1

Amylolytic and Lipolytic Activities of Bacillus from BataanMangrove

Cecile Angela D. Coronado

Heidi Rose B. Del Rosario

Annielyn R. Marcellana

Student Researchers

In partial fulfillment of the requirement

in the 2013 Regional Science Fair

Pitogo High School

Negros St., Pitogo, Makati City

Ms. Lilibeth S. Lumapas

Research Adviser

Ms. Teofila O. Zulaybar

Irene A. Papa

Research Consultants

2

October 02, 2013

ABSTRACT

This study was conducted to determine the amylase andlipase enzyme production capacity of bacteria under the genusBacillus. Mangrove soil samples were collected fromdifferent locations in Barangay Tortugas, Balanga City,Bataan from which bacteria were isolated and cultivated inSierra’s and Starch Agar media respectively. Theextracellular enzyme production of the isolates was observedvia direct agar plate assay. Amylase activity was detectedafter growth on soluble starch agar plates by a hydrolysiszone around the colony, using iodine staining. Lipaseactivity was detected after growth on peptone agar media byformation of whitish precipitate-zone using copper sulfatestaining. Screening of 35 Bacteria natural isolates fromBataan mangrove for specificity they exhibit relative to thereactions they catalyze, showed that most of the testedstrains exhibited both amylase and lipase enzyme activities,which illustrates their potential use in commercialapplications. However, plate-screening method is notquantitative because of poor correlation between enzymeactivity and colony to clear zone ratio. This screening studyopens an avenue to work with some of the potent strains foruseful product formation at large scale.

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Acknowledgement

The 2013 Science Investigatory Project entitled “Amylolytic and

Lipolytic Activities of Bacillus Isolated from Bataan Mangrove” would not

have been made possible without the following, for which it

is dedicated to:

All students, administration and staff of Pitogo High

School to whom this work is intended for;

Dr. Reynaldo S. Estacio, our Principal and Mrs. Imelda

D. Quiban, our Science and Technology Department Head,

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for their unending support and assistance to make this

project a reality;

Dr. Reynaldo Ebora, Director of the Institute of

Molecular Biology and Biotechnology, UP Los Banos,

Laguna, for allowing us to do our research work in the

institute;

The Bioinformatics And Drug Recovery Program -

Antibiotics Laboratory through Dr. Edwin P. Alcantara,

Program Leader; Ms. Teofila O. Zulaybar, MSc., Project

Leader; Mrs. Irene A. Papa, MSc., Study Leader; and

staff Mr. Alxis John C. Movida and Mr. Bernardo C.

Mercado for their efficient help in material preparation

and methodologies;

Ms. Lilibeth Sia Lumapas, our research adviser, who

greatly contributed in the realization and fulfillment

of what was once just a dream; for her subtle

assistance, guidance and motivations that led us to

finish our study;

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Our adviser and all our subject teachers, for their

valuable comments and understanding of our difficulties

encountered;

Our classmates and friends for their encouragement and

incitement to boost our self-esteem;

The people of Barangay Tortugas, Balanga City, Bataan,

for permitting us to have access to their mangrove

environment.

Our parents, brothers and sisters, for their moral

support, spiritual guidance, and unconditional love in

our research work;

And The Father Almighty, the Great Provider of our

wisdom and passion, the source of our strength and

determination.

Your contribution to this labor of love is highly

appreciated. Our sincerest thanks to all of you.

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TABLE OF CONTENTS

Abstract …………………………………………………………… i

Acknowledgement …………………………………………………… ii

Chapter 1: Introduction

Background of the Study …………………………………… 1

Objective of the Study …………………………………… 3

Significance of the Study …………………………………… 3

Scope and Limitations of the Study ……………………………

4

7

Review of Related Literature ……………………………

4

Conceptual Framework …………………………………… 8

Statement of the Null Hypothesis ……………………………

8

Definition of Terms …………………………………………… 9

Chapter II: Materials and Methods

Sample Collection …………………………………………

11

Preparation of Dilution Blank ……………………………

12

Isolation of Bacillus ……………………………………

12

Preparation of Culture Media ……………………………………

13

Lipase Assay ……………………………………………………

13

Amylase Assay …………………………………………… 13

Microscopic Assay ……………………………………………

13

8

Decontamination and Disposal ……………………………

14

Screening Procedure ……………………………………………

14

Analysis of extracellular Enzymes ……………………………

14

Chapter III: Results and Discussions …………………………… 16

Results ……………………………………………………16

Discussion ……………………………………………………18

Chapter IV: Summary, Conclusions and Recommendations

Summary and Conclusions ……………………………………20

Recommendations ……………………………………………20

Bibliography ……………………………………………………22

Appendix …………………………………………………………… 24

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CHAPTER I

Introduction

Background of the Study

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Enzyme mediated processes have been under usage from

ancient times, out of approximately 4000 known enzymes about

200 enzymes are of commercial usage. Majority of these

enzymes are microbial in origin, with the advancement of

technology, and wide application of these enzymes, many

enzymes are brought in to use by various industries (Tapasys

and Kynzang, 2007).

