Culturable

Heterotrophic Bacteria associated with a laboratory culture of

Botryococcus braunii, a colonial oil-producing green alga from Paoay Lake,
Ilocos Norte
Chapter 1

INTRODUCTION

1.1. Background of the Study

Botryococcus braunii (Kutzing,1849) is a colonial freshwater green alga, a
member of the Chlorophyceae (Chlorophyta) which is distinctive for its ability to
produce and accumulate oil from the long chain hydrocarbons found in lipid
substances. Oilcomprise 30-75% of B. brauniis biomass (Metzger et al., 1991;
Qin, 2005).

B. braunii is widely distributed in freshwater lakes, ponds or

reservoirs with various climates; continental, temperate, tropical, or alpine zones.

This global distribution is confirmed from the strains originating from Portugal,
Bolivia, France, Ivory Coast, Morocco, Thailand and including the Philippines
(Metzger et al., 1985). Blooms caused by microalgae such as B. brauniiin Paoay
Lake, Ilocos Nortehave been known to cause harmful effects on a variety of
freshwaterorganisms such as the fishes, zooplankton, and other species (Papa
et. al. 2008). Interactions between bacteria and algae are not well documented.
This algal species are likely to harbor unique populations of carbon utilizing
heterotrophic bacteria.

Hetetrophic bacteria, organic carbon-based compound utilizing microorganisms

thriving commonly in freshwaters, were shown to exert considerable influence,
antagonistic or beneficial, on B. braunii growth yield and hydrocarbon production
(Chirac et al., 2004). Bacteria can stimulate algal growth by releasing substances
such as vitamins and organic chelating agents (Haines and Guillard, 1974). The
presence of various microorganisms can influence not only the quantity of
hydrocarbons produced, but also their level in the algal biomass and their relative
abundance (Metzger et al., 2004).

With its richness in oil and ability to form rustic-colored blooms, B. braunii has
been proposed as a renewable source of liquid fuel for the present generation. In
view of its importance, this study aims in isolating heterotrophic bacteria
associated with Botryococcus braunii, which could have the potential of
enhancing or decreasing its hydrocarbon production.

1.2. Statement of the Problem

The study is conducted to isolate and identify the culturable heterotrophic
bacteria found having close association to green microalgae, Botryococcus
braunii, in Paoay Lake, Ilocos Norte.
It particularly seeks to answer the following questions:
1. What culturable heterotrophic bacteria can be found in association with
Botryococcus braunii?
2. What kind of medium can optimally support the growth of these
2

heterotrophic bacteria?
3. What are the characteristics of the isolated heterotrophic bacteria with
Botryococcus braunii?
4. What are the antibiotic properties found on these isolated freshwater
bacteria?

1.3. Objectives of the Study

This research aims:
1. To isolate and purify heterotrophic bacteria from Botryococcus braunii.
2. To formulate a medium based on examination of growth factor and
nutritional requirements that will promote optimal growth to the isolated
heterotrophic bacteria.
3. To identify and characterize the isolates through a series of morphological
and biochemical tests.
4. To test for antibiotic properties using the Burkholder plate assay.

1.4. Significance of the Study

In the present day, the world is running out of fossil fuels. The main problem is
that fossil fuels are limited and the demands of the people are increasing. As
different nations become more and more developed, an increasing amount of
fossil fuel is demanded. In order to solve this worldwide dilemma, natural
solutions like biofuels, which is found to be the best alternative to fossil fuels, are
considered. Much attention has been paid to microalgae with high oil productivity
3

such as Botryococcus braunii for they are found to be efficient sources of

biodiesel that are beneficial in the industrial field. The interaction of heterotrophic
bacteria with microalgae influences the algal biomass and the quantity of
hydrocarbons produced. Thus, leading to higher production of oil. However, there
are not many reports regarding specific types of bacteria that are associated with
Botryococcus braunii from other countries, and no information at all from
Philippine isolates.

1.5. Scope and Limitations

The scope of this study is on isolation and identification of the culturable
heterotrophic bacteria associated with Botryococcus brauniiin Paoay Lake, Ilocos
Norte. It also focuses on formulating a culture medium that will promote optimal
growth of the isolated microorganisms in vitro. Moreover, the isolated bacteria
are tested for antimicrobial properties through the Burkholder disk agar diffusion
assay.

This paper does not delve deeper into the details of the algal bloom and unusual
blooming coloration exhibited by Botryococcus braunii. In addition, the
identification of B. braunii will be conducted on-site through microscopy. Thus,
identification is limited only on the morphological and phenotypic basis and is not
tested on a molecular level.

