O1 O2 O3 O4 R X2 O1 O2 O3 O4 R X3 O1 O2 O3 O4 R X4 O1 O2 O3 O4 R X5 O1 O2 O3 O4 R X6 O1 O2 O3 O4 R X7 O1 O2 O3 O4 Legend: R – Randomization X – Application of treatments O1 – 1st observation at the instant O2 – 2nd observation on the 15th minute

O3 – 3rd observation on the 30th minute O4 – 4th observation on the 45th minute O5 – 5th observation on the 60th minute

B. Research Environment The research was performed at the residence house of the researcher at #3 Daisy St, Baptista Village, Calao East, Santiago City, Isabela and at the Science Laboratory of the Philippine Science High School-Cagayan Valley Campus, Masoc, Bayombong, Nueva Vizcaya from April 8, 2011 to November 14, 2011. Most of the materials and the equipment that were needed to perform the research are found within the premises of the researcher’s residence and at the laboratory. Some of the consultants of this research are members of the faculty and staff of PSHS-CVC and of the Marine Science Institute, University of the Philippines-Diliman Campus, Quezon City. C. Materials and Equipment The materials and equipment used in the making of the bioplastic resins were from the residence of the researcher. These include the metal spoons, the ceramic bowls, the Tupperware molds, the steamer, the stove and the liquefied petroleum gas. The natural algal polysaccharides were bought from a phycocolloids distributor in Manila and the glycerol was also bought in a Mercury Drug store in Manila.

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For the biodegradability test, the aluminum trays were bought from a grocery store in Santiago City. The loam soil was harvested also from the researcher’s residence house. The triple-beam balance that was used was borrowed from Roxas National High School. For the general chemical resistance test, the chemicals, the glass microwells, the glass medicine droppers and the analytical balance were from the Science Laboratory of PSHSCVC. Other materials such as pens, papers, scientific calculators and laptop were personal belongings of the researcher. Any other necessary materials and equipment that were used to perform the study are further discussed in the Research Method. D. Research Method This research about the development of biomass-based plastic resins from natural algal polysaccharides using steaming process has been accomplished using the procedures stated below. D.1.Bioplastic Resin Making The method that was used in making the bioplastic resins was based from the scientific blog of Brandon Sweeney about making bioplastics using potato starch (2008). However, there were some modifications used in this research such as the use of steam instead of direct heat in the heating of the raw bioplastic resin mixtures and the proportions of the ingredients in making the bioplastic resins. The natural algal polysaccharides which include the agar, alginate and carrageenan were bought from the Marine Resources Development Corporation, Inc. with the help of Dr. Marco Nemesio Montaño, a professor from the Marine Science Institute

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of the University of the Philippines, Diliman, Quezon City. A kilogram each of the powdered polysaccharides was delivered to the residence house of the researcher last April 8. The polysaccharides were then kept in tightly sealed plastic containers. After all the other materials that are needed to make the bioplastic resins have been prepared, the following bioplastic resin mixtures were made by mixing thoroughly all the ingredients until a single phase has been achieved. 1. 3 g agar + 5 mL glycerol + 30 mL distilled water 2. 3 g alginate + 5 mL glycerol + 30 mL distilled water 3. 3 g carrageenan + 5 mL glycerol + 30 mL distilled water 4. 1.5 g agar + 1.5 g alginate + 5 mL glycerol + 30 mL distilled water 5. 1.5 g agar + 1.5 g carrageenan + 5 mL glycerol + 30 mL distilled water 6. 1.5 g alginate + 1.5 g carrageenan + 5 mL glycerol + 30 mL distilled water 7. 1 g agar + 1 g alginate + 1 g carrageenan + 5 mL glycerol + 30 mL distilled water Each raw bioplastic resin mixture was then poured into separate molds. The molds were then covered tightly with an aluminum foil. Using a steamer, the raw bioplastic resin mixtures were then heated for about 15 minutes. After the said duration of time, the molds were then removed from the steamer and the aluminum foils were also detached from the molds. The molds were left first in a secured corner untouched by anyone to let the steamed bioplastic resin mixtures cool into a gel and not be contaminated by any foreign material. Twenty-four hours after the cooling process, the hardened bioplastic resin mixtures were removed from the molds. The removed bioplastic resin mixtures were then put into other open containers for about a week to let them dry further. After the drying process, the air-dried bioplastic resins were harvested from the

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containers. These bioplastic resins have been subjected into various tests to evaluate their quality and effectivity and to provide a scientific basis for their possible applications in the industry. D.2.Biodegradability Test The following procedures that have been made in the biodegradability test were based from the research of two Philippine Science High School – Western Visayas Campus scholars namely Julian Paolo Biyo and Jason Andrei Temelo entitled, “Effect of the Different Types of Substrates on the Biodegradability of SM Plastic Bags” (2008). However, there were some modifications used in this research such as the use of loam soil and open air only as substrates for the biodegradability test. The loam soil that was used in the biodegradability test was gathered from the residence house of the researcher in Santiago City, Isabela. Five hundred grams each of loam soil were put into seven 9” x 9” aluminum trays. One hundred mL of tap water were also added in the loam soil. Seven empty 9” x 9” aluminum trays were also used for the open air substrate. Each aluminum tray contained three 1.5” x 1.5” strips of a single type of a bioplastic resin. Before the bioplastic resins were immersed in the substrates, they were weighed first using a triple-beam balance. The measured mass was the initial weight of the bioplastic resin and was denoted by Wi. Every seven days, the bioplastic resins were recovered from immersion. Physical observations were also done to look for any signs and forms of biodegradation such as thinning, formation of holes and the presence of microbial colonies. After that, they were wiped with moist cotton films and air-dried for four hours. They were weighed again using a triple-beam balance. The measured mass by

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that time was the dry weight of the bioplastic resin and was denoted by Wf. For the whole duration of the experiment, there were six dry weights measured all in all. Using the initial and the dry weights of the bioplastic resin, the percent weight loss in each bioplastic resin has been calculated using the formula: percent weight loss = [(Wi - Wf) / Wi] * 100% Using these data also, the mean percent weight loss for each type of bioplastic resin has also been computed using the formula for average. Using the specifications set by the American Society for Testing Materials about the biodegradability of a material, the researcher was able to determine whether the bioplastic resins are biodegradable or not under controlled laboratory conditions. In this manner, the biodegradability of the bioplastic resins have been measured accurately and precisely but not that costly. D.3.General Chemical Resistance Test The following procedures that have been made in the general chemical resistance test were based from the scientific journals about corrosion monitoring of the Division of Alabama Specialty Products, Inc (2011). However there were some modifications used in this research such as the use of grinded bioplastic resins instead of a bioplastic resin film to provide more surface area for the reaction to take place. Three chemicals namely hydrochloric acid (HCl), sodium hydroxide (NaOH) and sodium chloride (NaCl) were used as the chemicals in the experimental group of this test method. Distilled water was also used as the control chemical in this test. Fifty milliliters each of HCl, NaOH and NaCl with a concentration of two molars were prepared by the

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researcher with the supervision of the laboratory assistant of PSHS-CVC and his research adviser. Using an analytical balance, 0.5 g of each type of grinded bioplastic resin was measured. The grinded bioplastic resins were then put in a microwell in a manner similar to Figure 3.2. Using a glass medicine dropper, a milliliter of a specific chemical was dropped onto the grinded bioplastic resins. The effect of the chemical to the bioplastic resin was observed at the instant the chemical was dropped and for every 15 minutes up to one hour the process has commenced. Any changes that the bioplastic has undergone due to the chemical dropped onto the surface of the bioplastic were considered a sign of corrosion. In this manner, the general chemical resistance of the bioplastics was measured very scientifically and practically. D.4.Disposal Method Most of the equipment and apparatus used in making the bioplastic resins, in the biodegradability test and general chemical resistance test were washed first with tap water and were autoclaved to prevent contamination and to prolong the shelf-life of the equipment and apparatus used. All the excess bioplastics, tested bioplastics and malfunctional bioplastics were composted at the residence house of the researcher. The excess HCl and NaOH solutions were allowed to participate in a neutralization reaction to form an aqueous solution of NaCl. The NaCl solutions were then diluted and thrown in the laboratory sink. Any other materials used in this research were segregated according to the disposal method designated by the local municipal government.

