Sample IMRAD

Biosorption Capacity of Chaetoceros calcitrans and Tetraselmis tetrahele on
Iron and Manganese
INTRODUCTION
Contamination of water supplies with metals is an area of concern both national
and international where the challenge to remediate is a must since metals are natural
components of the Earth's crust and are found in the environment only in moderate
amounts. However, “natural” contaminants can also have anthropogenic origins: in fact
human activities often cause the release of a large amount of naturally-occurring minerals
into the environment (Pinsino et. al, 2010). This is usually due to pollution related to
construction or iron and manganese mining.
According to the data of the Potential Minerals in Western Visayas, Capiz is
abundant in metals such as gold (Cuartero & Maayon), copper (Dumalag & Pilar),
chromite (Dumalag), manganese (Ivisan) and iron (Roxas City & Ivisan), such garnered
attention to mining industries and paved way for local officials to support for the “general
conduct of mining operation by the legally permitted mining company and was
strengthen by the proclamation of Provincial Ordinance (PO) No. 9 lifting of the 50-year
moratorium on large-scale mining in the province of Capiz. Such activity will make water
contamination inevitable.
Capiz is dubbed as the "Seafood Capital of the Philippines". It holds one of the
richest fishing grounds and is a major contributor in the aquamarine industry of the
Philippines. Farming and fishing are the primary sources of income for the people. The
combined natural bounty of land and sea sustain a vibrant food industry.
Biosorption Capacity of 2
In agricultural industry, iron and manganese serves as a micronutrient because
only small amounts are required to aid in normal plant growth. Plants do secrete acids
from the roots and when combined with too much iron, it lowers the pH of the soil.
Bronzing and stippling of leaves is a sign of toxicity among plants. Excessive manganese
concentrations in plant tissues can alter various processes, such as enzyme activity,
absorption, translocation and utilization of other mineral elements (Ca, Mg, Fe and P),
causing oxidative stress (Ducic and Polle, 2005; Lei et al, 2007).
Higher levels of iron do not dissolve in water. Fish and other creatures cannot
process all the iron they take in from water or their food. The iron can build up in
animals' internal organs, eventually killing them. Higher levels of iron in fish and aquatic
plants also have negative effects on the people or creatures consuming them. Marine
organisms can take up from the sea and from their diet these metals, which may consist
of particles in suspension or might be deposited in the sediment. Trace essential metals
are of environmental interest both as limiting nutrients (Fe, Zn, Mn, Cu, Co, Mo and Ni),
playing important roles in metal-requiring and metal-activated enzyme systems, and as
toxicants when present at high concentrations.
The presence of iron in potable water is rather unpleasant due to the bad odors it
spreads, its rusty taste and color, its feel on skin and hair, and its tendency to stain
clothing (Wageningen Univ., 2016). Furthermore, in humans, consuming large amounts
of iron can lead to a condition known as iron overload which when left untreated can lead
to hemochromatosis, a severe disease that can damage the body’s organs and gene
mutation (Garvin, 2015).

