Pasay City National Science High School

Pasay City National Science High School
2888 Vergel St. Pasay City
Potential of White Rot Fungi (Phanerochaete chrysosporium)

in Filamentous Green Algae (Spirogyra longata)
Growth Inhibition
A Research Study Presented to the

Science and Technology Department
Pasay City Science High School
In Partial Fulfillment
of the Requirements for the subject
Research Project
Presented by:
Barbosa, Jhoanna G.
Buhay, Jhulius Bernard P.
Libarra, Allyka Josh T.
Sayson, Neil Francis F.
G12 Del Mundo
Presented to:
Ms. Laurenna S. Caoctoy
Research Adviser
January 2020
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CHAPTER 1
Introduction
In general, our planet Earth is mainly composed of 75% water which

means that most parts of the world depend on aquatic environment to live and
survive. Since the past decades, issues in the water surfaces have arisen at much
higher rate that negatively affects the world water system (Sun et al., 2017). One
of those problems is the harmful algal blooms (HABs) that affects public health,
fish industry, and even desalination industry (Anderson et al., 2017). Most algal
bloom is resulted from eutrophication, which is the process wherein the bodies
of water have an excess dissolved nutrient (e.g. Nitrogen, Phosphorus, and
Potassium). In addition, eutrophication can be triggered by human activities.
That is why many researchers pursue this research problem to inhibit or control
the HAB to limit the negative effects of algal bloom to the water systems.
Furthermore, if most of the water environment is affected by HABs,

most of the people will suffer because of negative effects of HAB thus making
them harder to survive. For instance, since the Philippines is a tropical country
that has only two season, dry and rainy whereas algal bloom is prominent at dry
season or summertime (“Climate Change and Harmful Algal Blooms”, 2019),
then our country will have a higher chance of having HABs because of this
factor. Additionally, in the Philippines, some of the lakes, bays or fishponds are
continuously affected by HAB causing fish kills, nasty smell (“Laguna de Bay
turns green due to stinky but harmless algal bloom”, 2019), and ailments in
human health. Even though some human activities can cause HABs, they tried
to research for solutions by controlling algal blooms. That is why the researchers
from the Pasay City National Science High School pursue this type of issue to
possible solve the serious case of harmful algal bloom in the country.
Consequently, the researchers choose a kind of ligninolytic fungi that

can possibly degrade algae. Ligninolytic fungi, which has a common term as
Lignin degrading fungi, is one known type of fungi for its potential in
biodegradation and bioremediation (Cajthaml, 2014). One of its example is the
white rot fungi (Phanerochaete chrysosporium), which is a type of aquatic fungi
that is known for its degrading ability due to the secretion of enzymes during its
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metabolism generated by nutrient fatigue (Rodriguez-Couto, 2017). In the past

journals, P. chrysosporium is commonly used as biodegradation agent in some
pollutants because of its ability to degrade while nowadays, this type of fungi
can be used in biomineralization (Espinoza-Ortiz et al., 2017), biosorption
(Noormohamadi, 2018), biorefinery, etc. So, for this study, the researchers will
use the P. chrysosporium as growth inhibitor to a filamentous green alga
(Spirogyra longata), which can cause algal blooms, because of its natural
degrading ability in cellulose which is present in the green algae. Likewise, the
use of Spirogyra longata, commonly known as type of filamentous green algae,
has been introduced to carious researches like biogas production (Ramaraj et
al., 2015) and it is also been known as primary autotrophs in aquatic food webs
(Deb et al., 2019). Furthermore, the growth of this type of algae is quite
uncontrollable because of its mode of reproduction, which is either sexually
(through conjugation) or asexually (fragmentation) that can result to algal
blooms.
Moreover, this study introduces the potential ability of White Rot

Fungi (Phanerochaete chrysosporium) in filamentous green alga (Spirogyra
longata) as a growth inhibitor. The researchers choose Spirogyra longata to
control because it is one of filamentous algae that can be found in the
Philippines (e.g. Laguna de Bay) and filamentous algae are also known to cause
some algal blooms on the surface of the water. Furthermore, researchers choose
P. chrysosporium as growth inhibitor because it has also the presence of
cellulose (Rodriguez-Couto, 2017). In addition, WRF are a type of aquatic fungi
so it can be tested to filamentous algae which is commonly in bodies of water.
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Statement of the Problem
The main problem of the study is to determine the potential of white rot fungi
(Phanerochaete chrysosporium) in inhibiting the growth of filamentous green
algae (Spirogyra longata).
Specifically, the study will answer the following questions:
1. What are the morpho-anatomical characteristics of Spirogyra longata in

terms of cellular structure?
2. What is the optimum amount of P. chrysosporium to inhibit the growth of

