Chapter III METHODOLOGY

A. Research Design In this research, the following bioplastics from different combinations of seaweed polysaccharides will be made as the main experimental unit: 1. 6 g Floridean starch + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 2. 6 g agar + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 3. 6 g alginate + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 4. 3 g Floridean starch + 3 g agar + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 5. 3 g Floridean starch + 3 g alginate + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 6. 3 g agar + 3 g alginate + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 7. 2 g Floridean starch + 2 g agar + 2 g alginate + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water These bioplastics will undergo three tests in which the bioplastics¶ effectivity and quality will be determined. Biodegradability, tensile strength and general chemical resistance tests are the test methods to be performed. For each test, Completely Randomized Design will be used as a method in the assigning of the bioplastics in the different treatments and the observation of results. Each treatment in the test will have three replicates to maintain accuracy and efficiency of data.

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For the biodegradability test, the rate of biodegradation and the percent weight loss are some of the parameters to be measured. This will be done by the immersion of the bioplastics into different substrates for a duration of 180 days. The paradigm of the assignment of the bioplastics into the different substrates is shown in Fig 3.1. AR LS CS FW SW AS C1 E1 E1 E1 E1 E1 FS C2 E2 E2 E2 E2 E2 C3 E3 E3 E3 E3 E3 C1 E1 E1 E1 E1 E1 AG C2 E2 E2 E2 E2 E2 C3 E3 E3 E3 E3 E3 C1 E1 E1 E1 E1 E1 AL C2 E2 E2 E2 E2 E2 C3 E3 E3 E3 E3 E3 C1 E1 E1 E1 E1 E1 FS + AG C2 E2 E2 E2 E2 E2 C3 E3 E3 E3 E3 E3 C1 E1 E1 E1 E1 E1 FS + AL C2 E2 E2 E2 E2 E2 C3 E3 E3 E3 E3 E3 C1 E1 E1 E1 E1 E1 AG + AL C2 E2 E2 E2 E2 E2 C3 E3 E3 E3 E3 E3 C1 E1 E1 E1 E1 E1 FS + AG + C2 E2 E2 E2 E2 E2 AL C3 E3 E3 E3 E3 E3 Fig. 3.1. Paradigm of the Assignment of the Bioplastics in the Different Substrates Legend: FS ± Floridean starch AG ± Agar AL ± Alginate AR ± Air LS ± Loam Soil CS ± Compost Soil 24 FW ± Freshwater SW ± Seawater AS ± Activated Sludge C ± Control Group E ± Experimental Group

Multiple Time Series Design with Randomization will also be used as a pattern of observing the changes happening in the bioplastics physically and with the use of the said parameters. A 15-day interval will be adapted in the design to clearly show the trend of biodegradation. Table 3.1 shows the paradigm of the application of Multiple Time Series Design with Randomization in the observation of results in the biodegradability test.

Table 3.1. Multiple Time Series Design with Randomization R R R R R R R X1 X2 X3 X4 X5 X6 X7 O1 O1 O1 O1 O1 O1 O1 O2 O2 O2 O2 O2 O2 O2 O3 O3 O3 O3 O3 O3 O3 O4 O4 O4 O4 O4 O4 O4 O5 O5 O5 O5 O5 O5 O5 O6 O6 O6 O6 O6 O6 O6 O7 O7 O7 O7 O7 O7 O7 O8 O8 O8 O8 O8 O8 O8 O9 O9 O9 O9 O9 O9 O9 O10 O10 O10 O10 O10 O10 O10 O11 O12 O11 O12 O11 O12 O11 O12 O11 O12 O11 O12 O11 O12

Legend: R ± Randomization X ± Immersion of the bioplastics into the different substrates O1 ± 1st observation on the 15th day of immersion O2 ± 2nd observation on the 30th day of immersion O3 ± 3rd observation on the 45th day of immersion O4 ± 4th observation on the 60th day of immersion O5 ± 5th observation on the 75th day of immersion O6 ± 6th observation on the 90th day of immersion 25

O7 ± 7th observation on the 105th day of immersion O8 ± 8th observation on the 120th day of immersion O9 ± 9th observation on the 135th day of immersion O10 ± 10th observation on the 150th day of immersion O11 ± 11th observation on the 165th day of immersion O12 ± 12th observation on the 180th day of immersion For the tensile strength test, the tensile strength and the force vs. elongation curve will be determined to see if the bioplastics are strong and durable enough to withstand a tensile force. Fig. 3.2 shows the design of the experiment to be done in this test.

