1.

INTRODUCTION

1.1 Rationale

Algae are aquatic organisms that photosynthesize with the presence of light. They serve
as an ultimate source of both cellular and chemical energy for other organisms to which often
called primary producers. Generally, algae are categorized as macro-algae and algae.
Microalgae species can be found in a diverse environment, freshwater or marine environment
where they can grow abundantly. Microalgae require a wide variety of chemical elements but
most important are nitrogen and phosphorous for growth and reproduction (Cremen et. al., 2007).

Microalgae are the major food source for many aquatic organisms are then main live feed
component as it contains polyunsaturated fatty acids that can be used in hatchery operations.
They are also considered as bio-filters since they remove pollutants from wastewaters and are
good water conditioner. Potentially microalgae are good source for biofuel production because of
their high oil content and rapid mass production.

The aquaculture is a fast growing sector and constantly increasing its production.
Currently, microalgae are used in the aquaculture industry as a live feeds for all stages of
crustaceans, mollusks, zooplankton and some fish species especially during the larval stages.
The most frequently used microalgae are Chlorella, Tetraselmis, Scenedesmus, Pavlova,
Phaeodactylum, Chaetoceros, Nannochloropsis, Skeletonema and Thalassiora (Sirakov et. al.,
2015). The main application of microalgae in aquaculture is connected with their usage for feed
purposes.

Tetraselmis chuii is a green flagellated prasinophyte. They have an ovoid body shape
and a distinct curved body when viewed with sideways. Tetraselmis chuii is one of the species of
microalgae that is most extensively used in aquaculture and is considered to be an optimal
source of long-chain PUFA’s, and especially of eicosapentaenoic acid (EPA) (Meseck et. al.,
2005).

Meanwhile, Sargassum sp. are nutritious and rich source of bioactive compounds such
as vitamins, carotenoids, dietary fibers, proteins and minerals (Namvar et. al., 1987). Sargassum
sp. are sub-tropical and tropical brown macroalgae of shallow marine meadow (Yende et. al.,
2014). These can also be found in the coastal waters of Naawan, Misamis Oriental. Some studies
shown that it has good effects on the growth of the terrestrial plants such as beans and tomatoes
(Sutharsan et. al., 2014). Potentially, Sargassum sp. can be used to induce the growth of the
Tetraselmis chuii for mass production as proven to improve the population growth of
Skeletonema costatum (Nasmia et. al., 2017). The effect of seaweed liquid fertilizers (SLF) of
Sargassum wightii and Caulerpa chemnitzia on growth and biochemical constituents of Vigna
sinensis was studied. Among the two seaweeds tested, S. wightii exhibited better responses
(Sivasankari et. al., 2006)

This study was performed to evaluate and compare the effect of powdered Sargassum
used as fertilizer in the microalgae using the different treatments namely the Conwy, 200ppm,
400ppm and 800ppm of the powdered Sargassum in the population growth of Tetraselmis chuii.
The study used different concentrations to determine which concentration of powdered
Sargassum the Tetraselmis chuii grows best.

1.2 Statement of the Problem

Tetraselmis chuii is an aquatic algae that can be fed to larvae/juveniles of abalone,

oysters and even zooplankton. It requires fertilizer to increase their growth in terms of population.
Since that the fertilizers used in the MSUN-IFRD Phycology Laboratory of Naawan, Misamis
Oriental are expensive, hence, this study used powdered Sargassum sp. as an alternative
fertilizer which can be collected abundantly along the coastal waters of Naawan, Misamis
Oriental.

1.3 Objective of the Study

1.3.1 General Objective

This study deals in determining the population growth of Tetraselmis chuii using different
concentrations of powdered Sargassum as a fertilizer.

1.3.2 Specific Objective

To determine the growth rate of T. chuii cells for each concentrations of powdered
Sargassum.

To compare the population growth of T. chuii using the Conwy as the control and the
different concentrations of powdered Sargassum.

