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RESEARCH PLAN

Project Title: Soil Conditioning Ability of Humic Acid-producing Earthworm Gut

Microbial Isolates

Project Proponent/s: Katrina Deñoso

Ken Jover Tumalom Amparo

Matthew Dave Fraginal Bitancur

Project Adviser: Marion Rodrigo Diaz Mariñas

RATIONALE

Synopsis of the Background Study

1. Earthworms are called “soil engineers” since they contribute on the release of

available nutrients for other organisms and microorganisms and plants. (Kooch and

Jalilvand, 2008).

2. Earthworms cause major chemical, physical and biological effects on soil ecosystems

and making themselves as the important component of soil fauna. (Lavelle, et al,

2007).

3. Edwards and Bohlen (1996) stated that earthworms are part of pathogen reduction

systems and as a natural organic recycler of the soil.

4. According to Tan (2014) the structure, composition and properties of humic acid

(HA) greatly affects the crop yield and fertility of the soil as HA is an essential part

of the soil humus.

RESEARCH PROPONENTS: Katrina Deñoso, Ken Jover Tumalom Amparo & Matthew Dave Fraginal Bitancur
RESEARCH ADVISER: Marion Rodrigo Diaz Marinas
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5. Parle (1969) reported the fungal, bacterial and actinomycetic populations in three (3)

earthworms’ species such as: Lumbricus terristris, Alonga and Allobophora

calligonasa.

6. Humic substances (HS) in nutrient uptake have beneficial effects on the significant

inorganic components; nitrogen (N), phosphorus (P), and potassium (K). (Mylonas

and and Mc Conts, 1980).

SOCIETAL IMPACTS OF THE STUDY

1. Pedologists will gain such information and ideas about the use of organic soil

conditioner from the metabolites in the earthworm gut microflora.

2. Farmers will be able to have alternative soil conditioner that can enhance soil fertility

and will also help in replenishing the lost soil nutrients needed by the organisms that

uses soil as their source of nutrition and habitat

3. The use of organic soil conditioner instead of chemical-based and artificial soil

enhancers will be having a huge impact in the environment. Since this organic soil

conditioner, it won’t harm the environment than chemical-based.

4.

RESEARCH PROBLEMS

1. What are the bacterial species isolated from the different sections of earthworm gut,

namely;

a. foregut (FG);

b. midgut (HG); and,

RESEARCH PROPONENTS: Katrina Deñoso, Ken Jover Tumalom Amparo & Matthew Dave Fraginal Bitancur
RESEARCH ADVISER: Marion Rodrigo Diaz Marinas
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c. hindgut (HG)?

2. Which microbial isolates from the earthworm gut has the ability to produce humic

acid?

3. Will the humic acid-producing bacteria isolated from the various sections of the

earthworm gut affect soil samples by the following variables, namely;

a. pH level;

b. nitrogen (N), phosphorus (P), and potassium (K) content; and

c. water retention property?

HYPOTHESES OF THE STUDY

1. The bacteria isolated from the earthworm gut has the ability to produce humic acid

2. The humic acid-producing bacteria isolated from the earthworm gut has the ability to

affect the pH level, nitrogen, phosphorus, and potassium content, and water retention

property of the soil.

EXPECTED OUTCOME/S

This research study aims to accomplish the following outcomes, namely:

1. There is a certain bacterial isolate that has the ability to produce humic acid.

2. The nitrogen, phosphorus and potassium content, pH level and water retention property

of the soil will be affected by the humic acid-producing bacteria isolated from

earthworm gut.

RESEARCH PROPONENTS: Katrina Deñoso, Ken Jover Tumalom Amparo & Matthew Dave Fraginal Bitancur
RESEARCH ADVISER: Marion Rodrigo Diaz Marinas
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METHODOLOGY

Collection of earthworms

Dissection of earthworms

v
v
Bacterial culture

v
v
Identification of humic acid-producing microbial isolates

v
v
Applying the humic acid to soil samples

v
v
Data Gathering and Statistical Analysis

Collection of earthworms

An agricultural area at Prenza I, Marilao, Bulacan will be selected to serve as the

collection site of the earthworm. Using mustard extraction method, two (2) tablespoon or 1/3 cup

of powdered mustard thoroughly will be mixed into a one and half gallon distilled water. It will

then be slowly poured into the ground, Five (5) minutes will be elapsed before collecting all the

all earthworms that will come out to the surface. The earthworms will be temporarily stored in a

plastic container containing the soil where they came from.

