1

Chapter I

INTRODUCTION

A. Background of the Study

Mangroves are composed of medium height shrubs or trees established in intertidal

zones of estuaries, marshes, and deltas of tropical and subtropical latitudes. Approximately

one fourth of the world’s coastline is dominated by mangroves that are distributed in 112

countries and cover about 180,000 km2 of the globe’s surface in subtropical and tropical

regions (Latha & Mitra, 1998). Mangrove ecosystem is one of the most important ecosystems

and believed to be an important sink of suspended sediments. (Kathiresan & Bingham, 2001).

In these forests, mangrove trees catch sediment by their complex aerial root structure, thus

functioning as land builder (Holguin et al., 2001). They also generate considerable amount of

detritus such as leaf litter and woody debris hence constitute an ideal environment that

support or harbor diverse groups of marine animals, plants and microorganisms that are

widely acknowledged to be important elements in coastal ecosystems in the tropics (Holguin

et al., 2001). Mangroves preserve water quality and reduce pollution by filtering suspended

materials and by assimilating dissolved nutrients, stabilize sediments and protect the

shoreline from erosion.

In mangrove sediment communities, substantial fungal populations exist as part of the

vast microbial diversity involved in detritus processing (Abdel-Wahab, 2005). Marine fungi

occur in most marine habitats and generally have a pantropical or pantemperate distribution.

Marine fungi are major decomposers of woody and herbaceous substrates in marine

2

ecosystems. Their importance lies in their ability to aggressively degrade lignocellulose.

They may be important in the degradation of dead animals and animal parts. Marine fungi are

important pathogens of plants and animals and also form symbiotic relationships with other

organisms.

Yeasts are fungi that predominantly exist as unicellular organisms and at present there

are about 1500 recognized yeast species which are distributed between the ascomycetes and

the basidiomycetes (Kurtzman & Fell, 2005; Botha, 2011).Yeasts play a role in maintenance

of soil and sediment structure and aggregate formation. Also, Yeasts participate in soil

nutrient cycles and mineralization processes. On the other hand, yeasts serve as a nutrient

source for a diversity of soil predators and they have potential as plant growth promoters and

soil conditioners (Yurkov et al., 2012).

Anthropogenic activities on environment include impacts on biotic and abiotic

environments. In August, 2006, the oil tanker M/T Solar of Petron, carrying more than two

million liters of bunker fuel, sank off the southern coast of Guimaras causing oil spill that

travelled up through Guimaras and Iloilo strait. This oil spill greatly affected the marine

environment and mangrove reserves of the majority of the municipalities in Guimaras. The

mangroves and associated biota in mangrove forest of Panobolon Island, the sampling site of

this study is heavily impacted by the said oil spill.

Pollution has been implicated in the modification, increases or reduction of genetic

diversity in various organisms in mangrove coastal ecosystems (Latha &Mitra. 2004; Kokare

3

et al., 2004; Limtong et al., 2007). Due to the oil spill that happened, the mangrove habitats

face the dangers of losing some precious fungal resources. This study aims to isolate yeast

from the oil impacted and nonimpacted mangrove sediments along the coast of Panobolon

Island, Guimaras.

Independent Variable Dependent Variable

Fig 1. Conceptual Framework

B. Objectives

This study generally aimed to isolate yeast from the oil impacted and nonimpacted

mangrove sediments along the coast of Panobolon Island, Guimaras.

Specifically, this study aimed:

1. To determine the yeast count (CFU/g sample) isolated from the soil sediments

2. To identify the yeast species isolated

C. Hypotheses

In line with the objectives, the following hypotheses were drawn:

1. Yeasts will be isolated from the soil sediments taken from Panobolon Island, Guimaras

2. The site with growing mangroves (oil non-impacted) will yield the highest yeast count.

Surface Sediments taken

from Different Sites of

Panobolon Island in

Guimaras

Yeast Isolates

4

D. Significance of the Study

Results of this study will be highly useful for environmental science and will

highly prevent hazards brought about by oil spill and other anthropogenic activities and

pollution to areas where mangroves are grown and abundant. Health risks brought by

these activities will be minimized. Also, baseline information regarding the diversity of

yeasts in sediments found in mangrove areas will be provided.

E. Scopes and Limitations of the Study

This study determined the yeast isolated from the oil impacted and nonimpacted

mangrove sediments along the coast of Panobolon Island, Guimaras. Soil sediments taken

from three different sites in Panobolon Panobolon Island, Guimaras were used. Sampling

was done on September 7, 2013. Serial dilution and spread plate methods were employed.

Potato Dextrose Agar was used as media. Cultures were inoculated and incubated in the

laboratory. Incubation was done for 5-days. Laboratory work was conducted last

September 10, 2013 in the Microbiology Room of the University of the Philippines,

Miagao, Iloilo.

