Isolation, Identification and Optimization of Thraustochytrids in the Fallen Mangrove

Leaves of Brgy. Cayucyucan, Mercedes,

Camarines Norte, Philippines

I.H. Velasco1, M.E. Eborde1, A.M. T. Picardal1

School of Science and Technology, Centro Escolar University
ABSTRACT
The present study was undertaken to isolate, identify and optimize
thraustochytrids in the fallen mangrove leaves from Brgy. Cayucyucan, Mercedes,
Camarines Norte, Philippines. One Thraustochytrids CNT01 were isolated from fallen
mangrove leaves of in Brgy. Cayucyucan, Mercedes Camarines Norte, Philippines. It
was further studied for their optimal condition by biomass determination, result shows
that the highest biomass was seen at 6.0 pH level, 20C incubation temperature, 11%
glucose concentration and 25 ppt of salinity. The isolated Thraustochytrids
characteristic suggest that it belongs to the genus Schizochytrium sp.
Keywords: Thraustochytrids, Mangrove Ecosystem, Biodiversity

INTRODUCTION
Fungi and fungoid protist are
known to be an important degradation
agent in ecosystems, and their
importance has been extensively
investigated in terrestrial environments.
Less attention has been given to the
ecology of fungoid Protista, and
consequently
little
information
is
available
concerning
organic
degradation by their ectoenzymes.[1]
Yet, fungi are regarded to be more
important
than
bacteria
in
the
degradation
of
refractory
substrates.[2]Similarly, fungoid protists
play a role in decomposing and
mineralization of refractory organic
matter and thus enhance the carbon
cycle in coastal waters.[3]

Thraustochytrids are unicellular,

marine fungoid protists that are
ubiquitous in the sea water. They are
characterized by the presence of an
ectoplasmic net and non-cellulosic,
sulfrylated
scaled
cell
wall.[4]
Thraustochytrids are currently classified
under the Phylum Labyrinthulomycota of
the
kingdom
Protista
which
encompasses the diatoms, brown algae,
oomycetan
fungi
and
other
flagellates.[5]
Thraustochytrids
are
characterized
based
on
their
morphology and cell wall chemistry.
They are heterotrophic microorganisms
ubiquitously distributed in the marine
and estuarine environments. They are
common in mangrove swamps, algal

surfaces,
seawater,
and
marine
sediments.
Recently, they are also
associated with fallen leaves of many
mangrove species.
Thraustochytrids
play
an
important role in the aquatic food web
and represent a valuable source of
nutrients, such as polyunsaturated fatty
acids in the microbial loop in marine
sediments. Isolated Thraustochytrid is
known to produce a wide range of lipids
such as the docohexaenoic acid (DHA),
particularly an essential polyunsaturated
fatty acid (PUFA) of the omega-3 series.
SIGNIFICANCE OF THE STUDY
Limited studies were conducted
on Thraustochytrid species in the
Philippines so that findings in their
research can be meaningful to find
economic uses. The study provides an
information
about
the
isolation,
optimization, and identification of
Thraustochytrids
from
the
fallen
mangrove leaves in Brgy. Cayucyucan,
Mercedes,
Camarines
Norte,
Philippines. The study serves as a
reference in conducting a research
related to the isolation, optimization of
thraustochytrids from fallen mangrove
leaves of Brgy. Cayucyucan, Mercedes,
Camarines Norte, Philippines.
This
study can also provide baseline
information on the recent status of
Thraustochytrids
found
in
the
Philippines.
Thraustochytrids are known to
produce a wide range of lipids,
particularly
polyunsaturated
fatty
including the n-3 series, such as

docosahexaenoic acid (DHA) and

eicosapentaenoic acid (EPA).
This study is beneficial to the
poultry and aquaculture industry as a
promising alternative feed additive.
Thraustochytrids
also
have
an
antioxidant,
anticancer
and
immunomodulatory properties to human.
Therefore, it can also be beneficial for
the use of medicine.
OBJECTIVES OF THE STUDY
The study dealt with the isolation,
identification
and
optimization
of
Thraustochytrids from fallen mangrove
leaves of Brgy. Cayucyucan, Mercedes,
Camarines Norte, Philippines.
Specifically, the study considered
the following objectives:
1. To isolate Thraustochytrids in
the fallen mangrove leaves
from
Brgy. Cayucyucan,
Mercedes, Camarines Norte.
2. To determine
the optimal
condition that will favour the
growth
of
the
isolated
Thraustochytrids from fallen
mangrove leaves of Brgy.
Cayucyucan,
Mercedes,
Camarines Norte, Philippines.
3. To identify the isolated
thraustochytrids based on its
morphological characteristics.
MATERIALS AND METHOD
Collection of fallen mangrove leaves