1.4 Significance of the Study

Microalgae provide a vital role in the rearing of aquatic animals like molluscs, shrimp and
fish in the field of aquaculture. The purpose of this study was to determine the population growth
of Tetraselmis chuii using the different concentrations of powdered Sargassum until it reaches it
two successive declining densities. The result of the study evaluates the effectiveness of
powdered Sargassum as fertilizer and would determine what concentrations could attain the
highest peak population with the fastest growth rate and shortest time to double its population.
This study may guide on which concentration of powdered Sargassum can be best applied as
fertilizer in the Hatchery and Phycology Laboratory. The information generated will be useful in
the culture practices of Tetraselmis chuii using powdered Sargassum.

1.5 Scope and Limitations

The study was focused on the effects of different concentration of powdered Sargassum
on the Population growth of Tetraselmis until it reaches 2 consecutive declining at 6 hours
interval. The physico-chemical parameters that was checked before and after the culture period
were salinity and temperature respectively.

1.6 Definition of terms

Concentration is the amount of a specified substance in a unit of another substance.

(the free dictionary.com/concentration)

Exponential phase is a growth phase when the algal cells divide at an accelerating rate and
the population increases logarithmically which lasts for 4 or more days (Creswell,
2010).

Hemacytometer is an instrument for visual counting of the number of algal cells under a
microscope.

Lag phase is the first 2 to 3 days culture stage of microalgae when the cells become acclimated
to the new medium, grow, and begin cell division (Creswell, 2010).

Microalgae are unicellular aquatic plants (phytoplankton), the starting point of the aquatic food
chain. (http://medical-dictionary.thefreedictionary.com/Microalgae).

Peak day is the day with the highest population density during the culture period.

Peak densities is the maximum number of individuals per unit volume

Phytoplankton is a term used to describe plants that are so small that their movement is
primarily controlled by the motion of the water (Conte et al., 2001).

Population growth is the change in population over time and can be quantified as the change in
the number of individuals in a population using “per unit time” for measurement.

Senescent phase is when the cell density of the culture will decline in two consecutive times.

Tetraselmis chuii is a green microalgae that have an ovoid body shape and distinct curved body
when viewed sideways.

Pasteurization is a partial sterilization of a substance and especially a liquid (such

as milk) at a temperature and for a period of exposure that destroys
objectionable organisms without major chemical alteration of the substance.

Inoculant also known as soil inoculants are agricultural amendments that use beneficial
endophytes (microbes) to promote plant health. Many of the microbes involved form
symbiotic relationships with the target crops where both parties benefit.
(https://en.wikipedia.org/wiki/Mainpage)
Euryhaline is being capable of tolerating wide range of salt water concentrations.
(https://en.wikipedia.org/wiki/Mainpage)

Eurythermal able to tolerate a wide range of temperatures in the environment.

Disinfection is the destruction of pathogenic microorganism or their toxins or vectors by direct

exposure to chemical or physical agents.

Powdered sargassum are macroalgae processed into a powderized form.

Chlorination is one of many methods that can be used to disinfect water.

 II. REVIEW OF RELATED LITERATURE

Role of microalgae

Microalgae are used in mariculture as live feed for all growth stages of mollusks, for the
larval stages of crustaceans and some fish species and for zooplankton used in mariculture food
chains. They also are the primary diet of zooplankton reared as for late-larvae and juveniles of
some crustaceans and fish species (Brown, 1997). Algae are at the base of the entire aquatic
food chain, and support the production of renewable resources from fishing. Therefore it is not
surprising that the microalgae will compose the phytoplankton play a vital role in the rearing of
aquatic animals such as molluscs, shrimp and fish. Some of their properties also concern the
environment, supporting life in space and renewable energy production (Muller, 1977).
Microalgae have an important role in aquaculture as e means of enriching zooplankton for o-
feeding to fish and other larvae (Brown, 1986).

Life stages of microalgae

There are 5 phases of algal growth in batch culture 1 lag; 2 exponential; 3 declining
growth rate; 4 stationary; 5 death (Fogg and Thake, 1987). The growth of the microalgae needs a
variety of micro/macro elements which not only constitutes algae cells, but also help maintain
their metabolism system (Richmond, 1986).