5

CHAPTER II

REVIEW OF RELATED LITERATURE

YEAST

Yeast is a group of fungi in which unicellular form is predominant. As a group of

microorganisms yeasts have diverse distribution. They have been isolated from natural

substrates like leaves, flowers, sweet fruits, grains, fleshy fungi, exudates of trees, insect,

dung and soil. According to Rose and Harrison (1987-1993), yeasts play their role in the

dynamics of biological and chemical turnover in soil, plants, animals and water. There

are about 100 genera and 700 species of yeasts (Mushtaq et al., 2004).

Saccharomyces are known to be the most effective and most utilized

microorganisms for fermenting sugars to ethanol and traditionally have been used in

industry to ferment glucose based agricultural products to ethanol. Owing to its efficiency

in producing alcohol, Saccharomyces cerevisae is the most important commercial

microorganisms. Yeast is ubiquitous in the environment, but is most frequently isolated

from sugar rich samples. Some yeast strains are found in association with soil and insects.

Ethanol tolerance, sugar tolerance and invertase activities are some of the important

properties for use in industrial ethanol production (Jimenez and Benetez, 1986). Many

research workers found yeast in large numbers in a wide variety of natural habitats as

different as leaves, flowers, sweet fruits, tree exudates, grains, roots fleshy fungi, insects,

dung, soil (Chiranjeevi et al., 2013).

6

Some Common Yeast Isolates:

Fig. 2. Yeast Grown on Media

Fig. 3. Saccharomyces cerevisae, 100x

Fig. 4. Candida sp., 100x

7

Although yeasts are thought to be primarily degraders and utilize simple sugars,

the role of many, especially basidiomycetes, may be more complex, and their function in

soil ecosystems remains unclear. Identification of the yeast diversity in soil communities,

especially those with as few members as the polar deserts, is an important step in the

development of a model for food-web processes. Sites associated with seabirds and

marine mammals have higher nutrient inputs and human activity can disturb the soil and

inadvertently disperse or deposit non-indigenous microorganisms (Connel et al., 2008).

MANGROVES

Mangroves are coastal ecosystems, found in tropical and subtropical regions

around the world. They are found in the transitional zones between land, sea and rivers;

regarding their geographical distribution, mangroves are found in the Americas, Africa,

Asia and Oceania. Mangrove vegetation is found along 25% of the Earth’s coastlines and

75% of tropical coastlines. Mangrove sediments are the foundation for mangrove forests

and all that live in them. Life in mangroves requires special adaptations to survive in

areas that are periodically inundated with sea water. Mangroves can be seen as the thin

green line of vegetation around coasts and estuaries. Thin, because they account for less

than 1% of the world’s tropical forests and less than 0.4% of the total area of global

forests. They are present in 123 countries and territories, covering about 152,000 km2.

(Spalding et al., 2010)

Mangrove vegetation composition is controlled primarily by hydrology,

geography and climate of the region. Plant morphological adaptations such as aerial roots

8

facilitate life on and in the inter-tidal zone (Araújo et al., 1979). Mangroves can tolerate a

wide range of sediment types, temperature changes, nutrient, salinity and oxygen levels.

Mangrove plant species vary in their tolerance to these factors, forming characteristic

patterns or zones of vegetation. Spalding et al. describe 73 species that inhabit

mangroves. This seems to be a large number until a further look at their global

distribution is considered. Spalding et al. note that 62 species are found in the Indo-West

Pacific realm and only 12 species in the Atlantic Eastern Pacific realm.

Mangroves today are often found in or next to urban areas, where they are under

the constant impact of anthropogenic activities. The continued growth of urban areas

results in severe impacts on mangroves, modifying their hydrology, sediment and nutrient

dynamics (Lee et al., 2006). Small-scale changes in the physical structure of mangrove

forests can have significant effects on the diversity and abundance of wildlife in these

environments. Such modifications may affect food webs causing irrevocable damage;

which threatens their role as refuges, as nurseries, as well as foraging areas of marine

fauna (Skilleter et al., 2000). Human activities affect the functioning and normally

decrease the biodiversity of mangroves, thus leading to ecological imbalances and species

extinction. With the development of urban centers, there has been a reduction in the area

covered by mangroves. Irreversible damage occurs as a consequence of deforestation,

land-filling and reclaiming coastal land for industry, housing, tourism and ports.

According to Duke et al. , many mangroves are on the verge of extinction and are

expected to disappear from at least 26 of the 120 countries in which they are currently

found.