CHAPTER 2

Review of Related Literatures

2.1Botryococcus Braunii
Botryococcus braunii (Kiitzing, 1849), a unicellular photosynthetic microalga is a
member of Chlorophyceae (Chlorophyta). It is distinguished by its considerable
production of lipids and is widely distributed in many water bodies; freshwater
lakes, ponds o reservoirs, transient lakes or salty waters. This colonial microalga
is widespread in fresh and brackish waters of all continents (Chisti, 1980). The
cosmopolitan nature of the algae is confirmed by the strains originating in the
USA (Wolf et al., 1985a, b), Portugal, Bolivia, France, Ivory Coast, Morocco,
Philippines, Thailand, and the West Indies (Metzger et al., 1985a).

Botryococcus braunii, under light microscopy observations, the cells, are

generally pear-shaped or in pyriform, green in color, they are as if embedded
within a concave and are positioned around the border of the cluster. A cluster or
colony varies in size from 30nm up to 2mm and is held together by a matrix

infused with oil. Due to this cohesion, the colony appears to consist of single or
multiple cells. Ultra structural investigations show that the matrix consists of outer
walls originating from successive cellular divisions more or less closely pressed
to each other so these outer walls ensure colony cohesion. (Metzger, 1997)
Furthermore, the structural elements building up the matrix of the colonies in the
three races appear to consist of insoluble and chemically resistant biopolymers
arising from an extended cross-linkage of macromolecular lipids.

Image A shows Botryococcus braunii cells as seen under 40x Light Microscope (Sarada
et. al., 2007) Image B shows a microscopic image of the hydrocarbon oils are being
released as large droplets from the matrix (http://newenergyandfuel.com/wpcontent/uploads/2010/03/Botryococcus-braunii.jpg)

This bloom-forming algal flora dominates many water bodies. B. braunii is

uniquely characterized because of its ability to produce large amounts of
hydrocarbons. Such ability associated with the oil richness of this microorganism
-an oil content of 86 per cent of dry weight was reported by Brown et al. (1969)
from a bloom in a freshwater lake. Historically, the pioneering studies on
Botryococcus were concerned with the implication of this microalga in the

formation of some fossil materials ranging in age from Ordovician to present

(Cane, 1969, 1977; Cane and Albion, 1973) and exhibiting high petroleum
potentials. Later, fossil chemical compounds, specifically derived from B. braunii,
were found in high abundances in some crude oils (Moldowan and Seifert, 1980;
Brassell et al., 1986; McKirdy et al., 1986), or in some recent sediment (Huang et
al., 1996). Interest in modern B. braunii arose from the discovery in the 1960s,
that the living algae are able to synthesize high amounts of hydrocarbons (Swain
and Gilby, 1964; Maxwell et al., 1968), while it was considered until then that the
oil impregnating the matrix of the colonies consisted essentially of fatty acids
(Zalessky, 1926; Blackburn, 1936).

Three races of B. braunii have been documented, and these can be differentiated
on the basis of the characteristic hydrocarbon they are able to produce. Setting
hydrocarbon type as a standard to differentiate these three races was due to
small morphological differences existing between them and thus, the stains are
classified into three chemical races; A, Band L by reference to alkadienes,
botryococcenes and Iycopadiene, respectively (Metzger et al., 1985a, 1990).

The A race produces odd-numbered C25 to C31, n-alkadienes, and trienes.The

B race produces triterpenoid hydrocarbons known as botryococcenes (CnH2n
10, n = 30-37), apparently of isoprenoid origin (Chisti, 1980). The L race
produces lycopadiene, a C40 tetraterpene.

Regarding taxonomy, scientists Komarek and Marvan (1992) have proposed a

new taxonomy for Botryococcus, including 13 species. In this context they have
recently found (Metzger et al., 1997) that two strains they have classified in the L
chemical race are also considered as being Botryococcus neglectus (Jeeji Bai,
1996). Considering the great number of papers referring to the traditional name,
B.

braunii,

and

considering

the

difficulties

encountered

to

appreciate

morphological changes, not always stable under different culture conditions

(Plain et al., 1993), it is preferred however to maintain this species name, paired
with the appropriate epithet of the chemical race, A, B or L.

The A race produces odd-numbered C25 to C31, n-alkadienes, and trienes.

These linear olefins can constitute up to 61% of the dry cell mass of the green
active-state colonies (Gelpi et al., 1970). Two unusual hydrocarbons, C27H51
triene and C27H48 tetraene, were isolated from a B. braunii strain originating in
Lake Overjuyo, Bolivia.