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E. Statistical Tool The data that were gathered from the various tests were analyzed and interpreted using Two-Way Repeated Measures Analysis of Variance and Bonferroni Posttests via GraphPad Prism version 5.01 for Windows. Upon data analysis, the researcher also used the mean of the parameters to determine the overall effectiveness and quality of the bioplastic resins and the standard deviation in each set of data to find for any significant differences between and among the data gathered from each test method. In this manner, the inferential problems of the study were answered.

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Making the Bioplastic Resins Biodegradability Test General Chemical Resistance Test Disposal of Wastes Analysis and Interpretation of Data Figure. 3.3. General Research Method

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Chapter IV PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

The main purpose of this study was to synthesize and develop novel biomass-based plastic resins from natural algal polysaccharides and phycocolloids in such ways that the resulting products are cost-effective, highly efficient, environment-friendly and toxic-free. It sought to determine the biodegradability of the bioplastic resins in loam soil and open air within a time frame of 42 days and the general chemical resistance of the bioplastic resins in plain water and in acid, base and salt solutions for an hour. A. General Observations A.1.Raw Bioplastic Resin Mixtures The natural algal polysaccharides, which were in the form of a powder, have primarily very similar physical characteristics in terms of color, odor and particle size which made them very indistinguishable from each other. Table 4.1 shows the macroscopic properties of the powders of the natural algal polysaccharides. Table 4.1. Summary of the Physical Characteristics of the Powders of the Natural Algal Polysaccharides Natural Algal Color Odor Particle Size Polysaccharide similar to a almost close to agar light beige dried seaweed that of a or saltwater powdered milk similar to a almost close to alginate light beige dried seaweed that of a or saltwater powdered milk similar to a almost close to carrageenan pearly white dried seaweed that of a or saltwater powdered milk

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But when the natural algal polysaccharides were already mixed with an aqueous solution of glycerol, each substance acted so differently from each other which made them much easier to be distinguished. Table 4.2 shows the differences in the forms and colors of the various raw bioplastic resin mixtures. Table 4.2. Summary of the Differences in the Forms and Colors of the Raw Bioplastic Resin Mixtures Bioplastic Resin Mixture Form Color Ag agar suspension brownish-yellow Al viscous gum colorless Ca aggregate of hard gel granules white Ag + Al viscous gum brownish-yellow Ag + Ca aggregate of soft gel granules brownish-yellow Al + Ca viscous gel colorless Ag + Al + Ca viscous gel brownish yellow Legend: Ag - Agar Al - Alginate

Ca - Carrageenan

But despite of the differences in their forms and colors, there were also some similarities observed among the seven bioplastic resin mixtures which include uniform translucency, an odor similar to a dried seaweed and saltwater, a very slippery feel similar to a glue and the absence of any allergic skin sensitivity when touched. A.2.Steamed Bioplastic Resins After having been steamed the raw bioplastic resin mixtures for about 15 minutes, the steamed bioplastic resins showed a lesser degree of variation in terms of their macroscopic properties. The only difference observed already was their color. Table 4.3 shows the differences in the colors of the steamed bioplastic resins.

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Table 4.3. Summary of the Differences in the Colors of the Steamed Bioplastic Resins Steamed Bioplastic Resin Color Ag white Al colorless Ca colorless Ag + Al brownish-yellow Ag + Ca brownish-yellow Al + Ca colorless Ag + Al + Ca brownish-yellow Legend: Ag – Agar Ca – Carrageenan Al – Alginate Some similarities observed among the seven steamed bioplastic resins include partial opaqueness, an odor similar to a dried seaweed and saltwater, a squishy feel and a smooth, cold surface. A.3.Air-Dried Bioplastic Resins After a week of air-drying the steamed bioplastic resins, the air-dried bioplastic resins tend to have more similar macroscopic properties which include uniform translucency, absence of color and odor and a smooth, cold and wax-like surface. Moreover, thinning and shrinking were also observed in the air-dried bioplastic resins. B. Biodegradability Test One of the properties of the bioplastic resins that have been tested was the biodegradability. The percent weight loss of the bioplastic resins measured for every seven days was the major parameter in determining the biodegradability of the bioplastic resins in loam soil and open air within a time frame of 42 days. Additionally, some signs and forms of biodegradation observed in the bioplastic resins were also noted.

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B.1. Loam Soil After having been immersed in loam soil for 42 days the seven different types of bioplastic resins, results clearly showed a rapid biodegradation of the bioplastic resins. One factor that may have caused the rapid biodegradation of the bioplastic resins is the composition of the bioplastic resins. The natural algal polysaccharides namely agar, alginate and carrageenan are all organic substances including the glycerol plasticizer. Another possible factor is the substrate. Loam soil contains a variety of dead organic matter and nutrients, an abundance of microorganisms and of course, water. Agar, alginate and carrageenan are all polysaccharides which slowly absorb water causing them to swell up and break apart into smaller fragments making them easier to be digested by the microorganisms. The trend of the biodegradability of the bioplastic resins in loam soil is shown in Table 4.4 and Figure 4.1. Table 4.4 shows the computed mean percent weight losses of the bioplastic resins in loam soil and Figure 4.1 plots the computed mean percent weight losses of the bioplastic resins in a percent weight loss vs. time graph. Table 4.4. Mean Biodegradability of the Bioplastic Resins in Loam Soil Percent Weight Loss (in %) Bioplastic Resin PW7 PW14 PW21 PW28 PW35 Ag 7.33 14.00 26.67 42.00 55.33 Al 8.67 17.33 32.00 49.33 66.00 Ca 12.00 18.67 35.33 55.33 69.33 Ag + Al 9.33 14.67 32.00 46.67 65.33 Ag + Ca 8.00 14.00 31.33 48.00 64.67 Al + Ca 10.67 16.00 34.00 51.33 70.00 Ag + Al + Ca 8.67 14.00 31.33 48.67 68.00 Mean 9.24 15.52 31.81 48.76 65.52 Legend: Ag –Agar Al – Alginate

PW42 67.33 74.67 83.33 74.00 72.67 79.33 75.33 75.24

Ca – Carrageenan PWx – Percent Weight Loss at Day x

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30

Legend:

Ag – Agar

Al – Alginate

Figure 4.1 Mean Biodegradability of he Bioplastic Resins in Loam Soil Ca - Carrageenan

During the first seven days, the carrageenan only resin has the highest percent weight loss attained which was 12.00%. On the other hand, the agar only resin has the lowest percent weight loss attained which was only 7.33%. The mean percent weight loss of the bioplastic resins was 9.24% and the percent weight losses of the bioplastic resins were varied as much as 1.49%. During the 14th day, the carrageenan only resin again has the highest percent weight loss attained which was 18.67%. On the other hand, the agar only resin, the agar plus carrageenan resin and the agar plus alginate plus carrageenan resin tied for having the lowest percent weight loss attained which was only 14.00%. The mean percent weight loss of the bioplastic resins was 15.52% and the percent weight losses of the bioplastic resins were varied as much as 1.74%. During the 21st day, the carrageenan only resin still has the highest percent weight loss attained which was 35.33%. On the other hand, the agar only resin still has the lowest percent weight loss attained which was only 26.67%. The mean percent weight loss of the bioplastic resins doubled compared to the previous one which was already 31.81% and the percent weight losses of the bioplastic resins were varied as much as 2.51%. During the 28th day, the carrageenan only resin still has the highest percent weight loss attained which was 55.33%. On the other hand, the agar only resin still has the lowest percent weight loss attained which was only 42.00%. The mean percent weight loss of the bioplastic resins was 48.76% and the percent weight losses of the bioplastic resins were varied as much as 3.79%.