Spirogyra longata?
3. What are the effects of P. chrysosporium in the growth inhibition of

Spirogyra longata in terms of:
3.1 Total cell count;
3.2 Chlorophyll-a concentration; and
3.3 Algal cell morphology?
4. How does P. chrysosporium affect the growth inhibition of Spirogyra longata

in terms of total cell count, chlorophyll-a concentration, and algal cell
morphology?
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Objectives
The study aims to test the potential of White Rot Fungi (Phanerochaete
chrysosporium) in inhibiting the growth of filamentous green algae (Spirogyra
longata).
Specifically, the study aims to:
1. Determine the morpho-anatomical characteristics of Spirogyra longata in

terms of cellular structure.
2. Identify the optimum amount of P. chrysosporium to inhibit the growth of

Spirogyra longata.
3. Evaluate the effects of P. chrysosporium in the growth inhibition of Spirogyra

longata in terms of:
3.1 Total cell count;
3.2 Chlorophyll-a concentration; and
3.3 Algal cell morphology?
4. Assess the P. chrysosporium affect the growth inhibition of Spirogyra

longata in terms of total cell count, chlorophyll-a concentration, and algal cell
morphology.
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Hypotheses
Null Hypothesis:
White rot fungi (Phanerochaete chrysosporium) will not have a

significant effect in growth inhibition of filamentous green algae (Spirogyra
longata) in terms of terms of total cell count, chlorophyll-a concentration, and
algal cell morphology.
Alternative Hypothesis:
White rot fungi (Phanerochaete chrysosporium) will have a significant

effect in growth inhibition of filamentous green algae (Spirogyra longata) in
terms of terms of total cell count, chlorophyll-a concentration, and algal cell
morphology.
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Significance of the Study
An algal bloom is the rapid growth of algal cells to the extent that the
algae dominate the planktonic community. It is caused by the nutrient over-
enrichment, particularly phosphorus and nitrogen, in a body of water
(Nwabueze, 2011). Harmful Algal Blooms (HABs) is one of the major problems
that nearly every country located in Southeast Asia encounters every year,
especially, the Philippines. According to Poquita-Du & Todd (2015), the
country has experienced numerous HABs recurrences that were caused by
different algal species starting from the 1980s up until now.
Recently, there was a reported case of algal bloom in Laguna de Bay
from Philippine Daily Inquirer last June 2019, the Muntinlupa City Lake
Management Office (LMO) confirmed that the Spirogyra algae, locally called
as “liya”, was the cause of the algal bloom in the lake. In addition to this, nearly
8% of the total fish production in the Philippines comes from the fish pens in
Laguna de Bay, which is roughly 40% of the aquaculture production as stated
by the Food and Agriculture Organization.
Moreover, HABs have brought negative impacts not only to the aquatic
ecosystem in Laguna de Bay but also to the people living near the lake in terms
of their health, because referring to the Muntinlupa City Lake Management
Office’s (LMO) report, the stench produced by dried-up “liya” may cause
stomach aches, difficulty in breathing, and even respiratory tract infection and
asthma attacks. Furthermore, the algal bloom caused water service interruptions
in the Western part of Metro Manila and some parts in Cavite, and also resulted
to a drop in fish sales for the local fishermen and vendors.
The introduction of a new alternative way of mitigating and controlling
HABs in aquatic ecosystems will greatly contribute in preventing the negative
impacts brought by HABs in the environment and to the health of humans and
animals. In addition, the raw materials that the researchers will use in the study
are obtainable in their country.
The raw material that will be used for the growth inhibition of the algae
is a fungus, which is different from most of the researches that uses bacterial
inoculation as a solution for the management and mitigation for HABs. White
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Rot Fungi, a saprophytic fungus, which is widely known for its ability to
degrade whole wood components (i.e. cellulose, hemicelluloses and lignin) due
to its extracellular non-specific ligninolytic enzymes excreted during their
secondary metabolism (Rodriguez- Couto, 2017).
Since the researchers will use fungi as the growth inhibitor for algae,
the data that will be obtained from this study could be used as a guide for future
researches that are in line and related with the topic of this study.
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Scope and Limitations
The research study will mainly focus on determining the potential of