Excess manganese interferes with the absorption of dietary iron resulting to iron-
deficiency anemia. Symptoms of toxicity mimic those of Parkinson's disease (tremors,
stiff muscles) and excessive manganese intake can cause hypertension in patients older
than 40. Significant rises in manganese concentrations have been found in patients with
severe hepatitis and posthepatic cirrhosis, in dialysis patients and in patients suffering
heart attacks. Several studies have linked excessive manganese exposure may lead to
neurological disorders in children (Blaurock-Busch, 1997).
The search for new technologies involving the removal of toxic metals from waste
waters has directed attention to biosorption, based on the metal binding capacities of
various biological materials. Developments in the environmental biotechnology indicate
that bacteria, actinomycetes, fungi, yeasts, algae and seaweeds can remove heavy metals
from aqueous solutions by biosorption (Hydrometallurgy, 1997). In recent years, there
has been increasing interest in the use of biomass from microbial sources (Ahluwalia et.
al., 2007), particularly the microalgae to absorb heavy metal ions as part of remediation
efforts (Davis et. al, 2003) (Chojnacka et. al., 2004). The key advantage of micro algal
biomass is the wide range of functional groups present on variety of macromolecules,
including polysaccharides, proteins, peptides and nucleic acids, which accounts for the
majority of metal adsorption by ion- exchange mechanisms (Skowronski et. al., 2000)
(Mehta & Gaur, 2005). The scientific community has shown great interest in this
technique in view of the results obtained. Moreover, Algae are of special interest in the
search for and development of new biosorbents materials due to their high sorption
uptake and their ready availability in practically unlimited quantities in the seas and
oceans (Feng and Aldrich, 2004).
Tetraselmis tetrahele is a green microscopic algae with four flagellated
prasinophyte characterized by an ovoid body shape and a distinct curved body when
viewed sideways. The alga measures 10 – 16 µm in length, 8 - 11 µm in width and 4.2 –
5.0 µm in thickness (De la Peña & Franco, 2013). On the other hand,
Chaetoceros calcitrans has a cylindrical shape and a more or less equal extent in all
directions, and is a needle-shaped alga (Helcom, 2016). Since both are considered as
microalgae, generally, this study aims to find out the biosorption capacity of Chaetoceros
calcitrans and Tetraselmis tetrahele on Iron and Manganese. Specifically, it aims to
determine if there is significant difference in the amount of iron (mg/L) adsorp after 48
hours of exposure to the different treatments, to determine if there is significant
difference in the amount of manganese (mg/L) adsorp after 48 hours of exposure to the
different treatments and if there is a significant difference between the amount of iron and
manganese (mg/L) adsorp after 48 hours of exposure to the different treatments.
MATERIALS AND METHODS
Sterilizing Materials and Tools
All the materials used in this study, including all cleaned glasswares were packed
individually with old newspapers and were sterilized in an autoclave at 121ºC for 45 min
at 15 psi. while the plastic containers were cleaned thoroughly and were disinfected using
70% alcohol.
Preparing the Laboratory
The laboratory was cleaned and disinfected to ensure cleanliness.
Collection of Microalgae
Tetraselmis tetrahele and Chaetoceros calcitrans were obtained at the Phycology
Laboratory of SEAFDEC, Tigbauan, Iloilo.
Algal Culture
Eighteen (18) plastic containers were prepared. Each of the 9 plastic containers
was filled with 50 mL of Chaetoceros calcitrans and another 9 plastic containers was
filled with 50 mL Tetraselmis tetrahele. A beaker was used to measure the test samples
respectively. With the use of a pipette with tip, 0.5 mL of FeCl 3, Na2SiO3, Trace metal,
Vitamins stock, NaNo3, NaH2PO4, and Na2 EDTA was measured and added to every
container.
Figure 1: Measuring and Pouring of Microalgae

Photo taken by Ms. Lima Jocson
Microalgae Cell Count
Using pipette with tip 1 mL of each sample in every container were gathered and
placed in a clean glass slide and were sent to the laboratory. Using a light microscope, the
microalgae were analyzed and counted. The microalgae were allowed to grow for 7 days.
After 7 days a microalgae cell count was done again to insure growth and equal number
of microalgae present in every container. After the desired algal count was determined, it
was transported to the Chemistry Laboratory (ESEP Bldg) of Capiz National High
School, Roxas City.
Figure 2: Chaetoceros calcitrans Figure 3: Tetraselmis tetrahele