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During the 35th day, the alginate plus carrageenan resin attained the highest percent weight loss which was 70.00%; the carrageenan only resin which has 69.33% placed only the second. On the other hand, the agar only resin still has the lowest percent weight loss attained which was only 53.33%. The mean percent weight loss of the bioplastic resins was 65.52% and the percent weight losses of the bioplastic resins were varied as much as 5.21%. After the 42-day immersion of the bioplastic resins in loam soil, the carrageenan only resin again has the highest percent weight loss attained which was 83.33%. On the other hand, the agar only resin still has the lowest percent weight loss attained which was only 67.33%. The mean final percent weight loss of the bioplastic resins was 75.24% and the percent weight losses of the bioplastic resins were varied as much as 4.68% Some signs and forms of biodegradation observed during the 42-day immersion of the bioplastic resins in loam soil include the deformations of the edges and the surface of the bioplastic resins, the appearance of small holes, thinning and shrinking and of course, the presence of fungal and bacterial colonies in the outside and even in the inside of the bioplastic resins. B.2. Effect of Bioplastic Resin Type and Time on Loam Soil Biodegradability To determine the interaction of the bioplastic resin type factor and the time factor on the biodegradability of the bioplastic resins in loam soil, Two-Way Repeated Measures ANOVA and Bonferroni Posttests were the main statistical tools used. Table 4.5 shows the results of the Two-Way Repeated Measures ANOVA and Table 4.6 and Table 4.7 shows the results of the Bonferroni Posttests in the analysis of

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the percent weight losses of the bioplastic resins in loam soil during their 42-day immersion in loam soil. Table 4.5. Two-Way Repeated Measures Analysis of Variance on the Percent Weight Losses of the Bioplastic Resins in Loam Soil Source of Variation DF Sum of Squares Mean Square Interaction 30.0 339.2 11.31 Time 5.0 75100 15020 Bioplastic Resin Type 6.0 1102 183.7 Subjects (matching) 14.0 22.67 1.619 Residual (Error) 70.0 49.33 0.7048 Total 125.0 76610 Interaction accounts for 0.44% of the total variance (F = 16.04, DFn = 30 DFd = 70). The P value is less than 0.0001. If there is no interaction overall, there is a less than 0.01% chance of randomly observing so much interaction in an experiment of this size. The interaction is considered extremely significant. Since the interaction is statistically significant, the P values that follow for the row and column effects are difficult to interpret. The bioplastic resin type factor accounts for 1.44% of the total variance after adjusting for matching (F = 113.44, DFn = 6 DFd = 70). The P value is less than 0.0001. If the bioplastic resin type factor has no effect overall, there is a less than 0.01% chance of randomly observing an effect this big or bigger in an experiment of this size. The effect is considered extremely significant. The time factor accounts for 98.02% of the total variance after adjusting for matching (F = 21312.40, DFn = 5, DFd = 70). The P value is less than 0.0001. If the time factor has no effect overall, there is a less than 0.01% chance of randomly observing an effect this big or bigger in an experiment of this size. The effect is considered extremely significant.

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Matching accounts for 0.0296% of the total variance (F = 2.30, DFn = 14, DFd = 70). The P value equals 0.0118. If matching were not effective overall, there is a 1.2% chance of randomly observing an effect this big or bigger in an experiment of this size. The effect is considered significant. Table 4.6. Bonferroni Posttests on the Percent Weight Losses of the Soil Days Bioplastic Resin Type Pairs 7 14 21 28 Ag vs. Al ns *** *** *** Ag vs. Ca *** *** *** *** Ag vs. Ag + Al ns ns *** *** Ag vs. Ag + Ca ns ns *** *** Ag vs. Al + Ca *** ns *** *** Ag vs. Ag + Al + Ca ns ns *** *** Al vs. Ca *** ns *** *** Al vs. Ag + Al ns *** ns *** Al vs. Ag + Ca ns *** ns ns Al vs. Al + Ca ns ns ns ns Al vs. Ag + Al + Ca ns *** ns ns Ca vs. Ag + Al ** *** *** *** Ca vs. Ag + Ca *** *** *** *** Ca vs. Al + Ca ns ** ns *** Ca vs. Ag + Al + Ca *** *** *** *** Ag + Al vs. Ag + Ca ns ns ns ns Ag + Al vs. Al + Ca ns ns ns *** Ag + Al vs. Ag + Al + Ca ns ns ns ns Ag + Ca vs. Al + Ca ** ns ** *** Ag + Ca vs. Ag + Al + Ca ns ns ns ns Al + Ca vs. Ag + Al + Ca ns ns ** ** Legend: Ag – Agar Al – Alginate Ca – Carrageenan

Bioplastic in Loam

35 *** *** *** *** *** *** *** ns ns *** ns *** *** ns ns ns *** ** *** *** ns

42 *** *** *** *** *** *** *** ns ns *** ns *** *** *** *** ns *** ns *** ** ***

ns – Not Significant ** – Very Significant *** – Extremely Significant

During the first seven days, results of the Bonferroni Posttests show that most bioplastic resin type pairs have no significant difference between their percent weight losses in loam soil (i.e. agar only vs. alginate only resins). Furthermore, a few have

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extremely significant difference between their percent weight losses (i.e. carrageenan only vs. agar plus alginate plus carrageenan resins) and only two have very significant difference (i.e. agar plus carrageenan vs. alginate plus carrageenan). During the 14th day, results show that most bioplastic resin type pairs still have no significant difference between their percent weight losses in loam soil (i.e. alginate only vs. carrageenan only). Moreover, many pairs already have extremely significant difference between their percent weight losses (i.e. agar only vs. carrageenan only resins) and only the carrageenan only vs. alginate plus carrageenan resins has a very significant difference. During the 21st day, results show that most bioplastic resin type pairs already have an extremely significant difference between their percent weight losses in loam soil (i.e. agar only vs. agar plus alginate resins). Furthermore, many pairs have no significant difference between their percent weight losses (i.e. agar plus alginate vs. agar plus carrageenan resins) and only two have very significant difference (i.e. agar plus carrageenan vs. alginate plus carrageenan resins). During the 28th day, results show that most bioplastic resin type pairs still have extremely significant difference between their percent weight losses in loam soil (i.e. agar only vs. agar plus carrageenan). Moreover, a few pairs have no significant difference between their percent weight losses (i.e. alginate only vs. agar plus carrageenan resins) and only the alginate plus carrageenan vs. agar plus alginate plus carrageenan has a very significant difference. During the 35th day, results show that most bioplastic resin type pairs still have extremely significant difference between their percent weight losses in loam soil (i.e. agar