White Rot (Phanerochaete chrysosporium) Fungi as a growth inhibitor of
Filamentous Green (Spirogyra longata) Algae. In addition, the study aims to
determine the potential ability of Phanerochaete chrysosporium as a growth
inhibitor of Spirogyra longata as a possible alternative solution for controlling
and mitigating HABs rather than commercializing it. The Spirogyra longata
will be gathered directly from the Laguna Lake while the Phanerochaete
chrysosporium isolates will be bought from the National Institute of Molecular
Biology and Biotechnology in University of the Philippines Los Baños, Laguna.
Furthermore, the researchers will prepare a total of 3 setups for the
experimentation wherein each setup has a different ratio of fungi to algae (0:1,
1:1, and 2:1) and will be treated in the same environment to ensure consistency
throughout the experimentation. The setups will undergo laboratory tests to
determine the potential ability of Phanerochaete chrysosporium as a growth
inhibitor of Spirogyra longata in terms of total cell count via hemocytometer,
algal cell morphology via scanning electron microscopy, and chlorophyll-a
concentration.
The study is limited only in determining the potential ability of White

Rot (Phanerochaete chrysosporium) Fungi as a growth inhibitor of Filamentous
Green (Spirogyra longata) Algae. There are many ways of characterizing
Phanerochaete Chrysosporium to determine its potential ability as a growth
inhibitor of Spirogyra longata, however, the study will only focus on
determining the total cell count, algal cell morphology, and chlorophyll-a
concentration. Furthermore, the only conditions in the environment that the
researchers will control during the experimentation are the pH level,
temperature and light exposure. In addition, the researchers will have no control
over the impurities that might occur during the tests that will be done by experts
and conducted in different testing facilities that may affect the results. The study
will be conducted within the school premises and researchers’ vicinity and will
only last according to the allotted time given by the research adviser.
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Definition of Terms
Algal bloom is a relatively rapid increase in the population of a specific algae in

an aquatic system. It is the problem and the reason that the study will be
conducted. The study will give answer to the overgrowing of algae species.
Autotrophic are organisms that are able to make energy-containing organic

molecules from inorganic raw material. The filamentous green algae are an
autotropic organism which will be used in the study.
Biocontrol is a method of controlling a group of organisms by using another set

of organisms. It is the process in which the fungi will be used to control the
overgrowing of the algae which is covered by the study.
Biomass is a measure of the mass of the living component of soil organic matter.
It is used as a measurement to the growth of the algae.
Chlorophyll-a is a type of chlorophyll that is most common and predominant in

all oxygen-evolving photosynthetic organisms such as higher plants, red and
green algae.
Conjugation is a form of sexual reproduction of bacterial cells in which two

bacterial cells come together in a temporary fusion to transfer genetic material.
It is a reproduction process of filamentous green algae that occurs between its
algal cells.
Fragmentation is a form of asexual reproduction wherein a parent organism

breaks into fragments and individually grow into a new organism. It is also
another reproduction process that the filamentous green algae undergo.
Hemocytometer is a apparatus that is used for counting blood cells in

quantitatively measured volume of blood. It will be used as the measuring tool
to count the cells of the spirogyra longata before and after the inhibition process.
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Inhibition is the process of slowing or preventing a certain process, reaction or

function by a particular substance. It will be used as process to control the
overgrowing of the algae specie.
Lentic Environment refers to the terrestrial waters such as lake and ponds. In
this type of environment, freshwater filamentous green algae will be collected.
Morpho-anatomy is the study of anatomical forms and structures which is used

to differentiate species. This will be used as the basis to confirm and identify
the structure of the spirogyra longata that will be gathered from Laguna Lake.
Phanerochaete chrysosporium commonly known as the white rot fungi, is a

saprophytic fungus capable of the woody part of dead plants. This is the fungi
specie that will be used in the study as growth inhibitor to the algae.
Spirogyra longata commonly known as filamentous green algae, is a species of

algae that has spiral arrangement of chloroplasts which is commonly found in
freshwater environment. It will be used as the dependent variable in which its
growth will be inhibited by the fungi and it will be observed in the study.
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CHAPTER 2
Related and Review of Literature and Studies
This chapter presents the related literature and studies assimilated

after thorough and in-depth search done by the researchers. Several works of
authors locally and internationally were analyzed in order to formulate a solid
background of the present study. Only those that were relevant were studied.
Filamentous Green Algae (Spirogyra longata)
Figure 1 Microscopic image of Spirogyra longata