Enzymes are substances present in the cells of living

organisms in minute amounts and are capable of speeding up

chemical reactions (associated with life processes), without

themselves being altered after the reaction. They accelerate

the velocity of the reaction without necessarily initiating

it (Oyeleke and Oduwole, 2009).Microbial enzymes are

preferred to those from both plant and animal sources because

they are cheaper to produce, and their enzyme contents are

more predictable, controllable and reliable (Burhan et al.,

2003).These naturally occurring enzymes are quite often not

readily available in sufficient quantities for food

applications or industrial use. However, by isolating

microbial strains that produce the desired enzyme and

11

optimizing the conditions for growth, commercial quantities

can be obtained (Enzyme Technical Association, 2001).

Lipases are a group of hydrolytic enzymes that catalyze

the degradation of triacylglycerols to diacylglycerol,

monoacylglycerol, fatty acids and glycerols at the interface

between aqueous and the lipid phase (Veeraragavan 1990;

Thomsan, 1999). They are widely spread in nature, they were

isolated from different source such as plants animals and

microorganisms, Lipase have immense potential application in

various industries like cosmetic, food, detergent, paper and

pharmaceutical industries (Ashok, et al., 1999; Falony, et

al., 2006; Sharma, 2001; Gupta, 2004; Sierra,

1957).Enantiomers of lipase are used for resolution of chiral

drugs, Biofuels, personal products and flavor enhancers

(Priest, 1992).

Bacillus lipases attract attention because they have

unique protein sequences and many uncommon biochemical

properties (Kim et al., 2002). Lipases catalyze the hydrolysis

of triacylglycerols and are widely used in organic chemistry

due to its high selectivity and specificity and, therefore,

12

receive much attention because of its potential use in

industrial processes (Lesuisse et al., 1993).

On the other hand, the production of microbial amylases

from bacteria is dependent on the type of strain, composition

of medium, method of cultivation, cell growth, nutrient

requirements, incubation period, pH, temperature, metal ions

and thermostability (Pandey et al, 2002). The major amylase

produced by Bacillus is heat resistant, which is commercially

interesting because many processes require high temperatures,

so the thermosensibility ceases to be a limiting factor

(Konsoula and Liakopoulou-Kyriakides, 2006).

The isolation and characterization of novel hydrolyzing

enzymes from bacteria are still a highly active research

area, because bacteria have a higher growth rate than fungi,

leading to greater production of enzymes. Also, the habitat

of bacteria covers different environmental niches, which

favors the existence of versatile strains such as

thermophiles, psychrophiles, alkaliphiles, and acidophiles.

Hence, in the present study an attempt has been made to

isolate and characterize some of the Bacillus species from

13

local mangrove with a view to identify morphologically the

strains and to evaluate their enzyme production potentials.

Objective of the Study

This study aims to identify morphologically the bacillus

strain present in the mangrove soil of Barangay Tortugas in

Bataan and to evaluate their enzyme production potentials.

Specifically, the study aims to:

1. Isolate bacillus from Brgy. Tortugas, Balanga City,

Bataan mangrove forest soil

2. Determine if bacillus from Bataan mangrove exhibits

amylolytic and lipolytic activities

3. Determine the potential of bacillus isolated from Bataan

mangrove for enzyme production

Significance of the Study

This study has been made to isolate and characterize

some of the Bacillus species from local mangrove with a view

to evaluate their enzyme production potentials. The enzymatic

screening tests will generate information if there are

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enzymes present on the cells of bacillus isolated from Bataan

mangrove. Furthermore, the presence of these substances

would explain the importance of amylase and lipase activities

exhibited by bacillus strains. Consequently the information

would be useful for the preparation of future commercial use.

Scope and Limitations

This study is limited to the used of bacillus isolated

from Bataan mangrove and the evaluation of its enzyme

production potential.