9

Human activities impact on mangroves, especially those activities associated with

ports where diffuse oil and chemical spills are constant. Mangroves also receive

agrochemical runoff delivered by larger rivers and sewerage. Where shallow fresh water

meets deeper salt water and lower energy environments flocculation and sedimentation

occurs and chemical cycling occurs. Complex communities, of bacteria and fungi, can

biodegrade hydrocarbons in such environments. Microorganisms are often found in

complex communities called biofilms where different species degrade different types of

hydrocarbons found in petroleum. Sediments contaminated by oil and sewage can modify

the natural processes of decomposition. Hydrocarbon degradation is considerably faster

under aerobic conditions and covering sediments with heavy oil can quickly create

anaerobic conditions. Under anaerobic conditions, oil degradation is less efficient and can

result in the release of toxic sulfates (Holguin et al., 2001). LaMontagne et al. and others

(Girvan et al., 2005, Castle et al., 2006) have shown that the composition of inputs and

levels of hydrocarbons in sediments can influence the composition of bacterial

communities. Santos et al. published a review on the potential and challenges faced for

bioremediation of wetlands and mangrove sediment impacted by oil.

Marine sediments are inhabited by fungi. They are found in shallow coastal

sediments as well as in deep sea sediments and also are common in mangrove sediments.

Studies suggest that about half of the fungi in sediments are absorbed to sediment

particles and are difficult to detect. The remainder, are present in the interstitial water

between organic and inorganic sediment particles. Fungal numbers and biomass varies

10

with the type of sediment, with larger numbers seen in shallow coastal regions and

biomass decreasing rapidly with depth. Coarser sandy sediments have lower numbers of

fungi than do sediments of finer texture. Their primary role is considered to be the

mineralization of organic matter, and they are also a food source for benthic fauna

(Roitman et al., 1991).

The tropical micro-fungi represent a universe of unexplored biodiversity,

producing a wide range of enzymes that can degrade many types of organic and inorganic

substrates. Micro-fungi that have been found in mangrove forests are also diverse and are

present in this ecosystem as epiphytic, polisaprobial and pathogens in different organic

and inorganic substrates, in the sediment and on leaves, stems, fruits, roots and animals

(Hyde et al., 1997). Fungi including: ascomycetes, mitosporic fungi, basidiomycetes,

chitridiomycetes, myxocycetes, oomycetes, thraustochitrids and zygomycetes have been

reported as present in mangrove forests from around the World (Sridhar et al., 2005).

Many of these fungi are of terrestrial origin, and basidiomycetes, ascomycetes and

deuteromycetes are most predominant (Jones et al., 1997) however finer taxonomic

resolution at family, genus and strain levels is required.

ISOLATION FROM SOIL

Simple isolation of yeasts from soil does not a priori indicate that the organism in

question is indigenous. In a global study of soil bacterial diversity and richness, Fierer

and Jackson (2006) found that differences could best be explained by soil pH, with the

11

lowest levels of bacterial diversity in acidic soils. The bacterial diversity study found that

soils with pH values above pH 8.5 were rare, and the authors were not certain if the

bacterial diversity would plateau at near normal pH values or continue to rise with higher

pH values (Connell et al., 2008).

The population of yeast cells in soil is greatly dependent upon the type of

nutrients reaching them. Competition for nutrients is probably the single most important

factor in yeast ecology. Among physiochemical factors that affect the ecology of yeasts,

most important appear to be the energy sources, nutrients, temperature, pH value and

water (Mushtaq et al., 2004).

Soil is a very species-rich habitat containing all major groups of microorganisms

like bacteria, algae, protists and fungi. The great majority of fungal species have at least

some part of their life cycle in soil. The soil microcommunity plays a vital role for the

global element cycles and thus for life on earth, because 60–90 % of the whole terrestrial

primary production is decomposed in the soil, and furthermore many waste products of

human society are detoxified there. Fungi play a fundamental role for the functioning of

the ecosystem soil and due to their ability to decompose complex macromolecules like

lignin or chitin they are essential for making the nutrients like C, N, P, S available.

Moreover the fungal mycelium plays an important role for the stabilization of the soil

because it binds soil aggregates and thus reduces erosion and helps to increase the

waterholding capacity.

12

Soils also contain a lot of biotechnologically and pharmaceutically important

fungi; penicillin and cyclosporin are two well-known fungal products. The biological

diversity in soil is closely related to abiotic and biotic factors, but soil moisture is

generally assumed to be more important for microorganisms than temperature and pH-

value (Wuczkowski et al., 2004).

Soil biota interacts with aboveground ecosystem components and influence

ecosystem diversity, structure and functioning. Being a significant component of all

terrestrial environments, fungi have considerable impact on fundamental soil processes,

like decomposition, aggregation, nutrient release and nutrient storage (Yurkov et al.,

2012).