The B race produces triterpenoid hydrocarbons known as botryococcenes

(CnH2n10, n = 30 37), apparently of isoprenoid origin (Chisti, 1980).
Botryococcenes can exist as isomers with the same number of carbons but
different structures. In natural populations, botryococcene can constitute from 27
to 86% of the dry cell mass (Brown and Knights, 1969).The L race produces a
single hydrocarbon C40H78, a tetraterpene, known as lycopadiene(Metzger et
al., 1990). This hydrocarbon accounts for 2 to 8% of the dry biomass (Metzger
8

and Casadevall, 1987).

Even though these races have morphologies that are almost similar with each
other, sometimes, basing in hydrocarbon products alone can be rigorous and
complex. And so, under microscopy, these three races can be differentiated on
the basis of certain morphological and physiological characteristics. The cells of
the L race (8 to 9 m 5 m in size) are relatively smaller than the cells of races
A and B (13 m 79 m) (Metzger et al., 1988). The L race also contains less
pyrenoid (Metzger et al., 1988). Another difference among the races is the colony
color in the stationary phase. The B and L race algae turn red-orange and orange
brownish from green color of the active state. The A race alga turns pale yellow
from green. This difference is due to the accumulation of keto-carotenoids
(canthaxanthin, echinenone,adonixanthin, etc.) in the stationary phases of the B
and L races (Banerjee et. al, 2002).

Although

biochemically

different,

the

various

races

coexist

in

natural

environment. Mixed populations of alkadienes and botryococcene races have

been found inAustralian lakes (Wake and Hillen, 1981).

Table 1 summarizes the distinctive features of Races of B. braunii

Source: Banerjee et. al., 2002

In Table 1A are summarized the geographical sources from which B. braunii

samples have been collected and for some of them strains isolated, in relation to
the hydrocarbon type. (Metzger, 1998)

10

Source: Metzger et. al., 1998

11

2.2 Heterotrophic Bacteria associated with Botryococcus braunii

In the interaction between bacteria and microalaga, the role of the bacteria is
very important for they serve as the source of inorganic nutrients, they inhibit
algal growth and they regenerate organic and inorganic nutrients. But in some
conditions, alagaecide bacteria are present which directly kills the microalgae or
produces special compounds to lyse algal cells.

In the presence of bacteria, unicellular microalgae generally grow. When

Botryococcus braunii is massively harvested, the interaction between the
bacteria and the microalga tends to form a biofilm. The formation of biofilms in
some cases occurs in the microalgal cells (Rivas & et al. 2010). Probiotic biofilms
are formed when an interaction between bacteria and microalgae occurs. In
probiotic biofilms the bacteria and the microalgae tend to maximize every factor
that they dont have when they live separately. The biofilms have been known as
a reservoir of pathogenic bacteria. The bacteria found in biofilms are hard to
eliminate. Nitrogen is used in order to reduce the growth of pathogen bacteria. If
a biofilm will be eliminated it will just increase the occurrence of pathogenic
bacteria and a huge imbalance in nature will occur for the food chain of some
microorganisms will be diminished.

The presence of microorganisms has a huge effect on B. braunii growth and

hydrocarbon production; it can be advantageous or disadvantageous. The

12

presence of various microorganisms can influence not only the quantity of

hydrocarbons produced, but also their level in the algal biomass and their relative
abundance. However, their chemical structure is not affected (Chirac & et. al
,2004).Moreover, the association of bacteria for the production of large
hydrocarbon of B.braunii is not that essential.Stable association with various
bacterial species may increase and improve the supply of CO2.Increase of
growth rate indicates a beneficial relationship between the bacteria species and
the green alga (Banerjee,2002). Various microorganisms associated with B.
braunii involve mutualism and commensalism interaction.

There are some evidence found out the role of the bacteria to B. braunii is to
provide a source of inorganic nutrients, vitamins and creates a favorable
condition for its growth(Rivas,2010).Biofilms were detected and identified
together with the B.braunii through 16S rDNA sequencing. Specifically, the
species Rhizobium sp. and Pseudomonas sp. were detected and present in the
association of B.braunii(Rivas,2010).Banerjee,2002 was also able to detect the
presence

of

Arthrobacter

sp.,Cornyebacterium

sp.,Erwinia

sp.

and

Flavobacterium sp. in association.

The strain of Botryococcus braunii that belong to Race B harbored different

symbiotic bacteria that may help in production of potent renewable resources of
liquid hydrocarbon properties of the green alga(Tanabe,2012).The capability of
the bacteria to produce a hydrocarbon may contribute and converted to a bio13

diesel production and fuel efficiency.