The soil samples were collected from Brgy. Tortugas,

Balanga City, Bataan. The isolation of Bacillus and the

detection of enzyme production were conducted at the

Antibiotic Laboratory of the Bioinformatics and Drug Recovery

Program at the Institute of Molecular Biology and

Biotechnology (BIOTECH), University of the Philippines, Los

Baños, Laguna last August 26 to September 20, 2013 under the

supervision of Ms. Teofila O. Zulaybar and Mrs. Irene A.

Papa.

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Review of Related Literature

The use of enzymes in the diagnosis of disease is one of

the important benefits derived from the intensive research in

biochemistry since the 1940's. Enzymes have provided the

basis for the field of clinical chemistry.

All known enzymes are proteins. They are high molecular

weight compounds made up principally of chains of amino acids

linked together by peptide bonds. Enzymes can be denatured

and precipitated with salts, solvents and other reagents.

They have molecular weights ranging from 10,000 to 2,000,000.

Many enzymes require the presence of other compounds -

cofactors - before their catalytic activity can be exerted.

This entire active complex is referred to as the holoenzyme;

i.e., apoenzyme (protein portion) plus the cofactor

(coenzyme, prosthetic group or metal-ionactivator) is called

the holoenzyme.

One of the properties of enzymes that makes them

important as diagnostic and research tools is the specificity

they exhibit relative to the reactions they catalyze. A few

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enzymes exhibit absolute specificity; that is, they will

catalyze only one particular reaction. Other enzymes will be

specific for a particular type of chemical bond or functional

group.

Enzymes are catalysts and increase the speed of a chemical

reaction without themselves undergoing any permanent chemical

change. They are neither used up in the reaction nor do they

appear as reaction products. A theory to explain the

catalytic action of enzymes was proposed by the Swedish

chemist Savante Arrhenius in 1888. He proposed that the

substrate and enzyme formed some intermediate substance which

is known as the enzyme substrate complex.

Some of the typical applications include enzyme use in

the production of sweeteners, chocolate syrups, bakery

products, alcoholic beverages, precooked cereals, infant

foods, fish meal, cheese and dairy products, egg products,

fruit juice, soft drinks, vegetable oil and puree, candy,

spice and flavor extracts, and liquid coffee, as well as for

dough conditioning, chill proofing of beer, flavor

development, and meat tenderizing. Enzymes also play a

17

significant role in non-food applications. Industrial enzymes

are used in laundry and dishwashing detergents, stonewashing

jeans, pulp and paper manufacture, leather dehairing and

tanning, de-sizing of textiles, deinking of paper, and

degreasing of hides. (Oyeleke S.B., et.al. 2011).

The microorganisms of Bacillus genus are known to be one

of the most important sources of enzymes and other

biomolecules of industrial interest, being responsible for

the supply of about 50% of the market for enzymes (Schallmey,

et al., 2004). The world market for enzymes is estimated at

1.6 billion dollars, 29% for the food industry, animal feed

15% and 56% in other applications (Outtrup, 2002). Among the

different categories, the hydrolase enzymes are among the

largest of industrial application and, among these, the

alpha-amylase and beta-galactosidase have received special

attention (Konsoula, et al. 2006). These enzymes catalyze the

hydrolysis of starch and are produced by a wide variety of

microorganisms, however, for commercial applications they are

basically derived from the genus Bacillus (Schallmey, et al.,

2004). Finally, lipases catalyze the hydrolysis of tri-

18

acylglycerols and are widely used in organic chemistry due to

its high selectivity and specificity and, therefore, receive

much attention because of its potential use in industrial

processes (Lesuisse, et al, 1993).

Furthermore, Bacillus species are good secretors of

proteins and metabolites. Most species of Bacillus strains

have a high capacity to secrete a variety of extracellular

enzymes such as amylase, arabinase, cellulase, lipase,

protease, and xylanase, and these enzymes play important

roles in many biotechnological processes [Sinchaikul et al.,

2002;Cherry and Fidantsef, 2003].

Lipases are ubiquitous enzymes (Brockerhoff and Jensen,

1974; Borgstrom and Brockman, 1984; Desnuelle and Sjostrom,

1986; Wooley and Petersen, 1994) which are found in animals,

plants (Huang, 1993; Mukherjee and Hills, 1994) fungi (Iwai

and Tsujisaka, 1984) and bacteria (Brune and Goetz, 1992;

Jaeger et al., 1994; Jaeger and Reetz, 1998). Lipolytic

enzymes occur widely in nature but only microbial lipolytic

enzymes are commercially significant (Sharma et al., 2001).