Ten fallen senescent leaves were

collected randomly from the ground of
the mangrove site regardless of its
species
in
Brgy.
Cayucyucan,
Mercedes, Camarines Norte. The leaf
samples were placed in ziplock plastic
bags
for
the
isolation
of
Thraustochytrids. The sampling bags
were then placed in ice prior to
processing in the laboratory within 24
hrs. Along with these, the collection
sites pH, salinity and the temperature
were also noted
Preparation of Leaf Samples
Collected leaf samples were
washed three (3) times for an hour each
with natural seawater (NSW) in a 250ml
Erlenmeyer flask. It is supplemented
with antibiotics (0.3 mg/L penicillin, 0.3
mg/L Streptomycin, 0.3 mg/L Nystatin)
to prevent bacterial and mycelial
growth.The samples were set aside and
the three (3) washings are kept for
baiting.
Isolation of Thraustochytrids
For
the
isolation
of
Thaustochytrids, Sesame seed baiting
was used. The Sesame seeds were cut
into half and placed in an Erlenmeyer
flask and boiled for two (2) hours. After
boiling, the boiled sesame seeds were
divided into three parts and placed in
the 1st , 2nd and 3rd washing respectively
that was previously set aside and
incubated at 30C for 2 days. After
incubation, sesame seeds were placed

into the Glucose Yeast extract Peptone

Seawater (GYPS) agar composed of 3.0
g glucose, 1.25 g yeast extract, 1.25 g
peptone, 15.0 g agar and 1.0 L natural
seawater at pH 6.0 supplemented with
0.3 mg/L streptomycin and penicillin.3
These were incubated for 2-3 day. The
growth
of
Thraustochytrids
was
monitored by observing the plate under
the microscope. If the organisms are still
overlapping, subculture was done and
after 2 to 3 days incubation, they were
again observed under the microscope.
Subculturing continued until a pure
culture was obtained. This procedure
was done for the three washing.
Morphological Characterization
The plates were observed
microscopically
to
determine
the
organisms morphology by examining
the growth on all the sides of the
sesame seed. Likewise, samples were
taken and place on a slide for further
examination.
The
identification
of
Thraustochytrids were based primarily
on the microscopic and macroscopic
morphology.
Inocolum Preparation for Biomass
Production
The inoculum for biomass
optimization
was
prepared
by
inoculating the purified cultures of
Thraustochytrids into an Erlenmeyer
flask, containing 50 mL GYPS broth.

The broth was then incubated and

shaken at 200 rpm at 25- 300C for 2
days. After incubation, its density was
adjusted to 0.5 McFarland standards
with GYPS broth (sterile) to be used in
the succeeding experiment.
Parameters for Optimization
Growth of the Thraustochytrids
isolates were then tested under four
different
parameters
incubation
temperature, salinity, pH and glucose
concentration to determine their effects
on the biomass of the isolates. One (1)
mL of adjusted GYPS media was
inoculated with isolate in 100 mL
modified-glucose-yeast extract-peptone
broth composed of 10.0 g glucose, 10.0
g yeast extract and 1.0 g peptone in
1.0L NSW at pH 6.0.4 Three levels of
each parameter were tested:

pH
In the pH experiment, the initial
pH of the modified GYPS broth were
adjusted to different levels (pH 4, 5, 6,
7, 8 and 9) using 0.5 N KOH and O.5 N
HCl. It was measured using a calibrated
pH meter.
After pH adjustment, the broth
was then filtered, sterilized using a 0.45
mm-membrane filter and was incubated
for 2 days.
Glucose concentration
Lastly, for the effect of glucose
concentration, the amount of glucose in
the modified GYPS medium was
adjusted to different levels (7%, 9%, and
11%, w/v) while the incubation
temperature (30C); pH (6.0) and
salinity (50%NSW) were kept constant.