Image copyrights: Mona Hoppenrath Photo taken from: http://en.aquaculture.ifremer.fr/
Procurement of Iron and Manganese
100 mL each of iron chloride (1.25g/125 mL) and manganese sulfate (1.25g/125
mL) were purchased from the Central Science Laboratory of West Visayas State
University from Mr. Audie Suladay, Head of the Central Science Laboratory.
Experimental Design
The Pretest- Posttest design was used in this investigation. Spectrophotometry
analysis was used to determine the biosorption capacity of Chaetoceros calcitrans and
Tetraselmis tetrahele.
Pre Testing the Water Sample
Dilution of 1.25 mL iron/manganese in 498.75 mL Ozone Seawater was prepared
in all replicates. Ten (10) mL was taken from every sample and was placed in a sterile
plastic container where it was sent to MRWD Laboratory for spectrophotometry analysis
using the spectrophotometer machine (Spectronic 20, Milton-roy company).
Preparation of the Test Samples
The 490 mL water samples obtained in the pre testing was then added to the
prepared algal culture samples which contains 0.5 of each fertilizers + 50 mL of
Chaetoceros calcitrans and Tetraselmis tetrahele test sample respectively.
Preparation of the Different Treatment
Iron chloride and manganese sulfate were the 2 metals use in the study. There
were 2 treatments and a control: Treatment A. 50 mL Chaetoceros calcitrans + 490 mL
Ozonated seawater with Iron chloride / manganese sulfate (50 ppm) and Treatment B: 50
mL Tetraselmis tetrahele + 490 mL Ozonated seawater with Iron chloride / manganese

sulfate (50 ppm) and a Control - 500 mL Ozonated seawater. All treatments were done
in 3 replications and were treated for 48 hours.
Control Control
TB TB
TA TA
Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
Iron Manganese
Figure 4: Different Treatments

Photo taken by the Researcher
Gathering of Data
After 48 hours, the microalgae were separated from the solution using a 4x4 filter
paper. Ten (10) mL of each treated sample were transferred to sterile plastic containers.
The samples were sent to MRWD for spectrophotometry analysis using the
spectrophotometer machine (Spectronic 20, Milton-roy company).
Figure 5: Treated Samples after 48 Hours

Photo taken by the Researcher
Spectrophotometry Analysis
10 mL of each sample were transferred to a test tube. The test tube with iron and
manganese solution samples were subjected for spectrophotometry analysis using a
spectrophotometer machine (Spectronic 20, milton roy company), where one Iron MR no
1 tablet, one Iron MR no 2 tablet, one Manganese no 1 tablet and one Manganese no 2
tablet were crushed and mixed to dissolve in the treated samples. The samples were
allowed to stand for 10 minutes (Iron solution) and 20 minutes (Manganese solution) to
allow color development. Phot 39 (Iron) and Phot 20 (Manganese) were then selected in
the spectrophotometer. The results were then displayed as mg/L Fe and mg/L Mn.
The difference in the pre-test and post-test results were utilized as quantitative
data organized in table and were statistically evaluated.
Statistical Analysis of Data
One Way Analysis of Variance (ANOVA) and DMRT was utilized to determine
the comparisons between treatments and the control.
RESULTS
The means of the microalgae were compared to determine which of the samples
have the highest biosorptive capacity for Iron. Table I, showed that Treatment A:
Chaetoceros calcitrans got the highest mean of 0.1286 mg/L, followed by Treatment B:
Tetraselmis tetrahele with a mean of 0.115667 mg/L and the Control with a mean of
0.0000mg/L.
ANOVA at 5% level of significance, results revealed that the p-value (0.000) is
less than the α(0.05) thus rejecting the null hypothesis., therefore there is a significance
difference in the amount of Iron (mg/L) adsorp after 48 hours of exposure to
Chaetoceros calcitrans and Tetraselmis tetrahele. Moreover, Duncan's Multiple Range
Test has shown that the difference lies between Treatment A, Treatment B, and the
Control.
Table 1. Difference (mg/L) in the Amount of Iron Adsorp after 48 Hours of Treatment
Difference in (mg/L)
Replicates
Treatments 1 2 3 Mean ± SD
TA: Chaetoceros
0.128 0.126 0.132 0.1286a±0.0030
calcitrans
TB: Tetraselmis
0.111 0.117 0.119 0.115667b±0.004163
tetrahele
Control:
0 0 0 .000000c±.000000
Ozonated water
0.000 significant
*Mean with different letter superscript indicates significant difference
The means of the microalgae were compared to determine which of the samples
got the highest biosorptive capacity for Manganese. Table 2, showed that Treatment A:
Chaetoceros calcitrans got the highest mean of 0.02667 mg/L, followed by Treatment
B: Tetraselmis tetrahele, with a mean of 0.02317 mg/L and the Control with a mean of
0.0000mg/L.
ANOVA at 5% level of significance, results revealed that the p-value (0.0001) is
less than the α(0.05) thus rejecting the null hypothesis, therefore there is a significance
difference in the amount of Manganese (mg/L) adsorp after 48 hours of exposure to
Chaetoceros calcitrans and Tetraselmis tetrahele. Furthermore, Duncan's Multiple Range
Test has shown that the difference lies between Treatment A, Treatment B, and the
Control.
Table 2. Difference (mg/L) in the Amount of Manganese Adsorp after 48 Hours of