Retrieved from
http://bioweb.uwlax.edu/bio203/2010/fenske_megh/adaptation.htm
The genus spirogyra longata also known as filamentous green alga is

very common type of freshwater algae which can be easily found in freshwater
ecosystems such as streams, lakes and ponds. Additionally, it is photosynthetic
in nature and has a long bright grass-green filament that is composed of spiral-
shaped chloroplasts which makes it unique from other freshwater algae. It also
contains chlorophyll-a and chlorophyll-b pigments that makes it green in
appearance, although there is some spirogyra sp. that are colored yellow and
orange which is due to the presence of carotenoid pigments. Furthermore,
filamentous green algae are promising source of various components like
sugars, carotenoids, fatty acids, amino acids, toxins and even antioxidants
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(Kumar et. al, 2015). Since then, different researches were conducted using
spirogyra because of its diverse application in different fields such as
biotechnology, industries and food for aquatic organisms (Ramaraj, Unpaprom,
Whangchai, & Dusadee, 2015). According to the Editors of the Encyclopedia
Britannica (2015), spirogyra is consisting of thin unbranched chains of
cylindrical cells. In addition to that, spirogyra species can reproduce both
sexually through conjugation in which two filaments that are side by side are
joined by conjugation tubes forming a zygote, and asexually through
fragmentation reproduction of its filaments thus reproduction of filamentous
green algae is straightforward and can swiftly process. Specifically, spirogyra
longata undergoes to ladder-like conjugation, producing conjugation tubes by
both of its gametangia. Thus, spirogyra longata has a high reproduction rate
due to its reproduction method (Joseph, 2017).
Moreover, as presented in the study of Krupek et al. in 2015, there are

other physical variables (such as light, substrate type and the velocity of the
currents) that are directly accountable for the growth of diverse algae
community in lentic areas. Based on a study conducted by Ashok et al., the
dissolved oxygen by the filamentous green algae has an indirect relation with
the temperature of the environment. As the temperature increases, the solubility
of oxygen decreases resulting to deaths of aquatic animals due to loss of oxygen
(2015). In addition, the Lake Management Office also stated that deaths of algae
occur when temperature is high (above 30 degrees Celsius) (Cabalza, 2019). It
was also discovered in the study of Kumar et. al, that exposure of an alga
biomass to Ultraviolet rays will increase its biomass but can also result to algal
death which is responsible for the foul smell.
The rapid growth of algae causes harmful algal blooms and Harmful
algal blooms (HABs) affect not only the health of the citizens, but it also affects
the aquatic life and the fishery industries (Jia, 2013). A recent study was also
conducted on the shallow water areas of Lake Baikal, Russia that there are
heavily overgrowing filamentous green algae existing in the lake which affect
the reproduction of yellowfin sculpin. The overgrowth of the filamentous algae
results to restricting access to substrates that are used by yellowfin sculpin in
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egg laying, consequently resulting to decrease of reproduction rate of the

yellowfin sculpin (Khanaev, Dzyuba, Kravtsova, & Grachev, 2016).
Furthermore, the genus spirogyra is much more harmless than

cyanobacteria for the reason that cyanobacteria releases cyanotoxins in the
bodies of water affecting the lives of the aquatic organisms and even humans.
However, when filamentous green algae reproduce and rapidly grow thus
increasing its biomass, it will then be harmful to aquatic organisms.
In addition to that, a news from Philippine Daily inquirer that was

released on June 2019 stating that the on- going algal bloom releases bad smell,
which is caused by the spirogyra longata in Laguna de Bay, is not a threat to
the people nevertheless it can still harm aquatic animals in the Bay. The Lake
Management Office (LMO) of Muntinlupa City stated that the filamentous
green algae are beneficial because it is a natural food for fishes, nonetheless,
increase in biomass of spirogyra longata will harm the aquatic ecosystem in the
lake. Algal bloom of spirogyra causes a verdant odor but when it decomposes
it releases foul smell.
Due to the presence of heavily overgrown filamentous green algae and

other periphytic biomass, the production of water that is treated by water
treatment facilities decreased. The spirogyra longata obstructs filters in water
treatment systems which is a major problem and risk to the public health,
affecting our water reservoirs. In addition to that, it was also stated that the water
treatment facilities on Laguna de Bay doesn’t yet have a long-term solution for
the algal problem (Cabalza, 2019).
White Rot Fungi (Phanerochaete chrysosporium)
Figure 2 Microscopic image of P.

chrysosporium
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Retrieved from:
https://mycocosm.jgi.doe.gov/Phchr1/Phchr1.hom
e.html
Phanerochaete chrysosporium, a very known eukaryotic organism