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only vs. agar plus alginate plus carrageenan resins). Furthermore, a few pairs have no significant difference between their percent weight losses (i.e. alginate only vs. agar plus alginate plus carrageenan resins) and only the agar plus alginate vs. agar plus alginate plus carrageenan resins has a very significant difference. During the 42nd day, results show that most bioplastic resin type pairs still have extremely significant difference between their percent weight losses in loam soil (i.e. carrageenan only vs. agar plus alginate plus carrageenan resins). A few pairs have no significant differences between their percent weight losses (i.e. alginate only vs. agar plus algiante) and only the agar plus carrageenan vs. agar plus alginate plus carrageenan has a very significant difference. To know more on the significant differences between the percent weight losses in loam soil of the various bioplastic resin type pairs, refer to Table 4.6. Table 4.7. Bonferroni Posttests on the Percent Weight Losses of the Bioplastic Resins and the Mean Percent Weight Loss in Loam Soil Days Bioplastic Resin Type-Mean Pairs 7 14 21 28 35 42 Ag vs. Mean * ns *** *** *** *** Al vs. Mean ns ns ns ns ns ns Ca vs. Mean ** *** *** *** *** *** Ag + Al vs. Mean ns ns ns * ns ns Ag + Ca vs. Mean ns ns ns ns ns ** Al + Ca vs. Mean ns ns * ** *** *** Ag + Al + Ca vs. Mean. ns ns ns ns * ns Legend: Ag – Agar Al –Alginate Ca – Carrageenan Ns – Not Significant

* - Significant ** - Very Significant *** - Extremely Significant

During the first seven days, results of the Bonferroni Posttests show that most bioplastic resins have no significant difference between their percent weight loss and the

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mean percent weight loss in loam soil (i.e. alginate only resin). Furthermore, only the agar only resin has a significant difference and only the carrageenan only resin has a very significant difference. During the 14th day, results show that most bioplastic resins still have no significant difference between their percent weight loss and the mean percent weight loss in loam soil (i.e. agar plus alginate resin). Moreover, only the carrageenan has an extremely significant difference. During the 21st day, results show that most bioplastic resins still have no significant difference between their percent weight loss and the mean percent weight loss in loam soil (i.e. agar plus carrageenan resin). Furthermore, only the alginate plus carrageenan resin has a significant difference and both the agar only and carrageenan only resins have extremely significant difference. During the 28th day, results show that only a few bioplastics resins already have no significant difference between their percent weight loss and the mean percent weight loss in loam soil (i.e. agar plus alginate plus carrageenan resin). Moreover, only the agar plus alginate resin has a significant difference and only the alginate plus carrageenan resin still has a very significant difference. Also, both the agar only and the carrageenan only resins still have extremely significant difference. During the 35th day, results show that only a few bioplastic resins still have no significant difference between their percent weight loss and the mean percent weight loss in loam soil (i.e. alginate only). Furthermore, only the agar plus alginate plus carrageenan resin has a significant difference and the alginate plus carrageenan resin together with the agar only and the carrageenan only resins have extremely significant difference.

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During the 42nd day, results show that a few bioplastic resins still have no significant difference between their percent weight loss and the mean percent weight loss in loam soil (i.e. agar plus alginate resin). Moreover, only the agar plus carrageenan resin has a very significant difference and those resins having an extremely significant difference are still the same with the previous. To know more on the significant differences between the percent weight losses of the bioplastic resins and the mean percent weight loss in loam soil, refer to Table 4.7. B.3. Open Air After having been exposed in open air for 42 days the seven different types of bioplastic resins, results clearly showed a very slow biodegradation of the bioplastic resins. One factor that may have caused the very slow biodegradation of the bioplastic resins is the absence of water. The bioplastic resins were not put in a moist environment and so nothing would primarily break down or disrupt the polysaccharides’ chemical structure. Another is that agar, alginate and carrageenan do not spontaneously oxidize or combust into carbon dioxide and water even though there are enough oxygen molecules in the surroundings which may have induced aerobic degradation. The trend of the biodegradability of bioplastic resins in open air is shown in Table 4.8 and Figure 4.2. Table 4.8 shows the computed mean percent weight losses of the bioplastic resins in open air and Figure 4.2 plots the computed mean percent weight losses of the bioplastic resins in a percent weight loss vs. time graph.

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Table 4.8. Mean Biodegradability of the Bioplastic Resins in Open Air Percent Weight Loss (in %) Bioplastic Resin PW7 PW14 PW21 PW28 PW35 Ag 0.00 2.00 4.00 4.00 4.00 Al 0.00 2.00 4.00 4.00 4.00 Ca 0.00 2.00 4.00 4.00 4.00 Ag + Al 0.00 2.00 4.00 4.00 4.00 Ag + Ca 0.00 2.00 4.00 4.00 4.00 Al + Ca 0.00 2.00 4.00 4.00 4.00 Ag + Al + Ca 0.00 2.00 4.00 4.00 4.00 Mean 0.00 2.00 4.00 4.00 4.00 Legend: A – Agar Al – Alginate

PW42 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00

Ca – Carrageenan PWx – Percent Weight Loss at Day x

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40

Legend:

Ag – Agar

Al – Algiante

Figure 4.1 Mean Biodegradability of he Bioplastic Resins in Open Air Ca - Carrageenan

During the first seven days, the bioplastic resins manifested a zero percent weight loss. But during the 14th day, the bioplastic resins already have reached a mean percent weight loss of 2.00%. After the next seven days, the mean percent weight loss of the bioplastic resins doubled to 4%. The percent weight losses observed during the 14th up to the 21st day can be attributed to the constant thinning and shrinking of the bioplastic resins. But during the 28th up to the 35th day, the mean percent weight losses of the bioplastic resins remained still to 4.00% which can be attributed to the thinning and shrinking threshold of the bioplastic resins. During the last day of exposure, the mean percent weight loss of the bioplastic resins was only 6.00%. Thinning and shrinking were the only signs and forms of biodegradation observed during the 42-day exposure of the bioplastic resins in open air. C. General Chemical Resistance Test Another property of the bioplastic resins that was tested was their general chemical resistance. The chemicals that were used include distilled water and two molar aqueous solutions of hydrochloric acid, sodium hydroxide and sodium chloride. The effect of the chemicals to the grinded bioplastic resins for an hour was the major parameter in evaluating the general chemical resistance of the bioplastic resins. C.1. Hydrochloric Acid After having been exposed the grinded bioplastic resins to the hydrochloric acid solution, each type showed varying responses as time passed by. At the instant the hydrochloric acid was dropped to the grinded bioplastic resins, they have absorbed the chemical causing them to swell but they were not dissolved. There were no other changes observed in the physical properties of the bioplastic resins.

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During the first 15 minutes, white precipitates were observed at the bottom of the alginate-containing resins. Partial coagulation of particles was also observed for all the composite bioplastic resins and a viscous gum formation was observed for the alginate only resin. There was no effect observed for the agar only and carrageenan only resins. During the 30th minute, white precipitates were still observed at the bottom of the alginate-containing resins. Full coagulation of particles was now observed for all the composite bioplastic resins and a viscous gum formation was still observed for the alginate resin only. There was no effect still observed for the agar only and carrageenan only resins. During the 45th minute, drying and shrinking of all the bioplastic resins were now observed especially for the carrageenan only resin. The white precipitates observed for alginate-containing resins were already lost. Full coagulation of particles was still observed for all composite bioplastic resins and a viscous gum formation was still observed for the alginate only resin. Color changes were already observed for agarcontaining resins. During the 60th minute, drying and shrinking of all the bioplastic resins was still observed especially for the carrageenan only resin. The coagulation of the particles for all composite bioplastic resins resulted to a hard aggregate of grinded bioplastic resins. The viscous gum for the alginate only resin turned into a transparent film. Color changes were observed for agar-containing resins (brownish-yellow). C.2. Sodium Hydroxide After having been exposed the grinded bioplastic resins to the sodium hydroxide solution, each type showed varying responses as time passed by. At the instant the