Tetraselmis chuii

Tetraselmis chuii is a green flagellated prasinophyte characterized by an ovoid body

shape and distinct curved body when viewed sideways. The microalga measures 12-14
micrometer in length, 9-10 micrometer in width and belongs to the family Chlamydomonadaceae.
Most species are known form inshore marine environments, tide pools in particular but a few
freshwater species are also known (Bold and Wynne, 1995). Tetraselmis sp. are ideal for
aquaculture because they are euryhaline and eurythermal (Fabregas et. al., 1984).

Microalgae of the genus Tetraselmis are important for their use in the aquaculture as a
food source for several kinds of fish, shrimps and shellfish. They are cultured on a large scale,
being easily disseminated in the food chain (Costa and Franca, 1998).

Sargassum as fertilizer

Seeweeds are known as functional food because of their richness in lipids, minerals and
certain vitamins and also several bioactive substances like polysaccharides, proteins and
polyphenols, with potential medical uses. Chemical and nutritional composition of seaweeds
varies with individuals, species, habitats and maturity and depends on geographical origin or area
of cultivation, seasonal, environment and physiological variations and water temperature (Namvar
et. al., 2013). Seaweeds are good source of nutrients for the plants when added as fertilizer
(Kumari et. al., 2011). The liquid extracts prepared from S. wightii contains high levels of Mg, Na,
K, P, Fe, Cl, Zn, Cu, and N (Sivasankari et. al., 2006)

Aqueous extracts of Sargassum johnstonii at concentrations form 0.1 to 0.8% (w/v) that is
equivalent to 1-8 mgSWml-1 increased the rooting of Vigna mundo hypocotyl cuttings 2.4-3.2
times and reproductive parameters (flower number, fruit number an d fresh weight) of tomato
(Kumari et. al., 2011)

The effect of 20% (0.2 mgSWml-1) extract of Sargassum wightii gave 11% increase in seed
germination, 63% enhancement in number of lateral roots and 46% increase in shoot length of
Triticum aestivum, as compared to control (Kumar and Sahoo, 2011). Seaweed Liquid Extract
(SLE) at low concentration (1.5%) exhibited promoting effect on growth and yield parameters.
Differential responses in the content of photosynthetic pigments, protein, reducing sugar, ascorbic
acid and in the activity of nitrate reductase were also observed in the leaves of SLE treated
seedlings when compared to untreated seedlings. Higher concentrations (above 1.5%) of SLE
were found to show inhibitory effect (Vijayanad et. al., 2014).

As flower initiation and development are related not only to the physiological age of the
plants but also to their vegetative growth, superior reproductive performance and was observed in
plants treated with higher concentrations of seaweed extracts resulting in improved total fruit
production (Crouch and van Staden, 1992). The present study clearly indicates that aqueous
seaweed extracts is able to enhance overall growth in tomato plants as compared to the control
plants (Kumari et. al., 2011).
 3. MATERIALS AND METHODS

3.1. Study Area

The study was conducted at MSU-N IFRD CARES Naawan, Misamis Oriental. The study
was conducted for 7 days of culture.

Figure 1. MSU at Naawan Center for Aquaculture Research Enterprise and

Services(CARES).

3.2. Source of Experimental Specimen

Tetraselmis chuii is a green flagellated prasinophyte characterized by an ovoid body

shape and distinct curved body when viewed sideways. The micro alga measures 12-14
micrometer in length, 9-10 micrometer in width and belongs to the family Chlamydomonadaceae.
Tetraselmis sp. are ideal for aquaculture because they are euryhaline and eurythermal (Fabregas
et. al., 1984). Tetraselmis chuii was obtained from the Phycology Laboratory of MSUN IFRD
Naawan, Misamis Oriental. Approximately 1.5 L of concentrated sample was used for the study.
The quality and quantity of the cells Tetraselmis was examined and counted before the conduct
of the study.
 Figure 2. Tetraselmis chuii (www.google.com)

3.3. Cleaning and Disinfecting of Materials

The experimental materials used was 12 plastic containers with 1.5L water capacity. All
the materials needed like containers, air hose, air stone and graduated cylinder were cleaned
thoroughly following the protocols of the MSU-N IFRD Phycology Laboratory.

3.4 Disinfection of water

The chlorinated neutralized seawater was boiled, upon boiling point the sea water
extended for 15 minutes and the freshwater was extended for 30 minutes to kil the unwanted
organisms. The sterilized seawater and freshwater was cooled and transferred into the 15L
capacity plastic. This approach was followed to prevent contamination during the culture period.