Lipolytic enzymes are a versatile group of enzymes and also

19

express other activities like phospholipase,

isophospholipase, cholesterol esterase, cutinase, amidase and

other esterase type of activities (Svendsen, 2000). Lipolytic

enzymes are hydrolytic enzymes which hydrolyse triglycerides

to free fatty acids and glycerol (Sangeetha et al., 2011)

A simple, rapid, and precise method of determining

lipolytic potential of bacteria in

Vitro has been the subject of several investigations, and the

majority of methods developed so far prescribed media

containing various dyes in the presence of an emulsion of

some fatty substance. A change in the color of the dye near

growing colonies indicates lipolytic activity. Toxic

properties of these dyes, at concentrations needed for proper

detection of lipase action, have been one difficulty for

many, Recently, a promising method was developed by Sierra

who incorporated ester of polyoxyalkylene of sorbitan

(Tween); lipolysis was indicated when readily visible

crystals (presumably calcium salts) formed around colonies

growing on media containing these media (Sierra, 1957). He

20

pointed out several advantages of this method, including lack

of toxicity.

Microorganisms have become increasingly important as

producer of industrial

enzymes. Due to their biochemical diversity and the ease with

which enzyme concentrations may be increased by environmental

and genetic manipulation, attempts are now being made to

replace enzymes, which traditionally have been isolated from

complex eukaryotes. Starch degrading amylolytic enzymes are

most important in the biotechnology industries with huge

application in food, fermentation, textile and paper(Pandey

et al., 2000). Amylases can be obtained from several sources

such as plant, animal and microbes (Kathiresan and

Manivannan, 2006). Amylolytic enzymes are widely distributed

in bacteria and fungi. They are categorized in to exo-acting,

endo-acting and debranching enzymes. Among the amylases,

alpha-amylase is exo-acting whereas beta-amylase is endo-

acting enzyme.

Amylolytic strains of the genus Bacillus are a source of

starch-degrading enzymes of commercial interest (Debavov,

21

1982). Treating the bacillus strains on agar plates with

iodine reagent for 30 seconds led to the selection of clones

with increased amylolytic activity. This method is very

convenient for the screening of large numbers of amylolytic

strains. The first step for the isolation of potentially

useful strains is the screening for microorganisms able to

produce clear zones in the starch agar plates surrounding a

bacterial colony (Fogarty and Kelly, 1980). Though the

behavior in the liquid medium is the best indication for the

enzyme production, it is not widespread because it is time-

consuming and not suitable for large scale screenings. The

technique of iodine staining was the fastest compared to the

ethanol precipitation technique and the time-consuming low-

temperature precipitation at 5 "C. However, the use of the

iodine reagent to show starch degradation is considered a

destructive method since it kills the population (Seeley,

1960).

Conceptual Framework

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INDEPENDENT VARIABLES DEPENDENT VARIABLES

Figure 1: Paradigm of the Independent and Dependent Variables

Statement of Null Hypothesis

Based on the foregoing research problems, the following

hypotheses were formulated:

1. Bacillus isolated from Bataan mangrove do not exhibit

amylolytic and lipolytic activities

2. There is no significant difference in the media and

indicators used for enzymatic activities of bacillus

isolated from Bataan mangrove

BacillusCulturemedia

Enzymeproduction

23

Definition of Terms

Extracellular - is that part of a multicellular organism

outside the cells proper, usually taken to be outside the

plasma membranes, and occupied by fluid. Note that for

multicellular organisms, the extracellular space refers to

everything outside a cell, but still within the organism

(excluding the extracellular matrix). Gene products from a

multi-cellular organism are secreted from a cell into the

interstitial fluid or blood can therefore be annotated to

this term.

Enzymes -are large biological molecules responsible for the

thousands of chemical interconvertions that sustain

life. They are highly selective catalysts, greatly

accelerating both the rate and specificity of metabolic

reactions, from the digestion of food to the synthesis

of DNA. Most enzymes are proteins, although some catalytic

RNA molecules have been identified. Enzymes adopt a specific

3D structure, and may employ organic (e.g. biotin) and

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inorganic (e.g. magnesium ion) cofactors to assist in

catalysis.