under kingdom fungi, is a type of white rot fungi that is known for its
degradation ability to aromatic polymer lignin. These white rot fungi release
extracellular enzymes to break down lignin structures throughout its second
metabolism that is triggered by nutrient lethargy (Rodriguez-Couto, 2017).
For the past years, researches using this material is kind of a trend
because it can be used as a variable for biodegradation. The degradation ability
of Phanerochaete chrysosporium have been tested in synthetic dyes in
wastewater (K iran et. al, 2019), landfill leachate (Hu et. al, 2019), and even
pulping waste from paper industry which can greatly reduce the waste because
of the waste lignin the paper industry produces in large amounts (Venkateswara,
2018; Yang et al., 2019).
Moreover, this fungus with the help of immobilization can use for
bioremediation of composite-polluted wastewater that results in higher absorption
of pollutants in the water (Huang et. al, 2015). Also, recent research about the
Phanerochaete chrysosporium opens its potential to biosorption to heavy metals
such as nickel and cadmium (Noormohamadi et al., 2018). Additionally, these
white rot fungi also been experimented for biological augmentation machinery for
treating wastewater which results to improvement of reactor performance because
of shaping the microbial community composition which may help for future
researchers about the ecological race in sludge wastewater and toxicity
lessening of phenol wastewater (Hailei et. al, 2016).
Further studies about the Phanerochaete chrysosporium disclose its

ability on toxicity testing in some elements and heavy metals like the reaction
of the fungi to graphene oxide (GO) which gives toxicity level at maximum if
the concentration of the GO is higher resulting to the inhibitions of white rot
fungi’s activity (Xie et al., 2016). In addition, heavy metals like cadmium (Cd)
can significantly affect the ligninolytic enzyme activity of the Phanerochaete
chrysosporium if exposed at longer duration, resulting to the inhibition of its
15
extracellular secretions, but the good side to that study is the observation of
cadmium crystal particles, which concluded as a detoxification reaction of the
fungi in which the dissolved cadmium ions are precipitated that reduces result
toxicity and bioavailability (Chen et al., 2015). This study can help as a
reference for bioremediation of fungi against heavy metals. Contrary to the
previous study, other studies like Cao et al., 2017 introduced the biodegradation
of BDE-47 compound, that is also recognized as a toxic compound, using
Phanerochaete chrysosporium with the company of Cd2+ resulting to effective
degradation of bromodiphenyl ether (BDE-47) but reducing the amount of two
typical enzymes for lignin degradation, Lignin Peroxidases (LiP) and
Manganese Peroxidases (MnP), as the concentration of Cd2+ goes high. But
because the focus of the study is to inhibit the growth of algae by means of
biodegradation that has lignin present in algae system, the study of using P.
chrysosporium with Cd2+ can’t help the study to biodegrade the green algae
because of that reasoning.
Given some of its biological uses, P. chrysosporium have become a

standard organism for biodegradation because of its capacity to produce lignin-
degrading enzymes, which are the lignin peroxidases (LiP) and manganese
peroxidases (MnP) (Singh and Chen, 2008). Additionally, aside from the
capacity of P. chrysosporium to produce lignin degrading enzymes, it also
secretes extracellular cellulase complexes interdependently act to degrade
cellulose (Skyba, Cullen, Douglas, and Mansfield, 2016) which is helpful to this
study because the filamentous green algae have a presence of cellulose. In
addition, the same journal also proved that the gene expression of the P.
chrysosporium during degradation is affected by poplar wood substrate and its
incubation time. The study also determined some proteins that they called as
“proteins of unknown function” that may be a challenge for future research
about determining the proteins’ precise role in lignocellulose degradation
(Skyba, Cullen, Douglas, and Mansfield, 2016).
To improve more its potential, some researchers tend to deconstruct,

rearrange, and even adding some nanoparticles like silver nanoparticles, simply
AgNPs, at low doses (Huang et al., 2016) to influence the fungi’s ability to make
those enzymes to have better creation of stable lignin-degrading enzymes.
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These enzymes are important because it can degrade the lignin structure of an
organism. Some structures that lignin can be used for biofuel and bioenergy
which is really need nowadays because of shortage in nonrenewable resources.
Furthermore, the use of P. chrysosporium needed will be cultured on

potato dextrose agar (PDA) plates for 5 days at 4°C at the incubator, and then
subculture. After 7 days, the mycelial mats in the plate will be used for algicidal
experimentation.
Harmful Algal Blooms
Algae are considered as valuable indicators in aquatic ecosystems

because of their ability to respond quickly to species densities and compositions
that are caused by the changes in water composition (Deb, Saikia & Kalamdhad
2019). However, when algal cells proliferate or accumulate quickly, to the
extent that it dominates the planktonic community, can lead to a phenomenon
called algal bloom (Nwabueze, 2011). Algal blooms are often caused by
excessive nutrient input, particularly phosphorus and nitrogen (Nwabueze,
2011). According to Liu, Wang, Wei, Dong, & Hui (2012), algal blooms badly
damage the stability and balance of aquatic ecosystems, which brings negative
impact on aquaculture development, especially, the toxin-producing blooms,
often known as harmful algal blooms (HABs), can cause illnesses to humans
and animals.
Harmful algal blooms (HABs) are evident results of ecosystem

