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.

23
 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 CS – Compost Soil

AG – Agar FW – Freshwater

AL – Alginate SW – Seawater

AR – Air AS – Activated Sludge

LS – Loam Soil C – Control Group

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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

X O1 O1
R O1 O2 O3 O4 O5 O6 O7 O8 O9 O12
1 0 1
X O1 O1
R O1 O2 O3 O4 O5 O6 O7 O8 O9 O12
2 0 1
X O1 O1
R O1 O2 O3 O4 O5 O6 O7 O8 O9 O12
3 0 1
X O1 O1
R O1 O2 O3 O4 O5 O6 O7 O8 O9 O12
4 0 1
X O1 O1
R O1 O2 O3 O4 O5 O6 O7 O8 O9 O12
5 0 1
X O1 O1
R O1 O2 O3 O4 O5 O6 O7 O8 O9 O12
6 0 1
X O1 O1
R O1 O2 O3 O4 O5 O6 O7 O8 O9 O12
7 0 1
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

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O5 – 5th observation on the 75th day of immersion

O6 – 6th observation on the 90th day of immersion

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 + AG +
FS AG AL FS + AG FS + AL AG + AL
AL
E1 E1 E1 E1 E1 E1 E1
E2 E2 E2 E2 E2 E2 E2
E3 E3 E3 E3 E3 E3 E3
FB FB FB FB FB FB FB
Fig 3.2. Paradigm of the Tensile Strength Test

Legend:

FS – Floridean starch E – Experimental Group

AG – Agar FB – Force that caused breakage or

AL – Alginate fracture

C – Control Group

26
 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.

Legend:

rz – radius of the zone of corrosion

Stirrer Az – zone of corrosion

Corrosive
Chemical
Drop rz
Az
Bioplastic Surface

Fig 3.3. Measuring of the Zone of Corrosion

HCl H2SO4 NaOH KOH

E1 E1 E1 E1
FS E2 E2 E2 E2
E3 E3 E3 E3
E1 E1 E1 E1
AG E2 E2 E2 E2
E3 E3 E3 E3
E1 E1 E1 E1
AL E2 E2 E2 E2
E3 E3 E3 E3
E1 E1 E1 E1
FS + AG E2 E2 E2 E2
E3 E3 E3 E3
E1 E1 E1 E1
FS + AL E2 E2 E2 E2
E3 E3 E3 E3

27
 E1 E1 E1 E1
AG + AL E2 E2 E2 E2
E3 E3 E3 E3
E1 E1 E1 E1
FS + AG + AL E2 E2 E2 E2
E3 E3 E3 E3
C1 C1 C1 C1
PE C2 C2 C2 C2
C3 C3 C3 C3
Fig.3.4. General Chemical Resistance Test Paradigm

Legend:

FS – Floridean starch H2SO4 – 1M Sulfuric acid

AG – Agar NaOH – 1M Sodium hydroxide

AL – Alginate KOH – 1M Potassium hydroxide

PE - Polyethylene C – Control Group

HCl – 1M Hydrochloric acid E – Experimental Group

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

28
 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

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

29
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).

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% Na 2CO3 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).

30
 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

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

31
 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, “Effect of

the Different Types of Substrates on the Biodegradability of SM Plastic Bags” (2008).

D.2.i. Preparation of the Substrates

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

32
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.

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.

33
 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

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

34
 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

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.

35
 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.

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

36
 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.

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

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

Tensile Strength Test

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

General Chemical Resistance Test

Preparation of the Corrosive Application of the
Gathering of Data
Chemicals Treatments

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

Analysis and Interpretation of Data using ANOVA

37
 Fig.3.5.General Research Method

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