Treatment
Difference in (mg/L)
Replicates
Treatments 1 2 3 Mean ± SD
TA: Chaetoceros
0.0271 0.0259 0.027 0.02667a±0.0006658
calcitrans
TB: Tetraselmis
0.0249 0.0246 0.02 0.02317b±0.002747
tetrahele
Control: Ozonated
0 0 0 .000000c±.000000
water
0.000 significant
Table 3 showed that in the Iron (mean ± SD) column, Chaetoceros calcitrans got
the highest mean of all treatments with a value of 0.1290 mg/L, followed by Tetraselmis
tetrahele with a value of 0.1157 mg/L and the control got the lowest mean with value of
0.0000mg/L. While in the Manganese (mean ± SD) column, it showed that Chaetoceros
calcitrans got the highest mean of all treatments with a value of 0.02667 mg/L, followed
by Tetraselmis tetrahele with a value of 0.02317 mg/L and the control got the lowest
mean with value of 0.0000mg/L. Duncan’s Multiple Range Test has shown that the
difference lies between Treatment A (Iron), Treatment B (Iron), Treatment A
(Manganese), Treatment B (Manganese) and the Controls.
Table 3. Comparison of Iron and Manganese Biosorptive Capacity of Chaetoceros

calcitrans & Tetraselmis tetrahele.
Treatments Iron (mean ± SD) Manganese(mean ± SD)
Chaetoceros calcitrans 0.1290a±0.0030 0.02667a±0.0006658
Tetraselmis tetrahele 0.115667b±0.004163 0.02317b±0.002747
Control .000000c±.000000 .000000c±.000000
DISCUSSION
Biosorption is a technique that can be used for the removal of pollutants from
waters, especially those that are not easily biodegradable such as metals and dyes. A
variety of biomaterials are known to bind these pollutants, including bacteria, fungi,
algae, and industrial and agricultural wastes (Elsevier Inc., 2008).

Based on the results presented in tables 1, 2 and 3, there was a decrease in the
amount of Iron and Manganese after 48 hours of exposure especially using Chaetoceros
calcitrans. On the other hand, Tetraselmis tetrahele showed also biosorption potential.
The result was so promising since microalgae has the ability to tolerate those metals, has
high yields of recovery per unit mass and has a high specific outer area coupled with a
cell wall loaded with ionisable groups (Gakwisiri et. al, 2012).
Furthermore, microalgae has proven to possess high metal binding capacities due
to the presence of polysaccharides, proteins or lipid on the surface of their cell walls
containing some functional groups such as amino, hydroxyl, carboxyl and sulphate,
which can act as binding sites for metals (Priyadarshani et al, 2011).
Microalgae uptake metal, either actively (bioaccumulation) and/or passively
(biosorption)(Fourest and Roux, 1992). This is due to affinity of algal surfaces for heavy
metals leading to their adsorption and precipitation. The biosorption is passive non-
metabolic process of binding various chemicals on biomass (Volesky, 1990).
As the biosorption process involves in mainly cell surface sequestration, the
modification of cell wall can greatly alter the binding of metal ions. A number of
methods have been employed for cell wall modification of microbial cells in order to
enhance the metal binding capacity of biomass and to elucidate the mechanism of
biosorption (Monika, et. al., 2014). Of the many types of biosorbents recently
investigated for their ability to sequester heavy metals, microalgal biomass has proven to
be highly effective as well as reliable and predictable in the removal of heavy metals
from aqueous solutions (Priyadarshani et al, 2011).