Lipase - is an enzyme that catalyzes the

breakdown or hydrolysis of lipids or fats.  Lipases are a

subclass of the esterases. Lipases perform essential roles in

the digestion, transport and processing of dietary lipids

(e.g. triglycerides, fats, oils) in most, if not all,

living organisms. Genes encoding lipases are even present in

certain viruses. Lipases are ubiquitous enzymes which are

found in animals, plants fungi and bacteria. Lipolytic

enzymes have attracted much attention during the last decade

due to the diversity in their applications. They have been

utilized for the resolution of chiral drugs, fat

modification, synthesis of cocoa butter constituents, bio-

fuels, synthesis of personal care products, pharmaceutical

industry for the production of drugs and flavor enhancers

(Gerhartz, 1990).

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Amylase - is an enzyme that catalyzes the breakdown of starch

into sugars. Amylase is present in the saliva of humans and

some other mammals, where it begins the chemical process

of digestion. Foods that contain much starch but little

sugar, such as rice and potato, taste slightly sweet as they

are chewed because amylase turns some of their starch into

sugar in the mouth. The pancreas also makes amylase to

hydrolyze dietary starch which is converted by other enzymes

to glucose to supply the body with energy. Plants and some

bacteria also produce amylase. As diastase, amylase was the

first enzyme to be discovered and isolated by Anselme

Payen in 1833. All amylases are glycoside hydrolases and act

on α-1,4-glycosidic bonds.

Mangrove - is used in at least three senses: (1) most

broadly to refer to the habitat and entire plant assemblage

or mangal, for which the terms mangrove

forest biome, mangrove swamp and mangrove forest are also

used, (2) to refer to all trees and large shrubs in the

mangrove swamp, and (3) narrowly to refer to the

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mangrove family of plants, the Rhizophoraceae, or even more

specifically just to mangrove trees of the genus Rhizophora.

The term "mangrove" comes to English from Spanish (perhaps by

way of Portuguese), and is likely to originate from Guarani.

It was earlier "mangrow" (from Portuguese mangue or

Spanish mangle), but this was corrupted via folk

etymology influence of "grove”.

Lugol’s Iodine- is a solution that has been used as

diagnostic and preventive measures and as a disinfectant and

antiseptic. It is also used to test the presence of starch,

vagina cancer cell test, and thyroid function test.

27

CHAPTER II

Materials and Methods

Sample Collection:

Isolation of Bacillus from Bataan Mangrove was collected

by gently digging the soil in the mangrove forest of Barangay

Tortugas, Balanga City, Bataan. Approximately 400 g collected

soil was taken from different sites which were packed in 4 x

11inches sterile polyethylene bag with proper label. This

experiment was conducted for a month (August and September,

2013). The soil samples (numbered from 1 to 6) were air-

dried, sieved to remove the debris present on the soil and

transported to Antibiotics Laboratory of the Institute of

Molecular Biology and Biotechnology at UP Los Baños, Laguna

immediately for the microbial examination.

28

Preparation of Dilution Blanks:

Ten grams of the sample was transferred to a sterile 250

mL conical flask containing 90 mL of sterile 0.85% sodium

chloride solution and diluted logarithmically up to 10-5

level (five- fold dilution).

Isolation of Bacillus:

The nutrient media used for this study was a fresh

Nutrient Agar. For plating, 1 ml of the serially diluted

samples of soil was pipetted out into sterile Petri dish.

Then sterile media was poured into dishes aseptically and

swirled for a thorough mixing. After solidification the

plates were incubated overnight in an inverted position at

30°C. All the determination was carried out in duplicates.

The pure cultures/colonies were subjected for enzyme assay.

Preparation of Culture Media:

29

1. Starch Agar: Weigh 2.5 g of soluble starch; 0.43 g of

Pharmaceutical Agar; and 2 g of Nutrient Broth in 250 mL

distilled water. The solution was mixed thoroughly and

sterilized before pouring into plates.

2. Sierra’s Medium Agar: Weigh peptone 10 g; NaCl 5 g;

CaCl2.2H2O 0.1 g; agar 16 g in 1000 mL supplemented with

10 g of Tween 80.

Lipase assay:

For lipase activity, the strains were grown on Sierra’s

agar medium supplemented with 10 g of Tween 80.After

incubation at 300C for 5 days, the plates were flooded with

saturated copper sulfate solution. A formation of opaque

whitish zone (calcium precipitate) around the growing colony

was considered as positive.