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sodium hydroxide was dropped to the grinded bioplastic resins, they have absorbed the chemical causing them to swell but they were not dissolved. There were no other changes observed in the physical properties of the bioplastic resins. During the first 15 minutes, green precipitates were observed at the bottom of the agar-containing resins. Partial coagulation of particles was also observed for all the composite bioplastic resins and a viscous gum formation was observed for the alginate only resin. There was no effect observed for the carrageenan only resin. During the 30th minute, green precipitates were still observed at the bottom of the agar-containing resins. Partial coagulation was observed for the carrageenan only resin and full coagulation of particles was now observed for all the composite bioplastic resins. A viscous gum formation was still observed for the alginate resin only. During the 45th minute, drying and shrinking for all the bioplastic resins were now observed. Color changes were also observed; for agar-containing resins (greenish-brown) and for the remaining resins (white). Full coagulation of particles was already observed for composite bioplastic resins including the carrageenan only resin and a viscous gum formation was still observed for the alginate resin only. During the 60th minute, drying and shrinking for all the bioplastic resins were still observed. Color changes were still observed; for agar-containing resins (greenish-brown) and for the remaining resins (white). The coagulation of particles for the composite bioplastic resins and for the carrageenan only resins resulted to a hard aggregate of grinded bioplastic resins. The viscous gum for the alginate resin only turned into a transparent film.

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C.3. Sodium Chloride After having been exposed the grinded bioplastic resins to the sodium chloride solution, each type showed varying responses as time passed by. At the instant the sodium chloride was dropped to the grinded bioplastic resins, they have absorbed the chemical causing them to swell but they were not dissolved. There were no other changes observed in the physical properties of the bioplastic resins. During the first 15 minutes, trace amounts of green precipitates were observed at the bottom of the agar-containing resins. Partial coagulation of particles was also observed for all the composite bioplastic resins and a viscous gum formation was observed for the alginate only resin. There was no effect observed for the carrageenan only resin. During the 30th minute, trace amounts of green precipitates were still observed at the bottom of the agar-containing resins. Partial coagulation was observed for the carrageenan only resin and full coagulation of particles was now observed for all the composite bioplastic resins. A viscous gum formation was still observed for the alginate resin only. During the 45th minute, drying and shrinking for all the bioplastic resins were now observed. Color changes were also observed; for agar-containing resins including the alginate only resin (brownish-yellow) and for the carrageenan only resin (white). Full coagulation of particles was already observed for composite bioplastic resins including the carrageenan only resin and a viscous gum formation was still observed for the alginate resin only.

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During the 60th minute, drying and shrinking for all the bioplastic resins were still observed. Color changes were also observed; for agar-containing resins including the alginate only resin (brownish-yellow) and for the carrageenan only resin (white). The coagulation of particles for the composite bioplastic resins and for the carrageenan only resins resulted to a hard aggregate of grinded bioplastic resins. The viscous gum for the alginate resin only turned into a translucent film. C.4. Distilled Water After having been exposed the grinded bioplastic resins to the distilled water solution, each type showed varying responses as time passed by. At the instant the distilled water was dropped to the grinded bioplastic resins, they have absorbed the chemical causing them to swell but they were not dissolved. There were no other changes observed in the physical properties of the bioplastic resins. During the first 15 minutes, partial coagulation of particles was observed for all the composite bioplastic resins and a viscous gum formation was observed for the alginate only resin. There was no effect observed for the agar only and carrageenan only resins. During the 30th minute, partial coagulation was observed for the carrageenan only resin and full coagulation of particles was now observed for all the composite bioplastic resins. A viscous gum formation was still observed for the alginate resin only. There was no effect observed for the agar only resin. During the 45th minute, drying and shrinking for all the bioplastic resins were now observed especially for the agar only resin. Color changes were also observed; for agarcontaining resins including the alginate only resin (brownish-yellow) and for the

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carrageenan only resin (white). Full coagulation of particles was already observed for composite bioplastic resins including the carrageenan only resin and a viscous gum formation was still observed for the alginate resin only. During the 60th minute, drying and shrinking for all the bioplastic resins were still observed especially for the agar only resin. Color changes were also observed; for agarcontaining resins including the alginate only resin (brownish-yellow) and for the carrageenan only resin (white). The coagulation of particles for the composite bioplastic resins and for the carrageenan only resins resulted to a hard aggregate of grinded bioplastic resins. The viscous gum for the alginate resin only turned into a translucent film.

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Chapter V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

A. Summary Due to the very damaging and harmful effects of the improper use and disposal of conventional petrochemical plastic products to the environment, the researcher thought of a greener solution that would somehow lessen the impact of the said environmental problem and that is to synthesize and develop biomass-based plastic resins from natural algal polysaccharides and phycocolloids. It aimed to create good, environment-friendly, inexpensive and toxic-free bioplastic resins by utilizing agar, alginate and carrageenan as novel polymers for plastic products. The bioplastic resins were also tested for their biodegradability and general chemical resistance to provide a scientific basis for their plausible applications in the industry. The bioplastic resins were made by dissolving specific amounts of natural algal polysaccharides in an aqueous solution of glycerol, steaming the raw bioplastic resin mixtures and air-drying the steamed the bioplastic resins. For the biodegradability test, three replicates of each bioplastic resin were immersed in separate aluminum trays containing loam soil for 42 days and another three replicates of each bioplastic resin were exposed in open air for 42 days also. Every seven days, the bioplastic resins were harvested from their substrates to measure their dry weights, to compute for their percent weight losses and to observe some signs and forms of biodegradation. For the bioplastic resins immersed in loam soil, results show that the mean final percent weight losses of the bioplastic resins ranged from 67.33% to 83.33%. The mean

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final percent weight loss was 75.24%. Some signs and forms of biodegradation observed include the deformations of the edges of the bioplastic resins, the appearance of small holes, thinning and shrinking and of course, the presence of fungal and bacterial colonies in the outside and in the inside of the bioplastic resins. For the bioplastic resins exposed in open air, weight loss was only observed during the second week after exposing them in open air. The final mean biodegradability of the all the bioplastic resins was only 6%. Thinning and shrinking were the only observed signs and forms of biodegradation observed in the bioplastic resins. B. Conclusions This study has arrived at the following conclusions based on the analysis of the results. 1. The natural algal polysaccharides namely agar, alginate and carrageenan are efficient and effective polymers to create good, environment-friendly, inexpensive and toxicfree bioplastic resins. 2. The bioplastic resins are highly biodegradable and compostable in loam soil. Moreover, the bioplastic resins are also oxo-degradable. 3. The bioplastic resins are permeable and absorbent to chemical solutions but their qualities are not drastically and readily altered. C. Recommendations The researcher would like to propose the following recommendations to further improve the study: 1. the use of varying proportions of the natural algal polysaccharides, glycerol and distilled water,

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2. the use of glyceraldehydes as a plasticizer of the bioplastic resins instead of glycerol, 3. the application of pressure in the steaming of the bioplastic resins instead of heating only, 4. lengthening the time frame of the biodegradability test until the bioplastic resin reached a 100% percent weight loss, 5. the use of the amount of carbon dioxide released parameter as a measure of biodegradability instead of percent weight loss, 6. the use of varying concentrations of hydrochloric acid, sodium hydroxide and sodium chloride in testing the general chemical resistance of the bioplastic resins, 7. the use of other chemicals such as oils, bleaching agents, organic solvents and other inorganic salt solutions in testing the general chemical resistance of the bioplastic resins, 8. the addition of other substances and chemicals such as anti-microbial agents, casting compounds and waterproofing coatings in the bioplastic resins to further improve the properties and the quality of the bioplastic resins, 9. and the application of the bioplastic resins in the real-world as passive packaging materials, compost bags, insect-repellant patches and many more.