Cobalamin or also known as Vitamin B12 in alga can be acquire through

symbiotic relationship with bacteria. The role of vitamin B12 in the metabolism is
act as a primarily cofactor vitamin synthase. Some algae cannot produce their
own cobalamin,hence the bacteria will supply and associate it with algal cells. In
return, the bacteria derive its nutrition from the algae that produced during
photosynthesis.

2.3. Culture Media Development

The establishment of bacterial pure cultures is very important despite the
possibility of accessing the genetic information of organisms through genomics. It
is because the potential of any organism can best be achieved by having that
particular organism available for experimentation in the laboratory (Glckner and
Joint, 2010, Heidelberg et al. 2010).

Even in this age of high-throughput DNA sequencing, cultures are still essential.
They provide almost the only way to discover the physiology of microbes to
establish which organic substrates are used, to determine what secondary
metabolites might be released, or biotransformations might be possible. Indeed,
a culture is required for a full taxonomic characterization and to give a name to
an organism. So cultures remain an essential requirement, not only for
biodiscovery, but also for marine microbial ecologists if we are to understand the
14

role of microbes in the Earth System. (Joint, Muhling, and Querellou, 2010). In
lieu to this, a culture medium is used: it is a medium or composition of complex or
simple materials (i.e, nutrients, vitamins, etc.) which might be organic or
inorganic substrates that are the altered specifically to suit an organisms needs
for survival and growth.

The separation of microorganisms from their natural habitat and their cultivation
in laboratory or artificial conditions is termed in vitro in contrast with in vivo.
Culture media is developed to aid in the mission of arriving at a pure culture of a
bacterial species from mixed bacterial populations within biomass from the
environment and in animal samples. The composition must be precise and
tailored exclusively for the prerequisites of each species. This seems easy to do
but unfortunately, some bacteria are naturally resistant to culture in isolation on
usual or traditional media. This paves way to the limitations of culture media
development. Some bacteria termed as fastidious have complex growth
requirements including the need for very specific nutrients, temperature level,
incubation period and pH conditions. Kopke et al. (2005) investigated the effect
of different substrates and culture conditions on the growth of bacteria from
comparable samples of coastal sediments, and found that the various cultivation
approaches resulted in the isolation of different groups of bacteria specific to
each method, confirming the impact of cultivation conditions on the yield of
culture. In simple terms, if the requirements demanded by a bacterium for its
growth are not artificially fulfilled, some bacteria may not grow.
15

The focus is all about the development of culture media with respect to the
growth and survival of microorganisms. There are certain factors that cause the
effectiveness of deterioration of species growth in a culture medium. One of
which is the truth that a medium is really different from a species natural habitat.
Growth may be inhibited by bacteriocins released from other bacteria in a mixed
culture or by antibacterial substances present within the medium (Tamaki et al.,
2005). So in order to achieve the best approximation of the number of true
diversity of a given population, a technique of multiple methods of cultivation
should be employed. Another difference, Signalling molecules present only within
the natural habitat are thought to be essential for the growth of many bacteria
(Lewis, 2007; Nichols et al., 2008). In the absence of these beneficial interactions
and signals, some bacteria may struggle to grow in monoculture. Furthermore,
faced with an unfamiliar environment devoid of essential factors, bacteria may,
as a survival strategy, enter into a temporary state of low metabolic activity
accompanied by the inability to proliferate or to form colonies on culture media
(Barcina et al., 1990; Colwell, 2000; Lewis, 2007; Nichols et al., 2008), which
may be mistaken for a constitutional resistance to culture. These differences
have caused the inability to culture a diverse number of bacterial species.
Statistics revealed that there are currently estimated to be 61 distinct bacterial
phyla, of which 31 have no cultivable representatives (Hugenholtz et al., 2009).
With this in mind, developmental strategies regarding on culturing the
unculturable bacteria have been on the move these past few years particularly
16

in the field of environmental microbiology, as such those soil and aquatic

(freshwater and marine) inhabitants constitute the vast percentage of the
microbial population and also of the unculturable bacteria.

The majority of culture media used to date have been nutrient-rich. It is now
thought that these conditions may favor the growth of faster-growing bacteria at
the expense of slow-growing species, some of which thrive in nutrient poor
environments (Koch, 1997; Connon & Giovannoni, 2002), and may be inhibited
by substrate-rich conventional. Consequently, the use of dilute nutrient media
has led to the successful cultivation of previously unculturable bacteria from
various aquatic and terrestrial habitats (Watve et al., 2000; Connon &
Giovannoni, 2002; Rappe et al., 2002; Zengler et al., 2002). filtration methods
(Hahn et al., 2004), density-gradient centrifugation or elutriation and extinctiondilution whereby samples are diluted, ideally down to single cells, before their
culture in isolation (Watve et al., 2000; Connon & Giovannoni, 2002; Ben-Dov et
al., 2009; Song et al., 2009; Wang et al., 2009) were proven effective to actually
reduce the number of bacteria before cultivation.