Figure 3. Boiling of non-chlorinated seawater using kettle and electric (left); 15 liters
cooled boiled seawater (right).

3.5. Preparation of Powdered Sargassum

The Sargassum was collected from the coastal area of Pagawan, Initao, Misamis
Oriental. Those were washed with clean freshwater and air dried for 3 days. Moisture content was
removed by oven drying for 48 hours at 60⁰C. The oven dried Sargassum was finely ground using
a grinder (Erulan et. al., 2009).

Figure 4. Collection of Sargassum at Pagahan, Initao Misamis Oriental.

Figure 5. Washing of Sargassum with freshwater (left) and oven drying of Sargassum
(right).

Figure 6. Grinded powdered Sargassum (left) weighed in analytical weighing balance

(right).
3.6. Preparation of Stock Solution

Two hundred grams of powdered Sargassum was dissolved in 1L distilled water and the
contents was heated for 45 minutes at 60⁰C. After cooling, it was filtered with cheese cloth
(Erulan et. al., 2009). Different concentrations were prepared as 200ppm, 400ppm and 800ppm
using this formula:

V1= C2V2
C1

3.7. Algal culture

3.7.1. Qualitative analysis of starter using the binocular microscope

The Tetraselmis chuii starter was placed in an Improvised Counting Chamber and was
analyzed and under the microscope to determine the presence of contaminants that may cause
collapse during the culture period.

3.7.2. Initial stocking of Tetraselmis chuii

The initial density of Tetraselmis chuii was 50,000 cells/ml for all the experimental units.
The 1.5 liters plastic containers was filled with 1 liter of culture which was composed of boiled
water, Tetraselmis inoculum and the different concentrations of extracted powdered Sargassum.
The desired concentration of Tetraselmis chuii was determined using this formula:

V1= C2V2
C1

Where:

C1= Concentration of Tetraselmis chuii in the source cultrue

V1= Volume of culture

C2= Desired Concentration of Tetraselmis chuii

V2= Final volume of Tetraselmis chuii

A pure culture of Tetraselmis was obtained from the MSU-IFRD Phycolab. Tetraselmis
sp. were initially stocked at 50,000cel/ml in each culture containers.

3.8. Experimental Design and Treatments

Artificial light was added to allow photosynthesis of the organism. Aeration was
introduced to inject oxygen in the water. There were twelve 1.5L capacity containers, four
treatments with 3 replicates each used in the experiment. The T1- Conwy fertilizer as the control,
T2-200ppm, T3-400ppm and T4-800ppm of the powdered Sargassum were arranged in
Completely Randomized Design.

Figure 7. Culture set-up during the conduct of the study.

3.9. Monitoring

3.9.1. Collection of Samples

Fifteen samples were collected in every sampling time during the 210 hours culture
period. Daily sampling was done every 6 hours until the end of the experiment. The culture
medium was agitated to ensure the even distribution of the cells. A 10 ml sample was put in a vial
and added with a drop of Logul’s solution to immobilize and to stain the cells. Samples were then
brought to the laboratory for counting.

3.9.2. Counting of T. chuii population using hemacytometer

Samples were agitated thoroughly. A subsample was taken from the vial using a pointed
medicine dropper and loaded into the chamber of the hemacytometer covered with a cover slip.
The sample was allowed to settle for 2-3 minutes. Hemacytometer was then place on the
microscope stage and viewed in low power objective (10X). Counting was done in the four blocks
corners of the hemacytometer.
 1 2

3 4
2 1
Figure 8. Inner view of the Hemacytometer
(MicrobeHunter.com)

The two chambers of the hemacytometer were sampled to analyze to avoid bias. The
estimated population density of the T. chuii at the big blocks labeled 1,2,3 and 4 was calculated
and determined using this formula:

Population density = Total no. of cells counted X 10

(cells/ml) No. of Blocks (4)