imbalance which is the effect of numerous environmental changes. HABs are a
major problem for brackish, marine, and freshwater systems around the world,
particularly, coastal countries (Watson et a.l, 2015). Moreover, species that
cause HABs mostly comes from major taxonomic groups, including planktonic
cyanobacteria (cyanoHABs), invasive flagellates, eukaryotic plankton, and
filamentous chlorophytes (Watson et al., 2015).
In addition, some HABs that are considered toxic such as planktonic

cyanobacteria, often poses a serious threat to the health of humans and aquatic
ecosystem because of the toxins that they produce (Paerl et al., 2015). However,
17
filamentous chlorophytes are an example of HAB species that can be beneficial

both for aquatic organisms and humans because they serve as a source of food
and as a component in biological monitoring for evaluating water quality (Deb,
Saikia & Kalamdhad 2019).
With the numerous reports of occurrences and recurrences of HABs

across rivers, reservoirs, lakes, and other surface waters globally, this problem
has gained a lot of attention and therefore, lead many scientists and researchers
to conduct studies for its mitigation and management. According to Paerl et al.
(2016), the reduction of the nutrient inputs i] such as nitrogen (N) and
phosphorus (P) inputs are the primary focus in addressing mitigation processes
for HABs. However, climate change makes it more challenging to control
HABs because the increase in water temperature also led to the increase in
frequency of occurrences of cyanobacteria.
Mitigation and management approaches such as deployment of vertical

mixing, enhanced flushing in riverine waters and canals, and removal of
buoyant surface blooms by skimming, collecting, and concentrating, were all
reported to be successful. However, these physical approaches were only
successful and efficient in small ecosystems but not in larger reservoirs and
lakes (Paerl et al., 2016).
Biocontrol
Nowadays, Harmful Algal Blooms have gained more attention by

different researchers. Various strategies were conducted and experimented to
develop a method to control HABs and one of this effective strategy is by using
biocontrol agents. A similar study was conducted by Jia, Du, Fang, Zhao & Tian
(2013), discovered the effectivity of two specie of fungi -Trichaptum abietinum
and Lopharia spadicea as biocontrol agents to a variety of freshwater algal
species namely, Cyanophyta, Cholorphyta, Dinophyta, Cryptophyta,
Bacillariophta and Euglenophyta. In addition, wood-rot fungis like the
Trichaptum abietinum and Lopharia spadicea have algacidal abilities, it can
degrade the algal cells directly since the algal cells were enclosed by a mucous
18
membrane which is caused by the fungal mycelia. Thus, the two fungi
significantly inhibited different freshwater algae in growing except for the
Euglenophyta due to its capability to absorb some substances from the two fungi
(Jia, Du, Fang, Zhao & Tian, 2013).
Algal Biomass
Biomass determination is one of the most fundamental ecological

measurements. Although it is a static mea-security, biomass can provide
information about either the relative importance of a taxonomic unit within a
community (which has implications for competitive interactions and
biodiversity) or the influence of a community within an ecosystem. Benthic
algal biomass calculation is complicated by the fact that benthic algae typically
coexist as part of a complex assembly known as periphyton, biofilm, or the
German word "Aufwuchs". Benthic assembly evaluation of algal biomass is
confused by the presence of other organic components. If there is interest in the
role of algal assembly only, such as how much the benthic algae contribute to
the primary production of the total system or to the growth of a specific
invertebrate grazer, separating the algae from the other periphytic components
is critical (Steinman, 2017). In addition, microalgae are a group of
microorganisms in biotechnology processes that have various applications.
Biofuel processing, for example, is a promising use of microalgae that has
attracted considerable attention recently.
Scanning Electron Microscopy (SEM)

SEM is a method of microscopy which allows better resolutions than
of optical microscopes and is used for distinguishing the surface morphology,
and morphometry of the samples being studied (Santos, 2019). The scanning
electron microscope (SEM) may also be useful for revealing morphological
characteristics of isolated organisms and for diagnosis, but problems with
specimen preparation methods in the past have limited the use of SEM for
routine microbiology. It is used less commonly than transmission electron
19
microscopy for microbes such as viruses or bacteria, although it is an excellent

tool for studying ultrastructure (Golding, 2016). In this study, SEM will be used
to assert the structural differences of the algal samples before and after the
experimentation.
Total Chlorophyll-a Count
Figure 2 Chemical structure of Chlorophyll-a