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APPENDIX A Raw Data

Table A.2. Biodegradability Test: Soil Bioplastic Replicate Resin Type 1 2 Ag 3 Mean 1 2 Al 3 Mean 1 2 Ca 3 Mean 1 2 Ag + Al 3 Mean 1 2 Ag + Ca 3 Mean 1 2 Al + Ca 3 Mean 1 2 Ag + Al + Ca 3 Mean

Amount Left of the Bioplastic Resins Immersed in Loam

Wi 5.0 5.0 5.0 5.00 5.0 5.0 5.0 5.00 5.0 5.0 5.0 5.00 5.0 5.0 5.0 5.00 5.0 5.0 5.0 5.00 5.0 5.0 5.0 5.00 5.0 5.0 5.0 5.00

Wf7 4.6 4.6 4.7 4.63 4.5 4.6 4.6 4.57 4.4 4.4 4.4 4.40 4.5 4.6 4.5 4.53 4.6 4.6 4.6 4.60 4.4 4.5 4.5 4.47 4.6 4.5 4.6 4.57

Note: Accuracy level is set at ± 0.01 g.

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Amount Left (g) Wf14 Wf21 Wf28 Wf35 Wf42 4.3 3.7 2.9 2.3 1.6 4.3 3.6 2.9 2.2 1.7 4.3 3.7 2.9 2.2 1.6 4.3 3.67 2.90 2.23 1.63 4.1 3.4 2.5 1.7 1.3 4.2 3.4 2.6 1.7 1.2 4.1 3.4 2.5 1.7 1.3 4.13 3.40 2.53 1.70 1.27 4.1 3.2 2.2 1.5 0.8 4.1 3.3 2.3 1.6 0.9 4.0 3.2 2.2 1.5 0.8 4.07 3.23 2.23 1.53 0.83 4.2 3.4 2.7 1.8 1.3 4.3 3.4 2.7 1.7 1.3 4.3 3.4 2.6 1.7 1.3 4.27 3.40 2.67 1.73 1.30 4.3 3.5 2.6 1.8 1.4 4.3 3.4 2.6 1.8 1.4 4.3 3.4 2.6 1.7 1.3 4.30 3.43 2.60 1.77 1.37 4.2 3.3 2.4 1.5 1.0 4.2 3.3 2.4 1.5 1.1 4.2 3.3 2.5 1.5 1.0 4.20 3.30 2.43 1.50 1.03 4.3 3.4 2.6 1.6 1.2 4.3 3.4 2.5 1.6 1.2 4.3 3.5 2.6 1.6 1.3 4.30 3.43 2.57 1.60 1.23

Legend: Ag – Agar Al – Alginate Ca – Carrageenan

Wi – Initial Weight Wfx – Dry Weight at Day x

Table A.2. Biodegradability Test: Amount Left of the Bioplastic Resins Exposed in Open Air Amount Left (g) Bioplastic Replicate Resin Type Wi Wf7 Wf14 Wf21 Wf28 Wf35 Wf42 1 5.0 5.0 4.9 4.8 4.8 4.8 4.7 Ag 2 5.0 5.0 4.9 4.8 4.8 4.8 4.7 3 5.0 5.0 4.9 4.8 4.8 4.8 4.7 1 5.0 5.0 4.9 4.8 4.8 4.8 4.7 Al 2 5.0 5.0 4.9 4.8 4.8 4.8 4.7 3 5.0 5.0 4.9 4.8 4.8 4.8 4.7 1 5.0 5.0 4.9 4.8 4.8 4.8 4.7 Ca 2 5.0 5.0 4.9 4.8 4.8 4.8 4.7 3 5.0 5.0 4.9 4.8 4.8 4.8 4.7 1 5.0 5.0 4.9 4.8 4.8 4.8 4.7 Ag + Al 2 5.0 5.0 4.9 4.8 4.8 4.8 4.7 3 5.0 5.0 4.9 4.8 4.8 4.8 4.7 1 5.0 5.0 4.9 4.8 4.8 4.8 4.7 Ag + Ca 2 5.0 5.0 4.9 4.8 4.8 4.8 4.7 3 5.0 5.0 4.9 4.8 4.8 4.8 4.7 1 5.0 5.0 4.9 4.8 4.8 4.8 4.7 Al + Ca 2 5.0 5.0 4.9 4.8 4.8 4.8 4.7 3 5.0 5.0 4.9 4.8 4.8 4.8 4.7 1 5.0 5.0 4.9 4.8 4.8 4.8 4.7 Ag + Al + Ca 2 5.0 5.0 4.9 4.8 4.8 4.8 4.7 3 5.0 5.0 4.9 4.8 4.8 4.8 4.7 Note: Accuracy level is set at ± 0.01 g.

Legend: Ag – Agar Al – Alginate Ca – Carrageenan

Wi – Initial Weight Wfx – Dry Weight at Day x

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Table A.3. Biodegradability Test: Percent Weight Loss of the Bioplastic Resins Immersed in Loam Soil Percent Weight Loss (%) Bioplastic Replicate Resin Type PW7 PW14 PW21 PW28 PW35 PW42 1 8.00 14.00 26.00 42.00 54.00 68.00 2 8.00 14.00 28.00 42.00 56.00 66.00 Ag 3 6.00 14.00 26.00 42.00 56.00 68.00 Mean 7.333 14.000 26.667 42.000 55.333 67.333 1 10.00 18.00 32.00 50.00 66.00 74.00 2 8.00 16.00 32.00 48.00 66.00 76.00 Al 3 8.00 18.00 32.00 50.00 66.00 74.00 Mean 8.667 17.333 32.000 49.333 66.000 74.667 1 12.00 18.00 36.00 56.00 70.00 84.00 2 12.00 18.00 34.00 54.00 68.00 82.00 Ca 3 12.00 20.00 36.00 56.00 70.00 84.00 Mean 12.000 18.667 35.333 55.333 69.333 83.333 1 10.00 16.00 32.00 46.00 64.00 74.00 2 8.00 14.00 32.00 46.00 66.00 74.00 Ag + Al 3 10.00 14.00 32.00 48.00 66.00 74.00 Mean 9.333 14.667 32.000 46.667 65.333 74.000 1 8.00 14.00 30.00 48.00 64.00 72.00 2 8.00 14.00 32.00 48.00 64.00 72.00 Ag + Ca 3 8.00 14.00 32.00 48.00 66.00 74.00 Mean 8.000 14.000 31.333 48.000 64.667 72.667 1 12.00 16.00 34.00 52.00 70.00 80.00 2 10.00 16.00 34.00 52.00 70.00 78.00 Al + Ca 3 10.00 16.00 34.00 50.00 70.00 80.00 Mean 10.667 16.000 34.000 51.333 70.000 79.333 1 8.00 14.00 32.00 48.00 68.00 76.00 2 10.00 14.00 32.00 50.00 68.00 76.00 Ag + Al + Ca 3 8.00 14.00 30.00 48.00 68.00 74.00 Mean 8.667 14.000 31.333 48.667 68.000 75.333 Legend: Ag – Agar Al – Alginate

Ca – Carrageenan PWx – Percent Weight Loss at Day x

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Table A.3. Biodegradability Test: Open Air Bioplastic Replicate Resin Type 1 Ag 2 3 1 Al 2 3 1 Ca 2 3 1 Ag + Al 2 3 1 Ag + Ca 2 3 1 Al + Ca 2 3 1 Ag + Al + Ca 2 3

Percent Weight Loss of the Bioplastic Resins Exposed in

PW7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Percent Weight Loss (%) PW14 PW21 PW28 PW35 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00 2.00 4.00 4.00 4.00

Legend: Ag – Agar Al – Alginate

PW42 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00

Ca – Carrageenan PWx – Percent Weight Loss at Day x

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APPENDIX C Documentations

Figure C.1. A carrageenan only resin immersed in loam soil during the 21st day.