Temperature
For the effect of incubation
temperature on biomass, three different
temperature were tested (200C, 300C,
370C) while the pH (6.0); glucose
concentration (5%) and the salinity (24
ppt) were kept constant

Optimization parameter were

tested separately with the control
parameters
and
kept
constant
(temperature 30C; 24 ppt salinity; 5%
glucose; and initial pH 6.0).
Each
condition was done in triplicates and all
of the flasks were incubated and shaken
for three (3) days at 200 rpm.

Salinity

Biomass Quantification.

For the effect of salinity on

biomass, the amount of salinity in the
modified GYPS medium was adjusted to
different levels (20, 24, 30 ppt) while the
incubation temperature (30C); pH (6.0)
and salinity (24 ppt) were kept constant.

Ten (10) mL of the 3 day old

culture was placed in a pre-weighed
sterile Falcon tube.
Thraustochytrid
cells were harvested by centrifugation at
3500 rpm for 10 minutes.
The
supernatant was then discarded and the
cells were left to dry in an oven at 70C

for 2 days.5 The weight of the empty

Falcon tube was subtracted from the
weight of the Falcon tube with the dried
cells to obtain the dry cell weight
(DCW).This value is expressed as dry
cell weight in grams per 10 mL in
modified GYPS medium.
Growth Analysis under Optimum
Condition
The conditions of incubation
temperature, salinity, pH and glucose
concentration
where
the
optimal
biomass was produced as shown from
the results were used as the conditions
for the growth curve cultivation.
Cultivation for Growth Curve
Analysis.
Flasks containing 10 mL of the
modified GYPS broth adjusted to
optimal pH, glucose concentration and
seawater
concentrations
were
inoculated with one (1) mL of adjusted
GYPS (inoculum) and were incubated
under optimal temperature with constant
agitation.
Every day, two flasks were then
sampled for the optical density and
biomass determination. First, an aliquot
of the culture was taken for
spectrophotometric analysis of optical
density at 600-660 nm wherein the
blank used was sterile modified GYPS
medium.
A calibration curve comparing optical
density and biomass of the isolates was
prepared
using
data
from
this
experiment.

RESULTS AND DISCUSSION

Fallen mangrove leaves was
collected near the coast with low water
level at the mangrove community in
Brgy.
Cayucyucan,
Mercedes,
Camarines Norte, Philippines. The
collection site has the pH level of 6.0
and salinity of 24ppt.
Sesame seed baiting procedures
were used to isolate the thraustochytrid
in the collected mangrove leaves.
Different colonies grew near the sesame
seed bait and were observed under
compound microscope.
Thraustochytrids were seen using
the scanner magnification and low
power magnification. Spherical cells
which are translucent white in color and
other cell colonies with brown color were
observed attached at the seed surface.
The brown colonies were treated as
contaminants and the translucent white
colonies were identified as the isolate
and called as CNT01.
The isolate was subcultured onto
another plate to further purify the isolate
which is characterized with the pure
pale cream pus-like colonies in the plate
and spherical translucent white cells
under the microscope.
Subculturing
was repeated several times until pure
culture was obtained
Only one isolate was obtained
from the fallen mangrove leaves
samples.

Table 1. Morphological Characteristics

the Isolate

of

D
Plate 1. (A)Under LPO, (encircled) Baited
Thraustochytrid Isolate. BC-Brown contaminant
colonies. (B) Closer look of brown colonies (C)
Encircled, closer look of the CNT01 (D) Pus-like
Colonies of Thraustochytrid Isolate in a GYPS Agar.

the isolate formed a large

creamy translucent white colony on
GYPS media plate (See Table 1). The
cultures were also observed under
compound microscope at different
magnifications.
Photomicrographs of
the isolate showed cells that were tightly
attached forming a compact clustered
main body which is called the thallus.
(plate 2).
The dividing thallus came
from a single cell that continuously
divide into number of cells, such as
Tetrads (4 cells) and Octads (8 Cells),
until it forms a cluster composed of
numerous cells and during this time the
thallus was considered as mature.
(Plate 2) These dividing cells were form
into
spherical
to
irregular
spherical in shape that is due to the
continuous bipartition of the cells

Plate 2. (A) Thraustochytrids vegetative cells

under LPO. (B.) CNT01 cells under OIO; showing
(TH=Thallus),(T=tetrads) and (O=octads),(C=cluster)
and (MT= mature thallus) formation of the cells.