FS E1 E2 E3 FB

AG E1 E2 E3 FB

AL

FS + AG

FS + AL

AG + AL

FS + AG + AL E1 E2 E3 FB

E1 E1 E1 E1 E2 E2 E2 E2 E3 E3 E3 E3 FB FB FB FB Fig 3.2. Paradigm of the Tensile Strength Test

Legend: FS ± Floridean starch AG ± Agar AL ± Alginate C ± Control Group For the general chemical resistance test, the zone of corrosion will be measured so that the bioplastics ability to resist corrosive chemicals can be evaluated. Fig. 3.3 shows the process of dropping the chemicals onto the surface of the bioplastic to create the zone of corrosion. Fig. 3.4 shows the design of the experiment to be used in this test. 26 E ± Experimental Group FB ± Force that caused breakage or fracture

Legend: rz ± radius of the zone of corrosion Stirrer Corrosive Chemical Drop Bioplastic Surface Az ± zone of corrosion

rz

Az

Fig 3.3. Measuring of the Zone of Corrosion HCl H2SO4 NaOH E1 E1 E1 E2 E2 E2 E3 E3 E3 E1 E1 E1 E2 E2 E2 E3 E3 E3 E1 E1 E1 E2 E2 E2 E3 E3 E3 E1 E1 E1 E2 E2 E2 E3 E3 E3 E1 E1 E1 E2 E2 E2 E3 E3 E3 E1 E1 E1 E2 E2 E2 E3 E3 E3 E1 E1 E1 E2 E2 E2 E3 E3 E3 C1 C1 C1 C2 C2 C2 C3 C3 C3 Fig.3.4. General Chemical Resistance Test Paradigm 27 KOH E1 E2 E3 E1 E2 E3 E1 E2 E3 E1 E2 E3 E1 E2 E3 E1 E2 E3 E1 E2 E3 C1 C2 C3

FS

AG

AL

FS + AG

FS + AL

AG + AL

FS + AG + AL

PE

Legend: FS ± Floridean starch AG ± Agar AL ± Alginate PE - Polyethylene HCl ± 1M Hydrochloric acid B. Research Environment The study will be conducted in the Marine Science Institute of the University of the Philippines-Diliman Campus (UP-MSI) and in the Science Laboratory of the Philippine Science High School-Cagayan Valley Campus (PSHS-CVC) from April to October 2011. The materials needed to perform the study can be found in these agencies and some of the consultants of this research are the members of the faculty and staff of the agencies. C. Materials and Equipment Most of the materials to be used in this research will be borrowed from the Marine Science Institute of the University of the Philippines-Diliman Campus and the Philippine Science High School-Cagayan Valley Campus. These agencies are believed to have the best technologies and apparatus that can be used in this study. For the making of the bioplastics, seaweeds will be the main material to be used. The polysaccharides will be extracted using different solvents depending on the H2SO4 ± 1M Sulfuric acid NaOH ± 1M Sodium hydroxide KOH ± 1M Potassium hydroxide C ± Control Group E ± Experimental Group

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polysaccharide to be extracted. Cooking materials like spatula, casserole and stove will also be used in making the bioplastics. Weighing scales will be used in getting the mass of the ingredients needed to make the bioplastics and the mass of the bioplastics during experimentation. Graduated cylinders will be used for getting volumes and vernier caliper for lengths. Aluminum trays will be used to contain the different substrates. An Instron machine will be used in getting the tensile strength of the bioplastics. Other necessary materials needed to perform the research are further discussed in the Research Method. D. Research Method This research about the development of a biomass-based plastic from the natural polysaccharides of the selected Philippine marine seaweeds will be done using the procedures stated below. D.1. Bioplastic Making D.1.i. Harvesting the Seaweeds The seaweeds ± Gracilaria, Gelidium and Laminaria ± will be bought from the Farmer¶s Market in Cubao, Quezon City using Convenience Sampling. The seaweeds will be soaked in a saline solution and will be kept refrigerated first at a constant temperature of 20 to 25 oC. This will be used to maintain the freshness of the seaweeds. D.1.ii. Extraction of Polysaccharides The following procedures on how to extract the natural polysaccharides from seaweeds were based from the extraction processes of Prof. Marco Nemesio Montaño (2010).