13

Chapter III

METHODOLOGY

A. Sampling Procedure

The soil studied was oil impacted and non-impacted mangrove sediments taken

along the coast of Panobolon Island, Guimaras. There were three sampling sites (Figures

5-7). In site 1, the area was field with oil-impacted mangrove sediments. Dead mangroves

were present in the area. In site 2, the area was field with young mangroves. In site 3, old

mangroves were present. Sediments were taken using a trowel with a depth

approximately 5 inches below the surface. Samples were placed in a sterilized glass

bottles (recycled Gatorade bottles). The bottles were then placed on large containers

containing ice to preserve the samples. After sampling, bottles were placed in a

refrigerator in the Research Room (Chemistry) of UP Visayas, Miagao, Iloilo to preserve

the samples.

B. Sterilization and Disinfection of Materials

The materials used in this study were sterilized in an autoclave at 1210C for 15

min at 15 psi. These include all petri dishes, test tubes, pipette, beakers, and other

containers. Also, the working area inside the laboratory was cleaned and disinfected

using 70% isopropyl alcohol to avoid sample contamination and to maintain the area

sterile throughout the duration of the experiment.

14

Fig. 5. Site 1

Fig. 6. Site 2

Fig. 7. Site 3

15

C. Experimental Design

Completely Randomized Design (CRD) was used in the experiment. There were

three sampling sites and one blank. All set-ups were replicated thrice. Incubation was

done for five (5) days.

D. Cultivation Methods

All soil samples were cultured on plates of Potato Dextrose Agar with 2% NaCl.

The soil dilution and culturing technique with aseptic technique were used. Ten grams of

soil sample from each sampling site were weighed out with sterile precautions and

diluted with 100 ml water. Serial dilutions of 1/10, 1/5100 and 1/1000 with distilled water

were made. The soil dilutions were shaken using a mechanical vortex as each dilution

was made and were re-shaken briefly before inoculation.

Fig. 8. Agar plates with soil extracts

Inoculation was done using aseptic technique inside the laboratory hood. An

inoculum of 1.0 ml was put on each plate containing 25 ml of PDA with 2% NaCl and

spread with a glass spreader. Plates were incubated upside down at room temperature. A

16

blank set-up containing only 1.0 ml of distilled water in the culture media was made.

Cultures were incubated at room temperature for five (5) days before examination.

E. Screening of Yeast Isolate (not done)

After the incubation period, isolated colonies were inoculated again in PDA with

2% NaCl media. One colony will be systematically picked for structure analysis using a

compound light microscope. Skinner (1951) reported that by use of a shaking technique it

was possible to determine whether actinomycetes in soils occurred predominantly as

spores or as vegetative particles. It was hoped that the use of a similar shaking method

would give information upon the state of yeasts in soil.

F. Safety Precautions and Disposal

In order to ensure safety and to follow proper laboratory guidelines, masks,

surgical gloves, and laboratory gowns were worn throughout the duration of the

experiment. After the inoculation, the laboratory working area was disinfected using 70%

isopropyl alcohol. All the materials were again sterilized in an autoclave.

17

Chapter IV

RESULTS AND DISCUSSION

Yeast isolation from the oil impacted and non-oil impacted mangrove sediments along

the coasts of Panobolon Island, Guimaras was both successful and unsuccessful. During the

incubation, microbial growth was seen in all set-ups after the third day. However, the microbial

count turned out to be too numerous even at the 1/1000 dilution. Also, suspected yeasts grew in

swarm and were poorly isolated. Also, after the incubation period, growth of maggots inside the

plates was observed. This is probably because the area was not properly disinfected and even

though the plates were placed in a closed hood; it could have been more proper if there were

sealed with paraffin films.

Due to time constraints, second repetition of the inoculation procedure was not done.

Also, observation of the isolated microorganism under a microscope was not done in order to

determine if its structure is similar to that of some yeast organisms because of the fact that the

plates were contaminated by maggots.

On a theoretical basis, Menna (1957) was able to isolate different yeast strains from the

soils of New Zealand. The author also added that in set-ups that were acidified, yeast growth was

the highest. In a study of Chen, et. al. (2009), the researchers were able to isolate almost 109

marine yeast cultures from coastal waters of Northeatern Taiwan.

18

Chapter V

CONCLUSIONS AND RECOMMENDATIONS

Conclusions:

Microbial organisms can be isolated from soil sediments of both oil impacted and non-oil

impacted areas of Panobolon Island, Guimaras.

Yeast may have been one of those microorganisms that were able to be isolated from the

soil samples.

Recommendations:

Ensure proper aseptic technique while conducting the experiment.

Seal petri dishes properly in order to prevent contamination and growth of other

microorganisms (bacteria, maggots, etc.)

Use other media highly specific for the isolation of yeasts. For example, dextrose agar,

glucose-peptone agar, etc.

19

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