Optimization was performed to

determine
best
pH,
glucose
concentration, salinity and temperature
that will favor the growth of the isolate.
The optimum growth was measured by
biomass quantification, the higher the
biomass the more favorable the growth
is.
In pH, The isolate was subjected
to different pH such as 4, 5, 6, 7, 8, 9.
Based on the results of the optimization,
the highest biomass was seen at pH 6.0
and lowest was seen on pH 8.0.
In Glucose Concentration Isolate
was subjected to different levels of
glucose concentration such as 7%, 9%,
11%.
Based on the result of the
optimization, the highest biomass was
seen at 11% concentration and the
lowest at 7% concentration.

In Temperature, The isolate was

subjected
to
different
incubation
temperature such as 20C, 25C, 30C.
Based on the result of the optimization
the highest biomass was seen at 20C
and the lowest at 37C.
In Salinity, The isolate was
subjected to different levels of salinity
such as 20ppt, 25 ppt, and 30 ppt.
Based on the result of the optimization
the highest biomass was seen at 25 ppt
and the lowest at 30 ppt.
Based on the result of the
optimization the highest biomass was
seen at 6.0 pH level, 20C incubation
temperature, 11% glucose concentration
and 25 ppt of salinity. The lowest
biomass was observed at 8.0 pH level,
25C incubation temperature, 7%
glucose concentration and 30 ppt of
salinity. (figure,.1)

Figure 1. Growth of Isolate Under Different

Levels of pH, Salinity, Temperature, and Glucose
Concentration

Growth analysis was performed

to determine the growth curve or the life
cycle growth of the isolate; this will
determine the lag, log and stationary
phase growth of Thraustochytrids.
The biomass production of the
isolate was observed within a 5-day
cultivation period. Based on the result of
the average optical density (absorbance

at 660 nm) the highest biomass was

seen at Day 1 and Day 2. (Figure 2)

Figure 2. Growth Curve analysis

The growth curve data obtained

from the isolate showed different cell
growth. At 0 day the result shows its lag
phase, at day 1 and 2 the result show
the log or the exponential phase of the
organism where a small increase in the
organisms biomass and turbidity was
already noted.
However, the most
considerable increase in the organisms
biomass was observed upon sampling
at day 2 even if the organisms turbidity
increased slightly compared to the
previous sampling.
After about the
th
4 day of incubation, the organisms
growth started to slow indicating the end
of the log phase. After this stage of
growth, the organism starts to decline in
biomass and turbidity indicating its entry
unto the death phase of growth by the
6th day of the experiment. The stationary
phase cannot be determined exactly as
it may be somewhere between the 3rd to
5th day of growth.
The isolate was identified through
its morphological characteristic. The
color which appeared as white to palecream; the shape which shows cells

which are microscopically spherical and

that did not undergo repeated binary
division of vegetative cells but
propagate
solely
by
producing
zoospores; the thallus which are
characterized
by
the
successive
bipartition of the cells into tetrads and
octads prior to production of zoospore
(Plate 3) the growth parameters at pH
6.0, 11 % glucose concentration, 25ppt
salinity, and 20C temperature suggest
that the isolate belongs to genus
Schizochytrium.

Plate 3. (A) White to pale-cream colored

isolates. (B.) Thraustochytrids vegetative cells under
LPO showing thallus, tetrads, octads formation of the
cells which are microscopically spherical. (B)

CONCLUSION
Based on the results of the experiment,
Thraustochytrids can be isolated from
the fallen mangrove leaves of Brgy.
Cayucyucan, Mercedes, Camarines
Norte, Philippines and can be grown
best at pH 6.0 in 11% glucose
concentration with 25 ppt incubated at
20C. It is also concluded that the
Thraustochytrid isolated belongs to the
genus Schizochytrium.

RECOMMENDATION
Through
the
conclusions
obtained by the researchers, th following
statements are hereby recommended
for the future studies and researches:
1. To conduct more researches
of the mangrove areas in the
Philippines for the existence
of Thraustochytrids species in
the environment;
2. To
isolate
more
thraustochytrid strains from
the fallen mangrove leaves of
Brgy. Cayucyucan, Mercedes,
Camarines Norte, Philippines;
3. To improve and establish new
techniques for the isolation
and
preservation
of
Thraustochytrids;
4. To further identify the isolate
up to species level through
the use of DNA analysis;
5. To extract and analyze its
secondary metabolities, such
as, polyunsaturated fatty acid;
and
6. To conduct studies in other
economic
uses
of
Thraustochytrids.
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