3.10. Experimental data

Peak population in each treatment were determined according to the highest cell count
attained during the conduct of the experiment. Computations for the growth rate and population
doubling time were determined by getting the peak population and converting them to log values
and computed using this formula:
A. Growth rate (µ) (Chen, et. al, 2012)
µ= ((log₂(N₂)-log₂(N1))
(t-t₀)

Where :
µ= growth rate (division day)
N₁= initial population density
N₂= final population density
t₀= initial time
t= final time
B. To obtain measures on growth, velocity doublings per day were calculated as per
Herrero, et. al., (1991):
Doubling/hr= ln N(n)-ln N(i)
Ln2 (tn-ti)
 Where:
t¡= initial time
tn= peak time (expressed in hours)
N (¡)= initial population
N (n)= peak population

3.11. Physico-Chemical Parameters

An alcohol filled thermometer was used to measure the water temperature. This was
submerged in the water for one minute before reading. Checking of water was done before and
after the culture.
The salinity was measured using a refractometer. It was cleaned with sterilized
freshwater for calibration and to start the reading to zero and was wiped with tissue. A drop of
samples was then put into the refractometer to read the existing salinity.

3.12. Statistical Analysis

The daily population growth of Tetraselmis was counted and analyzed for seven days on
the basis of the number of individuals of the test organism was subjected to One-Way ANOVA
(Gomez and Gomez, 1984) using SPSS tools to determine the significant difference of each
treatments when p value is less than 0.05.
 4. RESULTS AND DISCUSSION

4.1. Population growth of Tetraselmis chuii

Phytoplankton comprises the base of the food chain in the marine environment.
Therefore, micro-algae are indispensable in the commercial rearing of various species of marine
animals as a food source for all growth stages of bivalve molluscs, larval stages of some
crustacean species, and very early growth stages of some fish species. Algae are furthermore
used to produce mass quantities of zooplankton (rotifers, copepods, and brine shrimp) which
serve in turn as food for larval and early-juvenile stages of crustaceans and fish. Algae are used
directly in the larval tanks, where they are believed to play a role in stabilizing the water quality,
nutrition of the larvae, and microbial control. (Coutteau et. al., 1994)

The mean peak population between and among treatments are shown in Table 1. The
highest mean peak density of 3,957,500 cells/ml was obtained by the T1-Conway fertilizer which
was the control followed by T4-800ppm concentration of powdered Sargassum (472,500
cells/ml), T3-400ppm (330,000 cells/ml) and the least density was T1-200ppm (295,000 cells/ml).
Statistical tests showed significant differences during the peak time between and among
treatments.

Table 1. Mean peak population of the four treatments at peak time.

Treatment Mean Peak Population (cells/ml) Peak Time (hr)
T1-Conway 3,957,500 144
T2-200ppm 295,000 138
T3-400ppm 330,000 138
T4-800ppm 472,500 138

The growth curve of the T. chuii using Conway, T1-200ppm, T2-400ppm and T4-800ppm
of concentrated powdered Sargassum water enrichments. The exponential phases were
observed up to 144th hour for Conway, and 138th hour for T1, T2, and T3. After this duration, the
cultures started declining up to the 210th hour. The study showed significant differences between
the treatments starting 78th hour or third day where T1 and T4 were significantly different than of
T2 and T3, however the T1 after 78th hour of the culture and beyond became significantly
different from all other treatments.
Figure 9. Mean cell population counts at every six hours interval.

The most important parameters regulating algal growth are nutrient quantity and quality,
light, pH, turbulence, salinity and temperature (Coutteau et. al., 1994). Enable of the T. chuii cells
to grow, the salinity and temperature of the medium was checked before and after the conduct of
the study. Marine phytoplankton is extremely tolerant to changes in salinity and Tetraselmis sp.
can be cultured in natural seawater between 15 and 36 ppt (Algal production, http://fao.org).
Based on polynomial regression data, optimal temperature of T. chuii cells growth was (Chen, et.
al., 2012)

The salinity before and after the conduct of the study ranges from 31-33 ppt and the
temperature where can be attested to pass the optimal salinity of the said organism. However,
the temperature of T. chuii cells before and after the conduct of the study ranges from 29-31 ⁰C
which could possibly be the factor the slow growth of some treatments. It can be observed that
the artificial light been used were not enough to produce heat allowing the organisms to grow. In
the growth curve that highest peak population was observed at the 6th day of culture were as
normally at the phyco-laboratory usually the exponential stage happens at the third day of culture.