According to Yalcin et al., (2007), algae are able to eliminate different forms of
heavy metals by chelating, making complexes, catalyzing or adsorption. Given a longer
time interval, more amounts of Iron and Manganese will be absorbed by the microalgae
as shown in the differences in the result of the samples in 48 hours of exposure.
CONCLUSION
Based from the data gathered, Chaetoceros calcitrans and Tetraelmis tetrahele
showed biosorption capacity on Iron and Manganese, thus, these two microalgae can be
used as a good phycoremediator on water contaminated with metals.
RECOMMENDATIONS
1. The Researcher recommends the use of Chaetoceros calcitrans and Tetraselmis
tetrahele on Iron and Manganese contaminated water.
2. The Researcher recommends allotting a longer time interval in the exposure of Iron
and Manganese on both microalgae.
3. Use other microalgae in treating Iron and Manganese contaminated water.
4. Test Chaetoceros calcitrans and Tetraselmis tetrahele in treating water contaminated
by other metals.
5. Design techniques in sequestering metals and its application in the future.
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fir (Pseudotsuga menziesii var. viridis and glauca) seedlings as affected by
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Bristol, England: Biopress”, Retrieved May 28, 2016
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ACKNOWLEDGEMENT
The researcher would like to extent their heartfelt gratitude to the following
persons for their support throughout the study:
To the almighty God, for giving the Researcher the strength and courage to finish
this study;
To Mrs. Ruth B. Barrios, Principal III, for allowing the Researcher to conduct this
study;
To Miss Lima Q. Jocson and Mrs. Selma P. Equibal, Research Advisers, for their
constructive criticisms and unceasing support during the conduct of the study;
To Engr. Wilhelmina S. Co, Research Consultant, for sharing her expertise and
insights;
To Dr. Evelyn Grace Ayson, Head of Research Division, SEAFDEC Tigba-uan,
Iloilo for allowing the Researcher to purchase the microalgae; Mrs. Kim Caumban,
Secretary of the Research Department for facilitating letter of the Researcher and Mrs.
Annie V. Franco, Senior Technical Assistant, of the Phycology Department for the help
extended in preparing and assisting the Researcher during the algal culture and for
making it possible to transport the microalgae;
To Mr. Audie Suladay, Head of Central Laboratory of West Visayas State
University for allowing the Researcher to purchase Iron and Manganese in aqueous metal
solution and for solving the amount needed for the study;
To Mr. Zaldy A. Lacson, Division Manager B- Water Resource Division of
MRWD the use of the laboratory and its equipment and Mrs. Victoria Pabelonia, Quality
Control Officer, for extending her precious time and expertise in testing the water
samples using spectrophotometer;
To Mrs. Judy B. Dunton, SSHT V- Science and Technology Department for
allowing the Researcher to use the Chemistry laboratory and its equipment during the
conduct of the study;
To Nong Jalem Paguiton and Nong Paul Ferrandiz, for helping out transport the
Microalgae;
To the Researcher’s parents, for their unconditional love, encouragement and
financial support.
Thank you very much.
The Researcher
APPENDICES
A. Raw Data and Statistical Analysis
B. Letters
C. Scanned Logbook
Appendix A
Raw Data and Statistical Analysis
Date of Sampling : 15-Aug-2016

Time of Sampling : 11:00 a.m.
Date of Analysis : 15-Aug-2016
Time of Analysis : 3:00 p.m.
Source : Ozonated water with Iron
Requested by : VIA FAITH S. BOLIDO
Amount of Iron (mg/L) in Water samples
Samples Parameters PNSDW Unit Results

Sample 1 Iron 1 mg/L 0.14

Source : Ozonated water with Manganese
Amount of Manganese (mg/L) in Water samples