Next, oligotrophic bacteria which require simplest substrates for survival are very
slow growing. Extended incubation times are a prerequisite for the cultivation of
such bacteria, with the added benefit that faster-growing members within the
mixed populations progressively die off over time, reducing the bacterial
competition. The culture of soil bacteria for up to 12 weeks has revealed
17

increasing colony counts and an increased recovery of rarely isolated strains with
time (Davis et al., 2005). Patience is indeed very important to achieve successful
results.

Nutrient alteration is another way to developing an effective culture medium.

Many bacteria have specific nutrient or chemical requirements for growth (Graber
& Breznak, 2005; Tripp et al., 2008). And so, past studies must be given attention
as to which is which and what kind of nutrition a bacteria needs. If it is a new
unculturable species, The characterization of phylogenetically related species
may provide clues to the metabolic requirements of organisms that are so far
resistant to culture. (Sait et al., 2002; Davis et al., 2005). Enrichment however
must always be applied with proper regulation.

Another way to improve the development is through the cultivation of helper

strains. Factors released from helper bacteria into the environment are often
growth-stimulatory for otherwise unculturable bacteria even in the absence of the
actual helper strain. Thus, the conditioning of media with spent culture
supernatants or cell-free extracts derived from helper strains has been used for
the growth stimulation of species such as Catellibacterium spp., Psychrobacter
spp., Sphingomonas spp. and Symbiobacterium spp. (Tanaka et al., 2004; Bae
et al., 2005; Kim et al., 2008a, b; Nichols et al., 2008).

To copy or and apply the species natural environment setting is the best way to
18

develop a culture media. In regard to this, sterile fresh- (Stingl et al., 2008; Wang
et al., 2009) and marine- (Rappe et al., 2002; Song et al., 2009) waters have
been used to culture previously uncultivated bacteria. The use of technique
combinations and correct environmental settings has been proven effective for
the development of culture media. This approach must be continually improved
for future culturing of diverse and as yet cultivated or unculturable bacterial life.

2.4. Burkholder Agar Diffusion Assay

Burkholder agar diffusion assay is mainly used to explore the bacteria-bacteria
antagonisms among different bacterial isolates and has been proposed as a
method of screening for antibiotic production of various isolated freshwater
bacteria. Burkholder assay was one of the methods employed to determine the
production of a pyrrole antibiotic by a marine bacterium (Burkholder et. al, 1966).

Rypien et. al, 2009 states that a single colony must be transferred to a broth, and
grown shaking at room temperature with the Optical Density at 600 nm (OD600)
being measured for each isolate before performing the antibiotic assay proper. A
soft agar lawn of bacteria was poured on an agar Petri plate. The lawn was
allowed to cool, then 10 ml of each producer isolate was spotted on to the agar in
a 3x3 or 7x7 Petri plates. Control producer spots consisted of 10 ml of marine
broth. Plates were incubated at 25C and 31C. After 96 h, the plates were
imaged with a transilluminator (Rypien et. al, 2009).

19

Antagonism was considered to occur when the diameter of the zone of inhibition
at least 1 mm greater than the diameter of the colony formed by the potential
producer (Rypien et. al, 2009). Interactions that demonstrated antagonism were
tested a second time. In cases of ambiguous results, the test was repeated a
third time. Many antagonistic bacteria are known to show induction or
enhancement of antibiotic production in the presence of competitors (Burgess et
al., 1999; Slatterye al., 2001; Trischman et al., 2004), suggesting that
antagonism plays a critical role in preventing invasion of bacteria, including
potential pathogens. Preliminary attempts to grow some strains in liquid shake
culture for production of the antibiotic were not entirely satisfactory, although
various media, different speeds of shaking, an forced aeration were employed
(Rypien et. al, 2009).

Selection of the type of antimicrobial assayto be performed is based on the kind

of organism being tested and source of the isolate i.e. blood, urine, freshwater
(Hudzicki, 2009). Most of the methods involve diffusion through solid or semisolid culture media to inhibit the growth of sensitive microorganisms
(Lertcanawanichakul et. al., 2008). The Burkholder agar diffusion method is
found to be the most ideal assay for freshwater algal isolates compared to its
other counterparts i.e. Kirby Bauerand Cross-Streak Method. Kirby Bauer
Method is a single disk diffusion method for antimicrobial susceptibility testingto
determine the sensitivity or resistance of pathogenic aerobic and facultative
anaerobic bacteria to various antimicrobial compounds in order to assist in drug
20

prescription for a patient (Hudzicki, 2009). Cross-streak method is more centered

on Actinomycetes(Lazzarini et. al, 2000),a group of Gram-positive bacteria,
which are commonly found in the soil playing a significant role in decomposition
(Velho-Pereira et. al, 2011). This method difficulty in obtaining quantitative data,
since the margins of the zone of inhibition were usually very fuzzy and indistinct
(Williston et. al, 1947). In addition, the streak non-uniformity caused a problem
with respect to quantifying the results (Williston et. al, 1947).