Amylase Assay:

Amylase activity was assessed by growing the selected

strains on starch-agar medium. After incubation at 300C for 5

30

days, the plates were flooded with 1% iodine in 2% potassium

iodide. The clear zone formed surrounding the colony was

considered positive for amylase activity.

Microscopic Assay:

The bacterial isolates were stained by Gram staining

(crystal violet for 1 min; iodine for 1 min; ethyl alcohol

for 30 s; and safranin for 30 s) and observed under a high

power magnifying lens in light microscope. Pure isolates were

streak lightly on a glass slide initially dropped with

sterile water. The slide was fixed under a flame before it

was viewed on a microscope.

Decontamination and Disposal:

Bacterial cultures, chemical media and all laboratory

glass wares and apparatuses that were in contact with the

test samples and test organisms were subjected to an

autoclave at a temperature of at least 121-132 °C for 30-40

minutes and cooled before it was discarded into a specified

trash bin. Other materials not in contact with any of the

31

microorganisms were cleaned thoroughly with detergent and

water. Working area was kept clean at all times with the use

of disinfectants like commercial Zonrox and 70% rubbing

alcohol.

Screening Procedure

All the 35 isolates were assessed for their

extracellular enzymatic activity. The strains were screened

on solid agar media at 300C in triplicates. Freshly grown

cultures of test bacteria were spot inoculated on starch agar

plates by the help of a sterile loop. Amylase activity was

observed by incubating the plates and exposing to iodine

vapors. Lipase activity of the isolates was observed by

incubating the plates and exposing to saturated copper

solution.

Analysis of extracellular enzymes: The extracellular

enzymes production viz. amylase, lipase were analyzed through

plate test and qualitative method by growing these individual

bacterial isolates in starch agar (amylase), and peptone agar

media (lipase). After 5 days of incubation at 30 °C, culture

32

plates were tested for enzyme activity by adding Lugol’s

iodine solution in amylase plates, saturated copper sulfate

solution in lipase plates. The clear zone formation around

the growing colony was considered as positive for amylase.

The lipase activity of bacterial isolates were determined

using a lipid source of Tween 80 on lipase test medium and

the formation of opaque whitish zone around the growing

colony was considered as positive (Booth, 1978).

Flow Chart of the Procedure

The procedure in conducting this research consists of

several steps. They are shown in the following methodology

flowchart:

33

CHAPTER

III

Results and Discussion

Results

All 35 isolates were screened for different

extracellular enzymatic activities. The strains were assessed

for enzymatic activity in terms of zone sizes (Table-1),

though enzymatic activities were observed at 300C.

Figure 2: Flowchart of Procedure

34

Legend: (+) Clear zone observed for amylase; whitish formation, opaque inAppearance observed for lipase.

Table 1: Lipase and Amylase enzyme activities of bacillus isolated fromBataan mangrove

Out of 35 bacterial isolates only 5 strains were screened as

a potent degrader of lipid by producing lipolytic enzyme and

showed a whitish precipitate lipolytic formation on Sierra’s

medium agar plate at 30 0C (figure 1) while there were 27

isolates showed a clear zone on Starch medium agar plate at

300C (figure 2).

Figure3: Screening of lipolytic enzyme producing whitish zone indicatesthe hydrolysis of copper sulfate as a result of lipase production

35

Figure 4: A starch agar plate showing amylase activity with clear white zoneat the center surrounding the single colony of Bacillus isolated

from soil sample collected from Bataan mangrove

Under microscopy, the morphology of the isolated strains

was revealed as rod-shaped cells (data not shown).Micrograph

images indicated that isolates no. 2, 4, 5, 6, 16, 23, 27 had

a similar long and straight rod shape (figure 3). But no

further confirmative tests were done to identify the isolates

up to species levels.

Figure 5: Gram stained of Bacillus isolates

Discussion

In this investigation we clearly recorded the occurrence

of bacillus strains, in the mangrove sites of Barangay

36

Tortugas in Bataan, with industrially important extracellular

enzymatic activities. In this regard, 77.14% of the isolates

showed extracellular amylolytic activity at 300C could be a

novel source for different industrial processes using amylase

as a catalyst. Starch degrading amylolytic enzymes are most

important in the biotechnology industries with huge

application in food, fermentation, textile and paper (Pandey

et al., 2000). Amylolytic enzymes are widely distributed in

bacteria and fungi. The microbial source of amylase is

preferred to other sources because of its plasticity and vast

availability. Microbial amylase has almost surpassed the

synthetic sources in different industries (Pandey et al.,

2000). However, it appears that 17.14%, a very low percentage

of bacillus associated with mangrove from Bataan showed a

potential for lipase production in vitro. This study indicates

that interaction of bacillus with Tween might be affected by

the medium. Furthermore, the smaller zone sizes on solid agar

plates are presumably because of low amounts of lipase

molecules released by the colonies (Kouker and Jaeger 1987).