Furthermore, as stated by Xin, et. al., (2010), a nutrient should be supplemented

sufficiently as it is considered a crucial factor to the performance of the growth of microalgae.
Maximum growth or cell density is one of the most important growth parameter (Chen, et. al.,
2011). Meanwhile the introduced treatment which according to Rajendran (2014) Seaweed
Extract Powder Fertilizer was known to have Nitrogen, Phosphorous, Potassium as well as trace
minerals like Zn, Mn, Mg, Fe, etc. It also contains natural plant growth material like auxins,
gibberlins and cytokinins.
 Several studies used seaweeds liquid fertilizer in some terrestrial plants such as Vigna
sinensis, the low concentration (20%) of aqueous extracts of S. wightii and C. chemnitzia
promoted the seedling growth including the parameters of shoot length, root length (6.42, 5.38
cm/seedling), fresh weight and dry weight, chlorophyll, carotenoids, protein content of shoot and
root. Among the two seaweeds tested, S. wightii exhibited better responses (Sivasankari, et. al.,
2006). Experiments were conducted on ground nut to study the potential red alga of H.
musciformis as a biofertilizer. The seeds were sown in soil and SLF were added to soil bed in five
different concentrations separately (1%, 2%, 4%, 6% and 8% w/v). The 2% concentration of
water extract showed better results of growth parameters, biochemical and pigments constitutions
(Selvam, et. al., 2014). Efficiency of the Seaweed Liquid Extract was observed by performing the
experiments at different concentration such as 0.2%, 0.4%, 0.6%, 0.8% and 1%. The Sargassum
tenerrimum as a fertilizer and also to improve the seed germination, growth, yield as well as
quality for better production and process (Sasikala, et. al., 2016).

The result shows that the highest mean peak density of 3,957,500 cells/ml was obtained
by the T1-Conway fertilizer which was the control followed by T4-800ppm concentration of
powdered Sargassum (472,500 cells/ml), T3-400ppm (330,000 cells/ml) and the least density
was T1-200ppm (295,000 cells/ml). Statistical tests showed significant differences of T1 to all
other. The exponential phases were observed up to 144th hour for Conway and 138th hour for T1,
T2, and T3. After this duration, the cultures started declining up to the 210 th hour. The study
showed significant differences between the treatments starting 78th hour or third day of the culture
and beyond. It can be observed that the Treatment 1 showed the highest peak population but
longest in doubling time at day 6 while all other treatments were observed to have its highest
peak density at day 5. A matter of 6 hours, the population of Tetraselmis chuii using of the
powdered Sargassum already declined unlike the Conway fertilizer. Depletion of nutrients by the
cultivation medium would cause starvation, stress and stunted reproduction of microalgae (Li, et.
al., 2012). Slow reproduction lowers cell density (Lananan, et. al., 2013). This could possibly be
one of the reasons of low cell density counts because of the depletion of nutrients after 7 days of
culture. Furthermore, the concentration of the powdered Sargassum might be not be enough to
provide best growth for the Tetraselmis chuii were we can observed that in many studies
conducted using the powdered sargassum as fertilizer to some other terrestrial plants most of
them promoted good growth at 0.2% concentration or even higher. The low concentration used in
this study possibly affected its growth as some studies used 0.2% where if converted into ppm it
is equal to 2,000ppm. Low concentration could possibly be the one of the factors that affects the
growth of the treatments.
4.2 Growth rate

Table .2 presents the growth rate among treatments. The T1-Conway obtained the
highest growth rate of 0.0456 followed with T4-800ppm, T3-400ppm and T2-200ppm of powdered
Sargassum (0.0245, 0.0206 and 0.0194, respectively). Statistical test shows that there is no
significant difference (p>0.05) in the growth rates among the treatments.

Table 2. Mean growth rate of cells in the four treatments.