21

Chapter 3

METHODOLOGY

Sampling Site: Paoay Lake, Ilocos Norte, Luzon, Philippines

The sampling site will be in Paoay Lake (Latitude: 18 7 16 N, Longitude: 120
32 18 E) situated in the Municipality of Paoay, Ilocos Norte. It is located 3
kilometers away from the sea in Suba.This inland lake is being bounded by the
towns of Suba in the north, Nanguyudanin the northeast, Pasil in the east,
Sungadan in the south and Nagbacalan in the north (Papa et.al., 2008). It is the
only lake of the province (Guerrero, 2001) having an area of 470 hectares.The
freshwater from the lake also supports farmers of the surrounding communities
and it serves as an irrigationsystem to water their crops. It has been declared as
a National Park by the government. In 2008, the massive bloom of Botryococcus
brauniin the lake has caused a decrease in dissolvedoxygen and might have
attributed to the toxic effects exerted by this green alga onthe other organisms
found in the lake (Papa et. al., 2008).

Figure 1.A. Map of the Sampling Site, Paoay Lake, Paoay, Ilocos Norte, Region
1, Luzon Island, Philippines (https://maps.google.com) B. Proposed collection
site. (https://images.google.com/paoay-lake)
22

Water Sampling. Collection of water sample will be done in a designated site of

algal abundance on Paoay Lake, Ilocos Norte. Water samples from different
depths (surface, 1 meter, 2 meters, 5 meters) (from your RRL at what depths are
they found?)will be collected using an improvised water sampling device made
from 2-liter plastic soda bottles. The 2-liter water samples will be passed through
a 25 m size nylon sieve to concentrate the phytoplankton to 20 ml. 10 mL of the
concentrated sample will be preserved in Lugol's iodine solution and the
remaining 10-ml will be used for on-site phytoplankton identification and isolation
of Botryococcus braunii and bacterial isolation.

Microscopic Identification of Botryococcus braunii and Isolation of

colonies. Collected specimens on-site will be immediately viewed under the
microscope using the Oil-Immersion Objective (OIO).

Table 3 shows the

guidelines for identification of the presence of Botryococcus braunii. Individual

colonies will be collected by pipetting with a fine-tipped plastic pipette and placed
in microcentrifuge tubes with 1-ml sterile saline. The separate pools of B. braunii
colonies will be prepared and used for bacterial isolation and also for culturing
the alga.

The bacterial isolation will utilize an oil-based, M9 Minimal Medium recipe as the
base medium and addition of a carbon source, Squalene (Sambrook, 2001) to
supply the necessary nutrients forthe bacteria present in the algae. Squalene, a
metabolite essential for sterol metabolism in all eukaryotes, will be the
23

preferred choice for the carbon source since it is commercially available and
biosynthesis of Botryococcene, the primary oil produced by B. braunii, is thought
to resemble that of squalene. (Okada et al., 2011). Both Botryococcene and
Squalene are of triterpenoid hydrocarbons. In extracting botryococcene,
expensive machineries and tools are in use and so with that squalene is the
recommended choice of oil because its economically practical.

Figure 1. shows the chemical structure of the C30 botryococcene, the primary oil
produced by B. braunii
(Molecular Formula: C30H50 Molecular Weight: 410.718 g/mol)
Source: http://pubchem.ncbi.nlm.nih.gov

Figure 2. shows the chemical structure of Squalene, the proposed carbon source similar
to the C30 botryococcene for the media to be used for bacterial isolation from B. braunii
(Molecular Formula: C30H50 Molecular Weight: 410.718 g/mol)
Source: http://pubchem.ncbi.nlm.nih.gov

For algal culture, the colony pools will be transferred to tubes containing different
concentration of f/2 medium (f/2, f/5, f/10) (Guillard and Ryther 1962). Intact
colonies will also be plated on water agar amended with f/2 medium. These will
be incubated under illumination at room temperature.

24

Table 3.