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For the extraction of the agar, 25 g of Gracilaria will be cooked in 5% NaOH solution in 90 degrees Celsius for an hour. The seaweed will then be washed in running tap water and it will be soaked in 750 mL of 0.5% HOAc for an hour. It will be washed again and will be boiled in one liter of distilled water. It will be blended and filtered in a filter bomb. The filtrate will be collected and will be frozen overnight. The next day, the gel will be thawed and the agar wafer will be dried. For the extraction of the alginate, 100 g of Laminaria will be soaked in a 0.1 M HCl solution overnight. It will be washed in a liter of 1% Na2CO3 solution. The seaweed will be blended and filtered in a filter bomb. The filtrate will be collected and precipitated with IsprOH that must be three times the volume of the filtrate. The gel will be dried and grinded. For the extraction of the Floridean starch, the Gelidium will be boiled in a liter of distilled water until the color of the water changes. The filtrate that settled at the bottom will be collected using a sieve. It will then be dried. D.1.iii. Preparation of the Bioplastics The method that will be used in making the bioplastics were based from a scientific blog of Brandon Sweeney (2008). The following bioplastic solutions below will be made by mixing the said ingredients thoroughly until a single phase is achieved. 1. 6 g Floridean starch + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 2. 6 g agar + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 3. 6 g alginate + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water

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4. 3 g Floridean starch + 3 g agar + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 5. 3 g Floridean starch + 3 g alginate + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 6. 3 g agar + 3 g alginate + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water 7. 2 g Floridean starch + 2 g agar + 2 g alginate + 5 mL glycerol + 5 g plaster of Paris + 60 mL distilled water Since there will be three replicates for each treatment, it would be better if they will be distinguished using a color code than by labeling them using a marker. For the first replicate of the bioplastics, 2.5 g of yellow food coloring will be added to the solution, blue food coloring for the second and red food coloring for the third. Each bioplastic solution will be put into a separate casserole and will be heated until a translucent viscous gel is formed. The gel will then be poured into a mold and will be put in a warm place for a day to dry. The following day, the bioplastics will be gathered and will be cut into 2´ x 3´ plastic strips. The bioplastics will be kept into a closed container until the tests will proceed to prevent it from biodegrading naturally. D.2. Biodegradability Test The following procedures to be made in the biodegradability test were based from the research of Julian Paolo Biyo and Jason Andrei Temelo entitled, ³Effect of the Different Types of Substrates on the Biodegradability of SM Plastic Bags´ (2008). D.2.i. Preparation of the Substrates

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Some of the substrates to be used in the biodegradability test such as loam soil, fresh water and sea water will be gathered from the appropriate places where they can be found. For the other substrates like compost soil and activated sludge, they will be made in the laboratory. The compost soil will be done by adding crushed, dried seaweed into the loam soil in a 1:4 ratio. On the other hand, the activated sludge will be made by mixing fresh water into the loam soil in a 2:3 ratio. Five hundred grams of a single substrate will be put into seven separate 7´ x 9´ aluminum trays. The reason why there are seven containers is that there are seven different types of bioplastics to be experimented and evaluated. Since there are five different substrates, a total of 35 containers will be used. Each container will have three different colors of a single kind of a bioplastic placed side by side. For the experimental group of this test, a total of 105 bioplastic strips will be used. For the control group of the biodegradability test, which is the exposure of the different bioplastics in the air, the plastic strips will be pinned in the four corners using sterilized thumb tacks. The strips will be placed horizontally in a wooden table. A total of 21 bioplastic strips will be used in the control group and for this test, 126 strips all in all will be used. Fig. 3.1 also shows the summary of the preparation of the substrates and the assignment of the bioplastics into the different substrates which includes the labels of the containers and the design of the biodegradability test. D.2.ii. Gathering of Data Before the bioplastics will be immersed into the different substrates, the several plastic strips will be weighed first using a weighing scale. This will be the initial weight (Wi) of the bioplastic.