Treatments Mean Growth Rate
T1-Conway 0.0456
T2-200ppm 0.0194
T3-400ppm 0.0206
T4-800ppm 0.0245

4.3 Population doubling time

The T2 with 200ppm concentration of powdered Sargassum has the shortest time
(0.0194 double its population of the Tetraselmis chuii and the longest time is in Conway fertilizer
(0.0457) as shown in Table 3. Statistical tests showed no significant difference between and
among treatments.

Table 2. Mean growth rate of cells in the four treatments.

Treatments Mean Growth Rate

T1-Conway 0.0457
T2-200ppm 0.0194
T3-400ppm 0.0206
T4-800ppm 0.0245
 5. SUMMARY AND CONCLUSION

The study was conducted at MSUN-IFRD CARES for 7 days or 210 hours as the culture
reached its successive declining densities. There were three treatments and a control which was
the Conway with three replicates each. The seawater was boiled and cooled. And so with the
materials been used were washed with soap and freshwater, later disinfected with boiled
freshwater.

After the preparation of the culture set up, the seawater was filled into the cultured
containers and the desired concentration of the algal starter. It was followed by the fertilizers
depending on the given concentrations in each treatments after which the aerators are installed,
salinity and temperature was checked before the start of the culture. Monitoring of the algal
population are done every day at 6am, 12pm, 6pm and 12am. Two ml of samples were taken
every sampling and subjected to counting under the binocular microscope at 10x objective.

As stated by Xin, et. al., (2010), a nutrient should be supplemented sufficiently as it is

considered a crucial factor to the performance of the growth of microalgae. Maximum growth or
cell density is one of the most important growth parameter (Chen, et. al., 2011). Depletion of
nutrients by the cultivation medium would cause starvation, stress and stunted reproduction of
microalgae (Li, et. al., 2012). Slow reproduction lowers cell density (Lananan, et. al., 2013).

The study showed significant differences between the treatments 0 hour to 78th hour of
culture where Treatment 1-Conway and Treatment 4-800ppm of powdered Sargassum are
significantly different from Treatment 1-200ppm and Treatment 2-400ppm. After 78th hour T1-
Conway showed significant differences among all other treatments. It can be observed that the
Treatment 1 or the Conway fertilizer showed the highest peak population but the longest in
doubling time because the nutrient availability is still enough to sustain until 6 days. Meanwhile,
powdered Sargassum having different concentration was observed to reach its exponential earlier
at day 5 a matter of 6 hours compared to the Conway fertilizer. It can be concluded that the
higher the concentration the higher the growth curve of the T. chuii cells.
 6. IMPLICATION AND RECOMMENDATION

This study on the population growth using different concentration of powdered

Sargassum as fertilizer on T. chuii which distinguishes what concentration could promote good
and high growth of population as alternative to Conway fertilizer. With the result presented, the
information could help the hatchery, fish pond operators and even in the pure culture of T. chuii
since Sargassum has anti-fungal and anti-bacterial factors which is one of the reasons of the
collapse of the culture as it was brought and cultured outdoor in the small scale culture. It is
suggested that the sulfated polysaccharide from Sargassum swartzii could be a better source of
natural antioxidant, as well as an antibacterial agent (Vijayabaskar, 2012). The result obtained
with methanol extract of Sargassum wightii whole plant was exhibited significant antifungal
activity (Vengadesan and John, 2014).

As stated by Xin, et. al., (2010), a nutrient should be supplemented sufficiently as it is

considered a crucial factor to the performance of the growth of microalgae. Efficiency of the
Seaweed Liquid Extract was observed by performing the experiments at different concentration
such as 0.2%, 0.4%, 0.6%, 0.8% and 1%. The Sargassum tenerrimum as a fertilizer and also to
improve the seed germination, growth, yield as well as quality for better production and process
(Sasikala, et. al., 2016). Depletion of nutrients by the cultivation medium would cause starvation,
stress and stunted reproduction of microalgae (Li, et. al., 2012).

As to the concentration of powdered Sargassum, the author suggests to increase its

population a compare to some terrestrial plants and to have a further study that would include the
Conway as a control since it is the one usually used in the phycology laboratory. Another study
that the author suggests is to study the effects of the concentrated powdered Sargassum
between refrigerated and not, and also between weeks before used as fertilizer. Nutrient content
could also be analyzed as a form of confirmation it has good effects towards cell development.