Furthermore, lipolytic surface-growing colonies may not

37

produce lipase when submerged in agar medium, and

constitution of the growth medium and/or the choice of

indicators used for detection of lipolytic activity could be

one of the contributing factors affecting its lipolytic

ability (Sierra, 1957).

CHAPTER IV

Summary, Conclusions and Recommendation

Summary and Conclusion

The present study mainly focuses on the lipolytic and

amylolytic activities of bacillus from Bataan mangrove.

Amylase activity was detected after growth on soluble starch-

agar plates by a hydrolysis zone around the colony using

iodine staining. Lipase activity was detected after growth on

peptone agar media by formation of whitish precipitate-zone

38

using copper sulfate staining. Screening of 35 Bacillus

natural isolates from Bataan mangrove for specificity they

exhibit relative to the reactions they catalyze showed that

most of the tested strains exhibited enzyme activity, which

illustrates their potential use in commercial applications.

Hence, Bacillus strains from Bataan mangrove exhibited

both lipase and amylase activities. Microscopic analysis

showed the strain was a Gram-positive, with rod-shaped cells;

some grew in short chain and some in long chain. However,

plate-screening method is not quantitative because of poor

correlation between enzyme activity and colony to clear zone

ratio.

Recommendations

The above investigation clearly revealed extracellular

enzymatic activity of bacterial strains isolated from a local

mangrove. A study such as this is a prerequisite for tapping

the biotechnological potential of the microbes from mangrove.

Hence, the following are highly recommended:

39

1. Confirmative tests should also be done to identify

properly the isolates up to species level based on

nucleotide homology and phylogenetic analysis using 16S

rDNA and gyraseA gene sequences, FAME analysis, and etc.

2. The study of other enzymes such as protease, cellulase,

gelatinase, casein hydrolase, lecithinase, chitinase and

pectinase should also be carried out.

3. Further characterization, physiologically and

biochemically, to confirm the identity of the bacillus

isolates like analysis of amino acids, lipids, phage

typing etc.

40

Bibliography

A. Books

Bennett, T. P., and Frieden, E.: Modern Topics inBiochemistry, pg. 43-45, Macmillan, London (1969).

Brune, K.A. - Goetz, F. 1992. Degradation of lipids by bacterial lipases. In VCH,

Germany, 1992.

Enzyme Technical Association (2001).Enzymes; A primer on useand Benefits today and

tomorrow. Washington, DC 200036 pp. 1-32

Fogarty, W. M., Kelly, C. T. - In: Economic Microbiology.Ed.: A. H. Rose,London: Academic Press: 1980, 115

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APPENDIX

A. List of Figures

Figure 1: Paradigm of the Independent and DependentVariables

Figure2: Flowchart of Procedure

Figure 3: Screening of lipolytic enzyme producing whitish zone indicates the

hydrolysis of copper sulfate as a result of lipase production

Figure 4: A starch agar plate showing amylase activity with clear white zone at the center surrounding the single colony of Bacillus isolated from soil sample collected from Bataan mangrove

Figure 5: Gram stained of Bacillus isolate

B. List of Tables

44

Table 1: Lipase and Amylase enzyme activities ofbacillus isolated from

Bataan mangrove

S.Y. 2013-2014 INTEL PHILIPPINE SCIENCE FAIR

RESEARCH PLAN 1A

Name: Cecile Angela D. Coronado Cluster: 2

Category: Life Science

Heidi Rose B. Del Rosario

Annielyn R. Marcellana

School address: Negros St. Pitogo, Makati City

45

Title of the Project:

Amylolytic and Lipolytic Activities of Bacillus Isolated From

Bataan Mangrove

Project Adviser: Ms. Lilibeth Sia Lumapas

Statement of the Problem:

This study aims to identify the amylolytic and lipolytic

activities of Bacillus isolated from Bataan Mangrove

Objective of the Study

This study aims to identify morphologically the bacillus

strain present in the mangrove soil of Barangay Tortugas in

Bataan and to evaluate their enzyme production potentials.