Guidelines for microscopic identification of the presence of

Botryococcus braunii
Description
Classification
Size
Colony color in
stationary
phase
Distinction
Appearance

Green microalgae, freshwater species and produce a massive hydrocarbon that

can be use for bio-diesel production.
Family Botryococcaceae
Race A & B 13m x 79m
Race L 8-9m x 5m
Race B and L red-orange and orange brownish from green color (log phase)
Race A pale yellow from green color
Rustic-yellow colored algal bloom

A colony surrounded by oil droplets.

A mass of cells.

A colony.

(Source: http://www.algaebase.org/search/species/detail/?species_id=27992) (Source: Sarada et. al, 2010)

A colony
(Source: Watanabe, 2007)

Cell Wall
Other Remarks

Colonies along the edge of a large mass.

(Source: Qin, 2011)

Relatively thick
Hydrocarbon oil found forming a droplet-appearance outside of the cell. Colonies
held together by a lipid biofilm matrix.

Homogenization of Algal Cells and Bacterial Enumeration and Isolation of

Heterotrophic Bacteria. A pool of 5-10 colonies in 0.5 ml saline will be
homogenized in a small flame-sterile porcelain mortar or glass homogenizer until
the homogenate shows colony breakage and effective cell dispersal upon
microscopic examination.
25

A loopful of the homogenate will be immediately streaked by multiple-interrupted

streaking on triplicate plates of 1/10 Tryptic Soy Agar (1/10 TSA) and the Oilbased Medium (OBM) that the group will formulate and test for bacterial isolation.

The homogenate will then be serially-diluted up to 10-7 in a 10-fold dilution series

with 4.5 ml saline. Aliquots of 0.1 ml from all dilutions will be spread-plated on
triplicate plates of 1/10 TSA and OBM.

Also, 0.1-ml aliquots will be inoculated in quadruplicates to 3-ml Tryptic Soy

Broth (TSB) and liquid OBM.

This Most-Probable-Number (MPN) method of

bacterial enumeration will be performed as a back-up for the plate-count method.

All the inoculated media will be incubated at room temperature and colony counts
will be performed on days 2, 5, 7 and 10. The MPN tube will also be observed
following the same time interval. The bacterial isolation outlined above will be
performed on a least two separate pools of B. braunii colonies.

Colony morphology assessment and preparation of stock cultures. The

plates that will harbor 30-100 colonies will further be assessed in terms of colony
morphologies. The colony types will be noted and the colony counts for each
type will be noted. Three colonies from each type will be streaked on 1/10 TSA
for purification and repeated streaking will be done until purity is achieved.
26

Isolated colonies will be inoculated to duplicate tubes containing 5-ml of 1/10

TSA soft agar (solidified with 0.3% agar). After the organisms have grown, the
cotton plugs will be replaced with sterile rubber stoppers to prevent the medium
from drying up for long-term (several months) storage at room temperature.

DNA isolation. From the same purification plates used above, a colony from
each type will be inoculated to 5-ml 1/4 strength TSB. After overnight incubation,
1.5 ml of each culture will be centrifuged at 8000 rpm for 10 minutes (room
temperature) and will be used for DNA isolation. A DNA isolation kit will be used
for genomic DNA isolation following manufacturer's instructions. Representative
colonies from each colony type will be picked and re-streaked to ten-fold diluted
TSA plate for purification (Poonguzhali et al., 2008).

Identification and Characterization of Heterotrophic Bacteria. Young cultures

(24-hour old on TSA) will be used for basic characterization. The isolates will be
characterized morphologically through Gram stain and endospore stain; and
biochemically through Oxidase test, Oxidative-Fermentative test, and the API
20E or 20NEkit.

Test for Antibiotic Properties. The identified bacteria will be screened for
antimicrobial properties using the Burkholder agar diffusion assays. This assay is
typically used to assess the presence of antagonistic interactions among bacteria
isolated within a certain community. In this assay, each isolate will be tested
27

against all the other isolates. Below is a description of the methodology.

A single colony from a 24-hour old plate will be transferred to a 5 mL TSB. The
optical density (OD600) will be measured for each isolate before the start of
assay. The absorbance level will be compared to the reference blank, as it
contains everything found in the sample except for the substance being
analyzed. An agar lawn of test bacteria will be poured using the spread plate
method. A small volume of broth culture, which is spread even over the surface
using a glass spreader will be inoculated on the designated culture medium. After
cooling, about 10uL -15uL of the producer isolate will be spotted on the agar
lawn. Plates will be incubated at 25C - 31C for 96 hours. The bacterium will show
antibiotic activity on the original agar plate spread with the water sample, as
demonstrated by the formation of zones of inhibition around individual colonies.
Antimicrobial metabolites will be considered to occur when the diameter of the
zone of inhibition will be at least 1 mm greater than the diameter of the colony
formed by the potential producer (Rypien, 2009).