Sample 1 Manganese 0.4 mg/L 0.03

Source : Ozonated water
Ozonated Water samples

Sample 1 mg/L 0.02
Sample 2 mg/L 0.02
Sample 2 mg/L 0.02

Source : Ozonated water with Iron (Chaetoceros calcitrans)


Source : Ozonated water with Iron (Tetraselmis tetrahele)


Source : Ozonated water with Manganese (Chaetoceros calcitrans)


Source : Ozonated water with Manganese (Tetraselmis tetrahele)
Amount of Manganese (mg/L) in Water samples


Source : Ozonated water
Ozonated Water samples

Sample 1 mg/L 0.02
Sample 2 mg/L 0.02
Sample 2 mg/L 0.02
Table 1. ANOVA Table on the Difference (mg/L) in the Amount of Iron after 48 Hours of
Treatment
TA 0.13 0.13 0.13 MEAN= 0.1287
TB 0.11 0.12 0.12 MEAN= 0.1157
(-) 0.00 0.00 0.00 MEAN= 0.0000
******************************************************************************
Treatment Mean
TA 0.1287
TB 0.1157
(-) 0.0000
******************************************************************************
: Sirichai Statistics Version 6.00 :
09-07-2016 20:17:48
Problem Identification: Procedure : Analysis of Variance I
---------------------------------------------------------------------------
Table.... Analysis of Variance
Source df SS MS F F.05 F.01 F-Prob
Treatment 2 0.0301 0.0151 1693.29 5.14 10.92 0.0000

Ex.Error 6 0.0001 0.0000
Total 8 0.0302 0.0038
GRAND MEAN = 8.14444455835554E-02
CV = 3.6607 %
LSD .05 = 5.95678586049769E-03
LSD .01 = 9.02403154265016E-03
*********************************************************
* DUNCAN'S MULTIPLE-RANGE TEST *
*PROBLEM IDENTIFICATION= *
*NUMBER OF MEANS= 3 *
*ERROR DEGREE OF FREEDOM= 6 *
*ERROR MEAN SQUARE= 8.88888518016153E-06 *
*STANDARD ERROR OF MEAN= 1.72132557255172E-03
***********************************************************
NAME ID MEAN RANKED AT PROBABILITY LEVEL .01
TA 0.1287 A
TB 0.1157 B
(-) 0.0000 C
MEANS NOT SHARING LETTER IN COMMON DIFFER SIGNIFICANTLY
BY DUNCAN'S MULTIPLE RANGE TEST.
TA 0.1287 A
TB 0.1157 B
(-) 0.0000 C

Table 2. ANOVA Table on the Difference (mg/L) in the Amount of Manganese after 48
Hours of Treatment
TA 0.03 0.03 0.03 MEAN= 0.0267
TB 0.02 0.02 0.02 MEAN= 0.0232
(-) 0.00 0.00 0.00 MEAN= 0.0000
******************************************************************************
Treatment Mean
TA 0.0267
TB 0.0232
(-) 0.0000
******************************************************************************
: Sirichai Statistics Version 6.00 :
09-07-2016 20:21:32
Problem Identification: Procedure : Analysis of Variance I
---------------------------------------------------------------------------
Table.... Analysis of Variance
Source df SS MS F F.05 F.01 F-Prob

Treatment 2 0.0013 0.0006 236.65 5.14 10.92 0.0001
Ex.Error 6 0.0000 0.0000
Total 8 0.0013 0.0002
GRAND MEAN = 1.66111112468772E-02
CV = 9.8225 %
LSD .05 = 3.25994687166202E-03