Retrieved from
https://pubchem.ncbi.nlm.nih.gov/compound/Chlorophyll-a
According to the U.S. National Library of Medicine, chlorophyll is a

green pigment found in most plants, algae, and cyanobacteria. Hans Fischer
elucidated the general structure of chlorophyll a in 1940, and by 1960, when
most of the chlorophyll a stereochemistry was established, Robert Burns
Woodward published a complete molecule synthesis as understood at that time.
Chlorophyll a is the plant's most abundant pigment (although not always in
algae), and its absorbance is therefore most commonly measured. However, the
absorption of chlorophylls b and c and other accessory pigments, as well as the
degradation products of these pigments, can also be measured (Steinman, 2017).
The chlorophyll-a count was used to determine the algicidal efficiency of the
fungi. The decrease of chlorophyll-a content of the samples indicated the
effectivity of the fungi as a biocontrol (Zeng, 2015). The chlorophyll count was
determined through extraction with 90% (v/v) acetone and determined using
modified standard methods. Following equation was used for the calculation of
the chlorophyll content using UV/VIS spectrophotometer.
20
CHAPTER 3
Materials and Methods
This chapter describes the details on the study of determining and

testing the potential of White Rot Fungi (Phanerochaete chrysosporium) in
Filamentous Green Algae (Spirogya longata) growth inhibition. The
experiment will be done by dividing the procedure into three phases as shown
in Figure 4.
Collection of Materials
Phase 1 • 18 g of Spirogyra
Gathering of Materials longata
• 450 mg of P.
chrysosporium
• 9000 ml of Deionized
Water
• 10 L of Jar
• Filter Paper & 1x1 nm
Sieve
Phase 2 Algal Identification
Algal Growth Inhibition
Algal Culture Maintenance
Growth Inhibition Test
Phase 3 Total Cell Count

Flowcytometry and
Hemocytometer Test, and Chlorophyll Concentration
Scanning Electron Microscopy
Algal Cell Morphology
Collection of Data
Figure 4 Schematic Diagram of the Methodology
21
Research Design
This study will determine and characterize the potential of white rot
fungi P. chrysosporium as a growth inhibitor of filamentous green algae
Spirogyra longata. Firstly, the algae samples will be collected in Laguna lake
and maintained under an autotrophic condition as described by Ramaraj et al.
(2015). Fungal strains will be obtained from University of the Philippines, Los
Baños. To determine the optimum amount of P. chrysosporium in inhibiting
Spirogyra longata growth, there will be one controlled set-up and two
experimental set-ups. To evaluate the algicidal efficiency of P. chrysosporium
against Spirogyra longata, the samples will be conducted at 48 hours (Munir et
al., 2015). All of the setups will be processed under the conditions of 25 °C, pH
7.0, and 12 h light/12 h dark cycle (Zeng, 2015). All the experiments will be
conducted in triplicate. After the experimentation, the biomass variation of
Spirogyra longata will be measured and compared to its weight before the
growth inhibition test. Several analytical testing will also be conducted such as
the Total cell count via Hemocytometer, Chlorophyll-a concentration, and
Scanning Electron Microscopy (SEM) for algal cell morphology.
Phase 1- Gathering of Materials
Spirogyra sp. samples will be collected at Laguna de Bay through the

Lake Management Office in Muntinlupa City. 1x1 mm sieve will be used to
remove the macro particles present and separate the macroalgae. The same
water from the sampling zone will be used as medium however, this time it will
be filtered through a 0.45 µm filter paper. P. chrysosporium will be obtained at
the Culture Collection of the National Institute of Biotechnology (BIOTECH)
at the University of the Philippines Los Baños (UPLB).
Phase 2- Experimentation
The algal samples will be identified through a Jar test and observation
under a light microscope, and authenticated at the National Museum of the
Philippines.
22
Algal Culture Condition and Maintenance
Spirogyra longata will be grown in autotrophic conditions of 10 L open

type outdoor jar. The jar containing 5 L working volume and 5 L base filled
with sterilized white sand.
Growth Inhibition Test
Liquid test will be conducted in a 1500mL flask containing P.

chrysosporium, and medium of Spirogyra longata culture. The experimental
set-ups are composed of 50 mg, and 100 mg P. chrysosporium to 2g/L Spirogyra
longata, respectively. The same amount algal culture but without the fungus
will be used as the control set-up throughout the experiment. To evaluate the
inhibitory effects of P. chrysosporium against the Spirogyra longata, the
samples for testing will be conducted at 48 hours (Munir et al., 2015). The
inhibition test is presumed to be managed under the conditions of 25 °C, pH 7.0,
and 12 h light/12 h dark cycle. The set-ups will be conducted in triplicate.
Table 1
P. chrysosporium to Spirogyra longata Proportions
Set-Up No. Fungi (mg) Algae (g) Medium (mL)
1 50 2 1000
2 100 2 1000
3 (Controlled) 0 2 1000
Phase 3 – Analytical Testings
Total Cell Count
Hemocytometer will provide the total cell count of the samples that the
researchers will need in order to analyze whether P. chrysosporium has been an
effective growth inhibitor for Spirogyra longata.
23
Chlorophyll-a Concentration
To acquire the algicidal efficiency of P. chrysosporium against the