28

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34

Appendices
Appendix I
Appendix I. Letter template for Field Work Requests and Permissions.
(Name of BFAR/DENR Official)
(Position)
(Complete office address)
Dear Mr/Ms/Mrs (name):
Warmest Greetings!
I am (name), a third year B.S. Microbiology student from the University of Santo
Tomas, Espana, Manila. My team and I are currently pursuing our undergraduate
thesis entitled: Culturable heterotrophic bacteria associated with Botryococcus
braunii, an oil-producing green algae, that would involve collection water sample
from the Paoay Lake.
In line with this, we would like to ask for permission, and as well as your
cooperation for the collection of Botryococcus braunii water sample from the
Paoay Lake since it is only in this lake that the species we are studying are
present. Rest assured that the data collected from this barangay will serve a
great purpose in the scientific community and would be of help in the betterment
of environment.
Hoping for your most favorable response from your good office.
Respectfully Yours,

Kimberly L. Po
09228229204
(Home address)

Gur-Kiratt K. Rai
(Contact #)

John Ryan P. Calicaran

(Contact #)

Angela Victoria A. Gutierrez

(Contact #)

35

APPENDIX II
(1x M9 minimal medium)
12.8 Na2HPO4.7H2O
3 g KH2PO4
5 g NaCl
1 g NH4Cl
0.24 MgSO4
0.11CaCl2
0.4% carbon source (Filter sterilized, added before pouring to plates or
transferring to tubes)
In 1 liter H2O

TrypticaseTM Soy Agar (ATCC Medium 18)

Composition per liter
Pancreatic digest of casein17.0 g
Agar....15.0 g
NaCl..... 5.0 g
Papaic digest of soybean meal..3.0 g
K2HPO42.5 g
Glucose..2.5 g
Use: For the cultivation and maintenance of a wide variety of heterotrophic
microorganisms. For the cultivation of a wide variety of fastidious and
nonfastidious microorganisms from clinical and nonclinical specimens. Also used
for the rapid estimation of the bacteriological quality of water.

36

37

APPENDIX III

PROPOSED BUDGET PLAN FOR RESEARCH WORK

An undergraduate research project on algal-bacteria interactions in Paoay Lake, Ilocos Norte
Project Period: February 2013 - February 2014


Budget Period: February 2013 - February 2014




Particulars
Breakdown Estimated Amount
LABORATORY MATERIALS:


Brand New & 2nd Hand Glasswares

5,000
Agar

8,000
Miscellaneous supplies

2,000
Identification (API 20E or 16s rRNA Sequencing)

10,000
Total (Laboratory Experiment Materials)

25,000

FIELD
WORK EXPENSES: (4D/3N)
Transportation:
Airfare (MNL-ILOCOS)
Bus (ILOCOS-MNL)
Van Rental w/ driver (within the city)
Tricycle and Taxi Fares
Total (Transportation Expense)

2,000 x 5
1,000 x 5

10,000
5,000
6,000
2,000
23,000

3,000 x 2 Days

Board
& Lodging:

1 Room (3 Nights Hotel Accomodation)
3,000 x 3 Days
Total (Board & Lodging Expense)


Food:
Food - Researchers (P200/meal x 3 meals x 4 days)
x 5 PAX
Food - Driver (P200/meal x 4 days)

Snacks (P100/snacks x 2 snacks/day x 4 days)
x 6 PAX
Total (Food Expense)

Miscellaneous:
Tips and Other Miscellaneous Expenses

Total (Miscellaneous)

Estimated Grand Total of Expenses

NOTE: The above expenses are mere estimates and

subject to change after confirmation with suppliers.

12,000
800
4,800
17,600

2,000
2,000

76,600

9,000
9,000

38

Recommendation for Pre-Oral Defense

The research proposal entitled Culturable heterotrophic bacteria associated

with Botryococcus braunii, an oil-producing green algae proposed by John
Ryan P. Calicaran, Angela Victoria A. Gutierrez, Kimberly L. Po and GurKiratt K. Rai in partial fulfillment of the requirements in Microbiology Seminar
601 (MicroSem 601) for the Degree of Bachelor of Science in Microbiology is
hereby recommended for Pre-Oral Defense.

Prof. THOMAS EDISON E. DELA CRUZ, Dr.rer.nat.

MicroSem 601 Adviser
Date: _______________________

Asst. Prof. MARY ANN G. SANTOS, Ph.D

Interim Research Adviser
Date: _______________________

Assoc. Prof. ALICIA ELY PAGULAYAN, M.Sc.

Chair
Department of Biological Sciences
UST- College of Science
Date: _______________________




39