LSD .01 = 4.93854640508831E-03
**********************************************************
*PROBLEM IDENTIFICATION= *
*STANDARD ERROR OF MEAN= 9.42023105541514E-04 *
***********************************************************
TA 0.0267 A
TB 0.0232 A
(-) 0.0000 B
TA 0.0267 A
TB 0.0232 B
(-) 0.0000 C
Table 3. Two Way ANOVA Table on the Comparison of Iron and Manganese
Biosorptive Capacity of Chaetoceros calcitrans & Tetraselmis tetrahele.
Sirichai Statistics Version 6.00 :
09-07-2016 21:02:56
Problem Identification : Procedure : Two Factors Factorial
---------------------------------------------------------------------------
Table
------------------------------------------------------------------------------------------
SOURCE df SS MS F F.05 F.01
------------------------------------------------------------------------------------------
Treatment 5 0.0502 0.0100 1761.55 3.11 5.06
A 2 0.0481 0.0240 4215.22 3.89 6.93
B 1 0.0000 0.0000 7.37 4.75 9.33
AxB 2 0.0021 0.0011 184.98 3.89 6.93
ERROR 12 0.0001 0.0000
TOTAL 17 0.0503 0.0030
------------------------------------------------------------------------------------------
Grand Mean = 0.0491 CV = 4.8655

FACTOR A FACTOR B
TWO WAYS TABLE
A/B B1 B2 AVERAGE
A1 0.13 0.12 0.12
A2 0.00 0.03 0.01
A3 0.02 0.00 0.01
AVG 0.05 0.05 0.05
***********************************************************
*PROBLEM IDENTIFICATION=FACTOR A *
*STANDARD ERROR OF MEAN= 9.74964244838934E-04
***********************************************************
A1 .12216666 A
A2 1.3500000 B
A3 .01158333 B

A1 .12216666 A
A2 1.3500000 B
A3 .01158333 B
***********************************************************
* *
*PROBLEM IDENTIFICATION=FACTOR B *
* *
***********************************************************

B1 5.0611111 A
B2 4.7555556 A

B1 5.0611111 A
B2 4.7555556 B

***********************************************************
* *
*PROBLEM IDENTIFICATION=INTERACTION AB *
* *
***********************************************************
TA1 .12866666 A
TB1 .11566666 B
TA2 2.7000000 C
TB2 2.3166666 C
TC2 000000000 D
TC1 000000000 D

TA1 .12866666 A
TB1 .11566666 B
TA2 2.7000000 C
TB2 2.3166666 C
TC2 000000000 D
TC1 000000000 D


Appendix B
Letters/Communications
Republic of the Philippines

Department of Education
Region VI- Western Visayas
DON MANUEL ARNALDO MEMORIAL SCHOOL
Brgy. Milibilii, Roxas City
April 27, 2016
RUTH B. BARRIOS
Principal III
DMAMS
Roxas City
Madam:
Good day!
I am Via Faith Bolido, Grade V pupil of this school. I would

like to ask permission from your good office to conduct a
scientific research study entitled” Iron and Manganese
Biosorption on Chaetoceros calcitrans and Tetraselmis tetrahele”
as an entry in the forthcoming Division Science Fair.
I am hoping for your approval regarding this matter.

May 5, 2016
Engr. WILHELMINA S. CO
MT-II
Capiz NHS
Roxas City
Madam:
Good day!
I am Via Faith Bolido, Grade V pupil of Don Manuel Arnaldo

Memorial School. I would to conduct a scientific research study
entitled” Iron and Manganese Biosorption on Chaetoceros
calcitrans and Tetraselmis tetrahele” as an entry in the
forthcoming Division Science Fair. In this connection , I would
like to solicit your expertise to be my research consultant to
shed light on this scientific study.
I am hoping for your positive response regarding this

matter.

July 15, 2016
DR. EVELYN GRACE D. AYSON

Head, Research Department
SEAFDEC
Tigbauan, Iloilo
Madam:
Good day!

like to ask permission from your good office to avail samples of
Chaetoceros calcitrans and Tetraselmis tetrahele from your
Phycology Department.

matter.
Approved:
DR. EVELYN GRACE D. AYSON

Head, Research Department

July 21,2016
JUDY B. DUNTON
Dept. Head, Sci. and Tech
Capiz National High School
Fuentes Drive, Roxas City
Madam:
Good day!

like to ask permission from your good office to use the Chemistry
Laboratory and its equipment. I am willing to abide to the rules
and regulations set by your institution.

matter.
Approved:
JUDY B. DUNTON
Dept. Head, Sci. and Tech

July 22,2016
ZALDY A. LACSON
Division Manager
MRWD
Roxas City
Sir:
Good day!

like to ask permission from your good office to use the
Laboratory and its equipment and to have the samples tested. I am
willing to abide to the rules and regulations set by your
institution.

matter.
Approved:
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Biosorption Capacity of Chaetoceros calcitrans and Tetraselmis tetrahele on

Iron and Manganese

Contamination of water supplies with metals is an area of concern both national

construction or iron and manganese mining.