Figure C.2. An agar only resin immersed in loam soil during the 28th day.

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Figure C.3. Alginate-containing resins immersed in loam soil during the 42nd day.

Figure C.4. A carrageenan only resin exposed in the open air during the 35th day.

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Figure C.5. The granulated bioplastic resins an hour after exposure to hydrochloric acid.

Figure C.6. The granulated bioplastic resins an hour after exposure to sodium hydroxide.

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Figure C.7. The granulated bioplastic resins an hour after exposure to sodium chloride.

Figure C.8. The granulated bioplastic resins an hour after exposure to distilled water.

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APPENDIX D Correspondence from Prof. Montaño

Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Friday, November 5, 2010, 4:59:04 AM Green seaweeds contain the highest amount of Floridean starch. Seaweeds contain polysaccharides (polymeric sugars). These polysaccharides are hydrolyzed into simple sugars and fermented to produce bioethanol. Problem is, the natural polymers from seaweeds cost much more than ethanol. Please surf the world wide web about seaweeds and their composition. On Fri, Nov 5, 2010 at 10:09 AM, Justin Richmond C. Domingo wrote: Sir: This is Justin Richmond C. Domingo, a third year student from Philippine Science High School-Cagayan Valley Campus. I am currently working on the conceptualization of my research paper about utilizing seaweeds as biomass-based plastics. I just want to ask if seaweeds contain starch and sugars that can be used as a polymer for my bioplastics. If yes, what are some of the seaweeds that can be found in the Philippines that contain the highest amounts of these starch and sugars. Thank you! Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Sunday, November 14, 2010, 8:29:17 PM Floridean starch is three-dimensional in structure. They are sulfated. They are usually extracted by water. You can determine the quantity by iodine titration. On Sun, Nov 14, 2010 at 10:31 AM, Justin Richmond C. Domingo wrote: Sir: I just want to ask on how to extract the Floridean starch from seaweeds. I also want to ask for the difference between Floridean starch of seaweeds and the plain starch from plants.

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Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Sunday, November 14, 2010, 8:29:17 PM Here is a simplified procedure of agar and alginate extraction. The powerpoint presentation is too large to be sent via e-mail. I will try to send it by parts. For the extraction of agar, cook 25 grams of dry Gracilaria in 5% NaOH solution in 90 oC for an hour. Wash in running water after. Soak in 750 mL of 0.5% HOAc for an hour. Wash again in running water. Extract with one liter of boiling water. Blend and filter in a filter bomb. Collect the filtrate and freeze it overnight. The following day, thaw the gel and dry the agar wafer. For the extraction of alginate, soak 100 grams of fresh brown seaweed in 0.1M of HCl solution. Wash seaweed with one liter of 1% Na 2CO3 solution. Blend and filter in a filter bomb. Collect filtrate and precipitate the sodium alginate with IsprOH. The volume of the IsprOH must be three times the volume of the filtrate. Dry and grind. On Sun, Nov 28, 2010 at 10:21 AM, Justin Richmond C. Domingo wrote: Sir: I just want to ask on how to extract agar and alginate from seaweeds. The agar and alginate will also be utilized to create the bioplastics since they are also similar to starch. I would also like to have a copy of your presentation about the different phycocolloids. I lost my notes during the internship program so as much as possible, I want to have a copy of that again because it would really help me a lot in the review of related literature and studies. Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Wednesday, January 19, 2011, 1:47:10 PM Alginic acid pH normally depends on the counterpart salt it has. It should be around pH 4 to 5. These polysaccharides found in seaweeds are safe when heated as long as the temperature is not burning hot. On Wed, Jan 12, 2011 at 5:59 PM, Justin Richmond C. Domingo wrote: Sir: I just want to ask if Floridean starch, agar, and alginate are safe when they are heated. The heating will be done because the bioplastics will be processed thermoplastically. In addition, I also want to ask the normal pH range of alginic acid. I think it would be better if I know it, because unknowingly while experimenting, I might spill some of those in my hands or to other equipments.

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Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Sunday, February 13, 2011, 8:21:23 PM 20-25 oC but it should be soaked in water. One should spray seawater to the seaweeds every 20 minutes. You may just put the seaweeds outside the refrigerator as long as there is enough air circulation within the surroundings. Constant spraying of seawater is also required to keep the air humid. On Sun, Feb 13, 2011 at 2:515 PM, Justin Richmond C. Domingo wrote: I would like to ask if what temperature range is required in the maintenance of the freshness of the seaweeds when kept in a refrigerator. Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Sunday, February 13, 2011, 8:21:23 PM Laminaria and Gelidium are temperate seaweeds. One would buy Gracilaria from Farmers’ Market in Cubao. They are “gulaman-dagat.” There should be lots of Sargassum in Leyte as a source of low-viscosity alginate. Try to find whether you can get Gelidelia acerosa. It is mostly found in the Pacific coast. Bolinao is not a good source of these seaweeds anymore because of nutrient contamination from fish farms. On Sun, Feb 13, 2011 at 2:15 PM, Justin Richmond C. Domingo wrote: Sir: I would like to ask how I can get seaweeds from MSI. I would also like to know if I will order directly from MSI or if I will go first in Bolinao to harvest the seaweeds that I need. If ordering will be preferred, how much does Laminaria, Gracilaria, and Gelidium cost.

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Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Friday, February 25, 2011, 9:02:35 PM Yes, you can use Gracilaria for the extraction of agar and Floridean starch. Sources of alginates are brown seaweeds. Please surf the Internet and look for pictures of tropical brown seaweeds. I am sure Sargassum is a very common seaweed. Just check first, before ordering Sargassum from Bolinao. To work in the lab, you have to inform your teacher and principal so that they can write a letter to our directress. Since you are not our student, there is a waiver that our lab is free from ligations. Furthermore, for the extraction of Floridean starch, our freeze dryer is not functioning anymore. We can just extract the agar. Come to think of it, why don’t you use gulaman bars of wafers sold in groceries as raw materials? Gulaman bars are food-grade agar. On Fri, Feb 25, 2011 at 12:28 PM, Justin Richmond C. Domingo wrote: Sir: I would like to ask if I can use Gracilaria for the extraction of both agar and Floridean starch if Gelidium will not be available. I would also like to ask if there are other sources of alginate which can be bought from the Farmers’ Market in Cubao. If none, can I just order Sargassum from MSI. Lastly, I would also like to ask for assistance for the extraction of the polysaccharides during my stay there in the coming summer when my letter of request will be approved.