Specifically, the study aims to:

4. Isolate bacillus from Brgy. Tortugas, Balanga City,

Bataan mangrove forest soil

46

5. Determine if bacillus from Bataan mangrove exhibits

amylolytic and lipolytic activities

6. Determine the potential of bacillus isolated from Bataan

mangrove for enzyme production

Statement of Null Hypothesis

Based on the foregoing research problems, the following

hypotheses were formulated:

3. Bacillus isolated from Bataan mangrove do not exhibit

amylolytic and lipolytic activities

4. There is no significant difference in the media and

indicators used for enzymatic activities of bacillus

isolated from Bataan mangrove

MATERIALS AND METHODS

Sample Collection:

47

Isolation of Bacillus from Bataan Mangrove will be

collected by gentle digging the soil in the mangrove forest

of Barangay Tortugas, Balanga City, Bataan. Approximately 400

g collected soil will be taken from different sites which

will be packed in 4 x 11inches sterile polyethylene bag with

proper label. This experiment will be conducted for a month

(August and September, 2013). The soil samples (numbered from

1 to 6) will be air-dried, sieved to remove the debris

present on the soil and will be transported to Antibiotics

Laboratory of the Institute of Molecular Biology and

Biotechnology at UP Los Baños, Laguna immediately for the

microbial examination.

Preparation of Dilution Blanks:

Ten grams of the sample will be transferred to a sterile

250 mL conical flask containing 90 mL of sterile 0.85% sodium

chloride solution and will be diluted logarithmically up to

10-5 level (five- fold dilution).

Isolation of Bacillus:

48

The nutrient media will be used for this study is a

fresh Nutrient Agar. For plating, 1 ml of the serially

diluted samples of soil will be pipetted out into sterile

Petri dish. Then sterile media will be poured into dishes

aseptically and will be swirled for a thorough mixing. After

solidification the plates will be incubated overnight in an

inverted position at 30°C. All the determination will be

carried out in duplicates. The pure cultures/colonies will be

subjected for enzyme assay.

Preparation of Culture Media:

3. Starch Agar: Weigh 2.5 g of soluble starch; 0.43 g of

Pharmaceutical Agar; and 2 g of Nutrient Broth in 250 mL

distilled water. The solution will be mixed thoroughly

and will be sterilized before pouring into plates.

4. Sierra’s Medium Agar: Weigh peptone 10 g; NaCl 5 g;

CaCl2.2H2O 0.1 g; agar 16 g in 1000 mL will be

supplemented with 10 g of Tween 80.

Microscopic Assay:

49

The bacterial isolates will be stained by Gram staining

(crystal violet for 1 min; iodine for 1 min; ethyl alcohol

for 30 s; and safranine for 30 s) and will be observed under

a high power magnifying lens in light microscope. Pure

isolates will be streaked lightly on a glass slide initially

dropped with sterile water. The slide will be fixed under a

flame before it will be viewed on a microscope.

Decontamination and Disposal:

Bacterial cultures, chemical media and all laboratory

glass wares and apparatuses that will be in contact with the

test samples and test organisms will be subjected to an

autoclave at a temperature of at least 121-132 °C for 30-40

minutes and will be cooled before it will be discarded into a

specified trash bin. Other materials not in contact with any

of the microorganisms will be cleaned thoroughly with

detergent and water. Working area will be kept clean at all

times with the use of disinfectants like commercial Zonrox

and 70% rubbing alcohol.

50

Screening Procedure

All the35 isolates will be assessed for their

extracellular enzymatic activity. The strains will be

screened on solid agar media at 300C in triplicates. Freshly

grown cultures of test bacteria will be spot inoculated on

starch agar plates by the help of a sterile loop. Amylase

activity will be observed by incubating the plates and

exposing to iodine vapors. Lipase activity of the isolates

will be observed by incubating the plates and exposing to

saturated copper solution.

Analysis of extracellular enzymes: The extracellular

enzymes production viz. amylase, lipase will be analyzed

through plate test and qualitative method by growing these

individual bacterial isolates in starch agar (amylase), and

peptone agar media (lipase). After 5 days of incubation at 30

°C, culture plates will be tested for enzyme activity by

adding Lugol’siodine solution in amylase plates, saturated

copper sulfate solution in lipase plates. The clear zone

formation around the growing colony will be considered as

positive for amylase. The lipase activity of bacterial

51

isolates will be determined using a lipid source of Tween 80

on lipase test medium and the formation of opaque whitish

zone around the growing colony will be considered as positive

(Booth, 1978).