According to the data of the Potential Minerals in Western Visayas, Capiz is

In agricultural industry, iron and manganese serves as a micronutrient because

of particles in suspension or might be deposited in the sediment. Trace essential metals

playing important roles in metal-requiring and metal-activated enzyme systems, and as

toxicants when present at high concentrations.

clothing (Wageningen Univ., 2016). Furthermore, in humans, consuming large amounts

mutation (Garvin, 2015).

deficiency anemia. Symptoms of toxicity mimic those of Parkinson's disease (tremors,

neurological disorders in children (Blaurock-Busch, 1997).

various biological materials. Developments in the environmental biotechnology indicate

from aqueous solutions by biosorption (Hydrometallurgy, 1997). In recent years, there

biomass is the wide range of functional groups present on variety of macromolecules,

oceans (Feng and Aldrich, 2004).

Tetraselmis tetrahele is a green microscopic algae with four flagellated

viewed sideways. The alga measures 10 – 16 µm in length, 8 - 11 µm in width and 4.2 –

calcitrans and Tetraselmis tetrahele on Iron and Manganese. Specifically, it aims to

hours of exposure to the different treatments, to determine if there is significant

manganese (mg/L) adsorp after 48 hours of exposure to the different treatments.

MATERIALS AND METHODS

Sterilizing Materials and Tools

Preparing the Laboratory

The laboratory was cleaned and disinfected to ensure cleanliness.

Tetraselmis tetrahele and Chaetoceros calcitrans were obtained at the Phycology

Laboratory of SEAFDEC, Tigbauan, Iloilo.

Figure 1: Measuring and Pouring of Microalgae

Microalgae Cell Count

School, Roxas City.

Figure 2: Chaetoceros calcitrans Figure 3: Tetraselmis tetrahele

Procurement of Iron and Manganese

The Pretest- Posttest design was used in this investigation. Spectrophotometry

Pre Testing the Water Sample

Dilution of 1.25 mL iron/manganese in 498.75 mL Ozone Seawater was prepared

using the spectrophotometer machine (Spectronic 20, Milton-roy company).

Preparation of the Test Samples

prepared algal culture samples which contains 0.5 of each fertilizers + 50 mL of

Chaetoceros calcitrans and Tetraselmis tetrahele test sample respectively.

Preparation of the Different Treatment

were 2 treatments and a control: Treatment A. 50 mL Chaetoceros calcitrans + 490 mL

mL Tetraselmis tetrahele + 490 mL Ozonated seawater with Iron chloride / manganese

in 3 replications and were treated for 48 hours.

Trial 2 Trial 3 Trial 1 Trial 2 Trial 3

Figure 4: Different Treatments

spectrophotometer machine (Spectronic 20, Milton-roy company).

Figure 5: Treated Samples after 48 Hours

manganese solution samples were subjected for spectrophotometry analysis using a

data organized in table and were statistically evaluated.

Statistical Analysis of Data

the comparisons between treatments and the control.

ANOVA at 5% level of significance, results revealed that the p-value (0.000) is

difference in the amount of Iron (mg/L) adsorp after 48 hours of exposure to

Chaetoceros calcitrans and Tetraselmis tetrahele. Moreover, Duncan's Multiple Range

ANOVA at 5% level of significance, results revealed that the p-value (0.0001) is

difference in the amount of Manganese (mg/L) adsorp after 48 hours of exposure to

Chaetoceros calcitrans and Tetraselmis tetrahele. Furthermore, Duncan's Multiple Range

Table 2. Difference (mg/L) in the Amount of Manganese Adsorp after 48 Hours of