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Every 15 days, the bioplastics will be weighed again. This will be the dry weight (Wf) of the bioplastic. There will be 12 dry weights to be measured. Before weighing, the plastic strips will be washed first in tap water and will be air dried so that foreign matters will be removed. Table 3.1 also shows the design to be used in the observation of the changes that will happen to the bioplastics during the biodegradation process. Using the initial weight and the dry weight of the plastic strips, the amount degraded, amount left and the percent weight loss can be calculated for a certain period of time. amount degraded = initial weight (Wi) ± final weight (Wf) amount left = initial weight (Wi) ± amount degraded percent weight loss = ( amount degraded / initial weight (Wi) ) * 100% Using these data also, the mean of the parameters can be also computed using the formula for average. When the bioplastics have passed the specification designated by the American Society for Testing Materials with regards to biodegradability, the bioplastics can now be considered biodegradable. D.3. Tensile Strength Test Using an Instron machine, the tensile strength of the bioplastics can be determined. The bioplastics will be placed in the claws of the machine and the Instron will generate a force that increases from time to time. The force will continue to increase until the sample breaks or fractures. This process will be repeated for three times and that

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correlates to the number of replicates which is three. There will be 21 bioplastics to be used in this test . Fig. 3.2 also shows the design used in this test. After the test, the machine will also generate a force vs. elongation curve. This will be used to determine the tensile strength profile of the bioplastic and the polyethylene plastics. The interpretation of the curve is discussed in Chapter II. To get the tensile strength, the force that caused the breakage or fracture will be divided by the original cross-sectional area of the plastic. Using the data gathered from the treatments, the mean tensile strength of the bioplastic can also be computed using the average formula. The tensile strength of the bioplastics will then be compared with the tensile strengths of the conventional plastics in Table 2.1 to see for the suitable applications of the bioplastics. D.4. General Chemical Resistance Test The following procedures to be done in the general chemical resistance test were based from the corrosion test methods of the Division of Alabama Specialty Products, Inc. (2011). D.4.i. Preparation of the Corrosive Chemicals Four corrosive chemicals will be used in this test. These are hydrochloric acid, sulfuric acid, sodium hydroxide and potassium hydroxide. A concentration of one molar will be prepared by adding one mole of the substance in a liter of distilled water. The solutions will be stirred thoroughly until a single phase is achieved. D.4.ii. Application of Treatments

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Using a glass stirrer, a drop of the corrosive chemical will be placed onto the surface of the bioplastic and the polyethylene plastic. This process is shown in Fig 3.3 and in Fig. 3.4, the design of the general chemical resistance test can be seen. For each chemical, 21 bioplastics and three polyethylene plastics will be used. All in all, 84 bioplastics and 12 polyethylene plastics will be used in this test. D.4.iii. Gathering of Data Before the chemicals will be dropped onto the surface of the plastics, the plastics will be weighed first using a weighing scale. This will be the initial weight (Wi) of the plastic. After the process of dropping the chemicals, the surface of the plastic will be corroded leaving a zone of corrosion. Its shape is like a circle and so its area can be calculated by getting the radius of the circle using a vernier caliper and evaluating it by the area formula of a circle. The plastics will be weighed again and this will be the final weight of the plastic (Wf). Using the initial and final weights of the plastics, the amount corroded, amount left and the percent weight loss can also be computed. amount corroded = initial weight (Wi) ± final weight (Wf) amount left = initial weight (Wi) ± amount corroded percent weight loss = ( amount corroded / initial weight (Wi) ) * 100% Using the data from the replicates, the mean of the parameters can also be computed using the formula for average. The data gathered from the bioplastics and the polyethylene plastics will be compared for comparison and to determine if there are significant differences.

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D.5. Disposal Method All the materials that will be used in this research will be washed first in tap water before disposal. The materials will be disposed according to the disposal method designated by the local municipal government. They will be segregated in terms of their biodegradability. The bioplastics will be composted and the corrosive chemicals will be thrown away with the supervision of an expert. Other equipment to be used in this research like those that will be used in making the bioplastics and the preparation of the corrosive chemicals will be sterilized using an auto clave after use.

E. Statistical Tool One-way Analysis of Variance (ANOVA) will be the main statistical tool to be used in this study. The significant differences of the parameters can be determined using this tool. a significant difference of 0.50000 will be used in this study. With the help of one-way ANOVA, the inferential problems can be answered.

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Bioplastic Making Gathering of Seaweeds Extraction of the Polysaccharides Preparation of the Bioplastics

Biodegradability Test Preparation of the Substrates Application of the Treatments Gathering of Data

Tensile Strength Test Placement in the Instron Machine Analysis of the Force vs. Elongation Curve

General Chemical Resistance Test Preparation of the Corrosive Chemicals Application of the Treatments Gathering of Data

Disposal Method Segregation of Waste Matters Composting of the Used Bioplastics Sterilization of Used Equipment

Analysis and Interpretation of Data using ANOVA

Conclusion Making

Fig.3.5.General Research Method

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