RESEARCH PROPONENTS: Katrina Deñoso, Ken Jover Tumalom Amparo & Matthew Dave Fraginal Bitancur
RESEARCH ADVISER: Marion Rodrigo Diaz Marinas
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Dissection of earthworms

The earthworms will be dissected aseptically by placing the earthworm ventral side up in

a sterile dissecting pan. The head and anus will be secured by a pin. The gastrointestinal (GI)

tract will be exposed by cutting through the earthworm’s muscular wall from clitellum towards

the anus. Different section of the GI tract will be isolated such as the foregut (FG), midgut (MG)

and hindgut (HG).

Bacterial culture

Using an inoculating loop, the gut content will be collected and streaked onto a petri dish

with Potato Dextrose Agar (PDA). Pure culture of microbes can be maintained using sterile

techniques. (Allan Jones, Rob Reed & Jonathan Weyers, 2003).

Identification of humic acid-producing microbial isolates

The morphological characteristics of the pure culture of microbes will be identified by

their size, form, margin, consistency and color. There will also be microscopic examination to

determine the motility and cell shape. Moreover, Gram staining will be performed to identify the

Gram + and Gram – bacterial isolates. Extraction of humic acid by weighing five (5) g of the soil

conditioner in 250 mL-cylinder, then add 100 mL of pyrophosphate solution on the sample that

will be close by a parafilm to isolate CO2 and the air from the sample then left for 18 hours. Add

25 mL of saturated sodium sulphate, then mix the mixture and left for 15 minutes. The mixture

will be filtered with discarding the first drops of the filtrate.

RESEARCH PROPONENTS: Katrina Deñoso, Ken Jover Tumalom Amparo & Matthew Dave Fraginal Bitancur
RESEARCH ADVISER: Marion Rodrigo Diaz Marinas
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Identification and Isolation of Humic Acid Producing Bacterial Isolate

To determine the humic acid content, add 5 mL of 1 N sulfuric acid solution to 25 mL of

the filtrate pipetted in a conical flask, then the pH of the solution will be adjusted to 1.3-1.5 using

the 1.0 N sulfuric acid solution. The acidified solution will be heated in a water bath at 70ºC-

80ºC until the HA begin to precipitate in a jelly-like shape. Allow the solution to cool down for 1

hour for completion of the HA precipitation that will be filtered in another conical flask, the

precipitate on the filter paper will be washed three times with the 0.1 N sulfuric acid solution to

kill the remaining fulvic acids. The precipitate on the filter paper will be dissolved by successive

washing with the warm 0.1 NaOH solution (40ºC-50ºC) to return the precipitate to the filtrate

(the total volume of NaOH solution is 50mL). The basic solution is allowed to cool and

transferred to 100 mL - volumetric flask and complete to the mark with distilled water. Ten (10)

mL of the previous solution will be pipetted in a conical flask. Potassium dichromate solution

(2.5 mL of the 1.0 N) and 5 mL of the concentrated sulfuric solution will be added to the conical

flask and will be left for 15 minutes. If the solution is dark oily, then it has a high organic acid

content and needs doubled amounts of the dichromate and sulfuric acid solutions to oxidize the

organic acids. The conical flask will be sealed tightly and will be left for 30 minutes, and then 50

mL of distilled water will be added to the former solution and 5 mL of 80% phosphoric acid

solution to clearly show the end point (the solution will be turning from dark-purple into green-

colored solution) upon addition of 0.5 mL diphenylamine indicator solution. The basic solution

will be titrated against the 1.0 N ferrous sulphate solution. The volume will be recorded which is

the sample volume (S). The blank solution is a mixture of 2.5 mL of the dichromate solution, 5

mL of the concentrated sulfuric acid solution and 5 mL of 80% phosphoric acid in a conical

flask. Distilled water (50 mL) will be added and the mixture allowed to cool down, then 0.5 mL

RESEARCH PROPONENTS: Katrina Deñoso, Ken Jover Tumalom Amparo & Matthew Dave Fraginal Bitancur
RESEARCH ADVISER: Marion Rodrigo Diaz Marinas
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of the diphenylamine indicator will be added. The solution was titrated against the 1.0 N ferrous

sulphate solution and the volume will be recorded as (B).The HA percentage will be calculated

in determination of carbon in humic compounds-determination (Walkey & Black,1934;

Jackson,2005).This procedure will be repeated three times.

Bacterial Preservation by Freeze Drying

The bacteria require a lypoprotectant to survive the freeze drying process. Reagent

eighteen (18) will be used to prepare the lypophilization medium. Add 0.75 of Typricase Soy

Broth, ten (10) gm sucrose and five (5) gm of Bovine Serum Albumin (BSA).Fraction V to one

hundred (100) mL deionized water.