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Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Sunday, February 27, 2011, 10:19:15 PM You can buy alginates from food suppliers. They are usually used in the making of dental impressions. I would not suggest the use of Sargassum to extract alginate because it produces very low-viscosity alginate and most likely degraded ones. They degrade easily. Try to look for Hormophysa triquetra. It produces high-viscosity alginate. Please ask your biology teacher on this seaweed. If ask dried Sargassum from us, we need money fro shipping charges from Tacloban to Manila. I would not advise drying the extract to recover the Floridean starch. The total volume of washings will be about five liters and you get only less than 5%. I suggest you surf the net for the procedure to extract the agar so we can discuss. Your teacher must be able to guide you on this. Even my undergraduate students come to me for advice not to get instructions and information. On Sun, Feb 27, 2011 at 12:23 PM, Justin Richmond C. Domingo wrote: Sir: I just want to ask if I can just dry the Floridean starch without using any freeze dryer. I would also like to know if the air-dried Floridean starch and the freeze-dried Floridean starch have differences. I would also like to know if there are some commercial alginates available in the market. If Sargassum will be used to extract the alginate, are there facilities in MSI used to extract alginate. Lastly, if I will be ordering Sargassum from Bolinao, how much would it cost?

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Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Sunday, March 6, 2011, 8:46:18 PM Just wait for the reply of our directress. We might be able to supply with Floridean starch and alginate but due to time constraints, you might just use agar and alginate from commercial sources. We will try to ask for sources of commercial alginates and agar. Usually a kilo would cost PhP 1,500.00. Floridean starch is not usually isolated because it is very soluble in water. You can use carrageenan but it might cost you more. Just make sure you have the procedures right. On Sun, Mar 6, 2011 at 5:38 PM, Justin Richmond C. Domingo wrote: Sir: I have already sent the letter of request to the directress of MSI. I am expecting that it will be approved by Dr. McGlone. If it will be approved, can I just buy your agar, alginate and Floridean starch supplies? How much would it cost? If you do not have supplies, what could be the substitute for the polysaccharides? Can I just use carrageenan also as a raw material? Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Monday, March 7, 2011, 7:12:34 PM The polysaccharides will be in powdered or wafer form. On Mon, Mar 7, 2011 at 4:58 PM, Justin Richmond C. Domingo wrote: Sir: Thank you for the information you have given me during my last consultation with you. It is okay if the Floridean starch is not isolated because it will also be dissolved in water. But the thing is that, how can I measure the amount of Floridean starch dissolved in the solution. If I will be buying the polysaccharides, will they come in powder form or in a solution? I think it would be better if they are in powder form.

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Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Tuesday, March 8, 2011, 7:19:57 PM I will give the estimates when I am back in Manila on Monday. Remember that we do not have Floridean starch. I was suggesting you that try carrageenan instead of Floridean starch. On Tue, Mar 8, 2011 at 4:24 PM, Justin Richmond C. Domingo wrote: Sir: I would like to order 700 grams of each polysaccharide—agar, alginate, and Floridean starch—that I will be using. If possible, I want it to be manufactured in your agency or any other agencies MSI can fully and truly trust. Since this is a research, it would be better if I will be using materials of high-quality. After delivery, I will also give the payment immediately. Since I will buy materials, can you please send me the cost analysis because I will also need it in my research. Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Wednesday, March 23, 2011, 7:46:23 PM After discussing with my staff and some of the faculty, we would like to know about the following: 1. methodology of your research 2. equipment and materials in your research (we might not have the equipment and materials you need in our lab) 3. duration of your stay in the lab 4. Will a faculty member going to supervise your experiment? On Tue, Mar 22, 2011 at 9:03 PM, Justin Richmond C. Domingo wrote: I would like to know how my letter of request going on is. I would like to hear some updates about it.

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Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Thursday, March 31, 2011, 8:27:22 PM One cannot just buy 700 grams of the polysaccharide. It has to be at least a kilo or more. I requested the supplier that we buy only a kilo each. They agreed since it is for research. Usually, they sell a 25 kg sack of the polysaccharide. I will go through your methodology this weekend and give you feedbacks. My trips to the south for carrageenan processing will take most of my summer vacation. In fact, most of my vacation. These are the prices of the polysaccharides. 1. agar – PhP 1,100.00 2. alginate – PhP 650.00 3. carrageenan – PhP 700.00 On Thu, Mar 31, 2011 at 2:14 PM, Justin Richmond C. Domingo wrote: Sir: As part of my research about the bioplastics from algal polysaccharides, I will no longer be conducting my research there. My research teachers suggested me that it would be better if I will just be ordering 700 grams of each polysaccharide from your agency. Another is that, one of the tests that I will be conducting is the biodegradability test and it will take me approximately six months. According to them, I must already start my research this coming April so that I can already finalize my data. They also said that I may be joining research contests next school year. I already sent you the copy of my research. Some parts have not yet been updated such as the use of carrageenan instead of Floridean starch and that the research will already be conducted in our residence house in the Science Lab of our school. This month, I am already planning to start my research. I have already informed you that I need 700 grams of agar, alginate and carrageenan. I think it would be better if they come already here after ten or 12 days, if possible. But the thing that concerns me most is the payment of the products. How would that work? By the way, my address is #3 Daisy St., Baptista Village, Calao East, Santiago City, Isabela 3311.

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Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Friday, April 1, 2011, 8:06:07 PM I will order the polysaccharides on Tuesday since my contact at Marcel Trading left for China yesterday. He should be back by Tuesday morning. My comments on your methodology are as follows: 1. You might like to dissolve or you can suspend the algal polysaccharides first. 2. Always remember that alginate gives a very viscous solution without heating. 3. When the Ca2+ ions touch base with alginate, this results in a gel that is irreversible even with heating. 4. Most of your examples of seaweeds are of temperate variety. 5. You might like to read about interactions of polysaccharides with other gums and ions. Good luck in! On Fri, April1, 2011 at 2:10 PM, Justin Richmond C. Domingo wrote: Sir: Okay, I will just order one kilo of each polysaccharide. But how could I give the payment for the products and when will you deliver them to me? Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Wednesday, April 6, 2011, 8:31:24 PM The polysaccharides will be delivered tomorrow and will be sent via LBC. If you have any PNB or LandBank account, you can deposit the reimbursements in my savings account. On Wed, Apr 6, 2011 at 9:21 AM, Justin Richmond C. Domingo wrote: Sir: How could I give the payment of the polysaccharides to you safely?

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From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Friday, April 8, 2011, 7:51:43 PM I already sent the algal polysaccharides early this afternoon. Please inform me when you get them. I will be in Cebu for the Philippine Chemistry Congress for a week. On Fri, Apr 8, 2011 at 9:37 AM, Justin Richmond C. Domingo wrote: Sir: I just want to know if you had already delivered the polysacchrides. Re: Seaweeds as Bioplastics From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Tuesday, April 12, 2011, 8:24:07 PM I will get to you on this next week. I am still in Cebu for the Chemistry Convention. On Tue, Apr 12, 2011 at 8:52 AM, Justin Richmond C. Domingo wrote: Sir: I already received them. Tomorrow, I will be paying the polysaccharides. May I ask your PNB account number, sir, so that I can deposit immediately the payment in your account. From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Friday, April 15, 2011, 8:16:42 PM I am still in Cebu for the Philippine Chemistry Congress. You may send the payment to my PNB account: Marco Nemesio Montaño PNB, UP Campus, Diliman, Q.C. S/A: 393041000022 Wishing your experiments go well! On Fri, Apr 15, 2011 at 6:31 AM, Justin Richmond C. Domingo wrote: Sir: Can I just ask again for your PNB account number?

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From: Cokie Montaño To: Justin Richmond C. Domingo Sent: Saturday, May 28, 2011, 8:45:12 PM I bought the polysaccharides from the Marine Resources Development Corporation, Inc. Through my friends there, I was able to buy less than a 25 kg sack. They sell only 25 kg of each polysaccharide. On Sat, May 28, 2011 at 12:42 PM, Justin Richmond C. Domingo wrote: Sir: May I just ask the name of the company where you bought the polysaccharides.

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