Spirogyra longata, the total number of chlorophyll-a will be measured as
follows:
R= (Co-Ct) /Co x100
Where R is the percentage of chlorophyll-a content (%), Co is the initial

chlorophyll-a content (μg/L), and Ct is the chlorophyll-a content after treatment
(μg/L). All of the experiments described above were carried out in triplicate,
and the data are reported as the averages of three separate experiments ± SD (n
= 3) (Zeng, 2015).
The method will be done using a centrifuge and a spectrophotometer at

the De La Salle University, Taft Ave., Malate, Manila.
Scanning Electron Microscopy
The algal cell morphology and microstructural changes of the Spirogyra

longata treated with and without P. chrysosporium will be analyzed by scanning
electron microscopy (SEM) in De La Salle University, Taft Ave., Malate,
Manila.
Collection of Data
The alginate-based aerogel foam concrete will undergo physical and

chemical performance test to determine its ability. Quantitative analysis results
will be obtained on the result of thermal conductivity test, as well as the density
and compressive strength tests. One-way Analysis of Variance (ANOVA) was
used to determine whether there are significant differences between the thermal
conductivity, and density and compressive strength of the concrete.
24
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Potential of White Rot Fungi (Phanerochaete chrysosporium)

A Research Study Presented to the

In general, our planet Earth is mainly composed of 75% water which

Furthermore, if most of the water environment is affected by HABs,

Consequently, the researchers choose a kind of ligninolytic fungi that

metabolism generated by nutrient fatigue (Rodriguez-Couto, 2017). In the past

Moreover, this study introduces the potential ability of White Rot

Statement of the Problem

Specifically, the study will answer the following questions:

1. What are the morpho-anatomical characteristics of Spirogyra longata in

2. What is the optimum amount of P. chrysosporium to inhibit the growth of

3. What are the effects of P. chrysosporium in the growth inhibition of

3.1 Total cell count;

3.2 Chlorophyll-a concentration; and

3.3 Algal cell morphology?

4. How does P. chrysosporium affect the growth inhibition of Spirogyra longata

Specifically, the study aims to:

1. Determine the morpho-anatomical characteristics of Spirogyra longata in

2. Identify the optimum amount of P. chrysosporium to inhibit the growth of

3. Evaluate the effects of P. chrysosporium in the growth inhibition of Spirogyra

3.1 Total cell count;

3.2 Chlorophyll-a concentration; and

3.3 Algal cell morphology?

4. Assess the P. chrysosporium affect the growth inhibition of Spirogyra

White rot fungi (Phanerochaete chrysosporium) will not have a

White rot fungi (Phanerochaete chrysosporium) will have a significant

Significance of the Study

Scope and Limitations

The research study will mainly focus on determining the potential of

The study is limited only in determining the potential ability of White

Algal bloom is a relatively rapid increase in the population of a specific algae in

Autotrophic are organisms that are able to make energy-containing organic

Biocontrol is a method of controlling a group of organisms by using another set

Chlorophyll-a is a type of chlorophyll that is most common and predominant in

Conjugation is a form of sexual reproduction of bacterial cells in which two

Fragmentation is a form of asexual reproduction wherein a parent organism

Hemocytometer is a apparatus that is used for counting blood cells in

Inhibition is the process of slowing or preventing a certain process, reaction or

Morpho-anatomy is the study of anatomical forms and structures which is used

Phanerochaete chrysosporium commonly known as the white rot fungi, is a

Spirogyra longata commonly known as filamentous green algae, is a species of

Related and Review of Literature and Studies

This chapter presents the related literature and studies assimilated

Filamentous Green Algae (Spirogyra longata)

Figure 1 Microscopic image of Spirogyra longata

The genus spirogyra longata also known as filamentous green alga is

Moreover, as presented in the study of Krupek et al. in 2015, there are

egg laying, consequently resulting to decrease of reproduction rate of the

Furthermore, the genus spirogyra is much more harmless than

In addition to that, a news from Philippine Daily inquirer that was

Due to the presence of heavily overgrown filamentous green algae and

White Rot Fungi (Phanerochaete chrysosporium)

Figure 2 Microscopic image of P.

Phanerochaete chrysosporium, a very known eukaryotic organism

Further studies about the Phanerochaete chrysosporium disclose its

Given some of its biological uses, P. chrysosporium have become a