The samples dispensed into tubes will be froze in a -80ºC freezer. The samples will be

kept frozen until they are connected to a manifold.

The samples will undergo the sublimation process using the lyophilizer, condenser tube

and the vacuum attached to a manifold. When the sublimation process is done and the samples

are dried, acetylene torch will be used to seal the tubes. The sealed tubes containing the freeze-

dried samples will be stored 4ºC in the dark.

Applying the humic acid to soil samples

Before applying the humic acid, collection of soil samples from the Marilao riverbank,

industrial site, agricultural land, pastureland and residential area respectively. The said soil

samples will be subjected to determination of the pH level, nitrogen (N), phosphorus (P), and

potassium (K) content and water retention property. The soil samples will be sent to the Bureau

of Soils and Water Management for the pre-testing of NPK and pH level. The humic acid

RESEARCH PROPONENTS: Katrina Deñoso, Ken Jover Tumalom Amparo & Matthew Dave Fraginal Bitancur
RESEARCH ADVISER: Marion Rodrigo Diaz Marinas
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isolated from the earthworm gut bacterial isolates will be applied as the soil conditioner. The soil

samples will be treated for two weeks before conducting the same test to determine the

aforementioned variables. The soil samples will be sent again to the Bureau of Soils and Water

Management for the post-testing of NPK and pH level.

DATA GATHERING

The bacterial isolate with the ability to gather all the results from the experiment and it

will be subjected to descriptive and inferential statistics to determine the difference between the

pH level, nitrogen (N), phosphorus (P), and potassium (K) content and water retention property

of the various soil samples treated with humic acid isolates.

RISK AND SAFETY

Name of reagent/ Classification of Risks Biological Containment

substance/ material material

Bacterial isolates BSL-1 Risk group contains Work is done on an open

biological agents that bench or in an appropriate
from gut pose low risk to biosafety hood.
personnel and the Standard microbiological
microflora environment. These practices are used when
agents are highly working in the laboratory.
unlikely to cause Decontamination can be
disease in healthy achieved by treating with
laboratory workers, chemical disinfectants or
animals or plants. by steam autoclaving.
Lab coats and gloves are
required.
The laboratory work is
supervised by an
individual with general
training in microbiology
or a related science.

RESEARCH PROPONENTS: Katrina Deñoso, Ken Jover Tumalom Amparo & Matthew Dave Fraginal Bitancur
RESEARCH ADVISER: Marion Rodrigo Diaz Marinas
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Aseptic Techniques

1. Heating inoculating loops and forceps in a Bunsen burner/flame. This is an effective

way of sterilization because no microbes can survive to naked flame. (Jones, Reed &

Weyers, 2003).

2. A sterile workplace will be prepared using a standard procedure.

3. Before starting any sterilization procedures in your work area, wash the work area

thoroughly with bleach.

4. Arrange all the supplies needed for the procedure on the laboratory bench near the

sterile field. Make sure all the materials are properly labeled.

5. Safety Considerations on materials/ equipment to be used:

Name of Safety Considerations Risks

Material/Equipment
1. Use of Bunsen Never leave open flame unattended, not Open flame,
burner even for a few seconds. serious burns
Since burns are among the most common
accidents, make sure that face, clothing,
and hair are not above or near the opening
of the burner.
Remove flammable and combustible
materials from the vicinity of the flame.
A fire extinguisher should always be kept
nearby.
The tube connecting the Bunsen burner
should be regularly checked.
2. Inoculation Loop Inoculation loop should be sterilized first Contamination
using a Bunsen burner to avoid
contamination.
3. Petri Dish For all manipulations of cultures in petri Contamination of
dishes, lids should be lifted as short a time the
as possible. Lids are never to be placed on materials/substances
the bench top. Do not walk around the inside the dishes
room with an open plate. As with all other
media, do not breathe on open plates.

RESEARCH PROPONENTS: Katrina Deñoso, Ken Jover Tumalom Amparo & Matthew Dave Fraginal Bitancur
RESEARCH ADVISER: Marion Rodrigo Diaz Marinas
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DISCUSSION OF RESULTS

The soil-conditioning ability of humic acid-producing bacteria isolated from

earthworm gut affect, replenish and improve the soil properties such as; pH level, nitrogen (N),

phosphorus (P), and potassium (K) content and water retention property and after applying the

soil conditioner it can sustain life of plants again and other organisms living in the soil.

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RESEARCH ADVISER: Marion Rodrigo Diaz Marinas
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RESEARCH PROPONENTS: Katrina Deñoso, Ken Jover Tumalom Amparo & Matthew Dave Fraginal Bitancur
RESEARCH ADVISER: Marion Rodrigo Diaz Marinas