Republic of the Philippines

Department of Education
NATIONAL CAPITAL REGION
SCHOOLS DIVISION OF LAS PIÑAS CITY
LAS PIÑAS CITY NATIONAL SCIENCE HIGH SCHOOL
Carnival Park St. Bf Resort Village, Talon II, Las Piñas City

RESEARCH PROPOSAL
Synthesizing Earthworm (Lumbricus Terrestris) Slime for Biogas Conversion through
Anaerobic Digestion as a Substitute for Natural Gas Supply

By Group No. 6

Researchers: Daisy
B. Aguilar
Luccia Isabelle K. Gabor
John Richard Olete
 A. INTRODUCTION

I. RATIONALE

In the current age, oil and fossil fuels serve as a power player for the industries that
bound the planet. Although, the usage of both continues to damage the environment, as well as
the lives of the public from increasing the prices of products and needs, to destroying the
condition of our planet. Citizens face multiple environmental and economic crises, and they
will continuously do if these issues persevere from our own hands.

Threatening environmental issues such as climate change and global warming continuously
happen, as long as the amount of greenhouse gas emissions is constantly increasing. “The
carbon dioxide emissions of fossil fuels are projected to increase by 1% in 2022, hitting a new
record of 37.5 million tonnes” (Nature Publishing Group 2022), which is truly an alarming rise
in carbon dioxide emissions. The relentless expansion of oil usage is a factor contributing to the
hike of greenhouse gas emissions, and it is predicted to drive the rate by a 6% increase compared
with 2021 (Nature Publishing Group 2022).

The wide use of oil and gasoline also contributes to economic issues such as inflation since
the demand for oil has increased since the pandemic (Patterson and Goldfarb 2022), making it
far from affordable for the public.

II. SCIENTIFIC BASIS

“Earthworm slime is primarily composed of nitrogen, an essential natural element found in

amino acids. Nitrogen is vital for growth and reproduction among flora and fauna. In some
species of burrowing worms, their slime is used to firm the walls of their burrows, keeping them
from collapsing” (Whelan 2017). Though these slime excretions not only serve as a potent
fertilizer or a lubricant for their locomotion to ease through their surroundings, but its nitrogen
components help in creating efficient and healthy compost pits.

Aside from nitrogen (N), traces of potassium (K) and phosphorus (P) were also
discovered with the former containing 1.85-2.25% and latter of a rate of 1.55-2.25% per kg/ha
annually (Sinha, Rajiv, Herat, et al. 2009). It also accommodates micronutrients, beneficial soil
microbes, and “plant growth hormones and enzymes”.

III. IMPORTANCE OF THE STUDY

 The study that is presented is essential to our environment. Most devices, machines,
factories, vehicles, and such use fuel oils that release harmful gases that cause pollution and
other environmental risks. This study promotes the use of safe biogas that gradually reduces
climate change and greenhouse gas emissions to provide a safe and prudent nature. Over and
above that, this can also be practical to residential services as basic need for daily life not only
because it can be an alternative for other fuels ranging from home heating to automotive fuels
but moreover, it can be used as a fertilizer and also helps in generating indoor light which
consumes less electric cost.

In businesses and corporations, since oil is the major factor of why products are quite costly
today — causing them to have low business income, with the biogas, both the consumers and the
retailer will be at advantage. Low price of products would benefit the consumers and in branch
of that, high income and profit would satisfy the retailers plus in developing their performance
and in gaining more revenue.

Furthermore, in economic factors, biogas is a potent engine for economic growth especially
in rural communities that lack economic possibilities. This biogas also has many significant
economical benefits, and this incorporates the local work inventions in specialized, assembling
and development or exchanges. Monetary improvement creates enormous amounts of interest in
rustic networks — since cheaper products are more likely to be sold. In addition, biogas
technology can also help in formation of valuable results from squanders, going out as a huge
financial multiplier.

IV. THEORETICAL FRAMEWORK

The relationship between the nitrogen component of earthworm slime and the required
nitrogen composition for the production of biogasses enables the earthworm slime to be a
potential product to be used to convert it into biogas. The environmental
benefits of earthworm slime such as enriching the soil, serving as a vital medium responsible
for growth for instances of life, increasing soil nutrient unit, conduct better soil drainage
attribute and steadily anchored soil framework — hence a salient module for our ecosystem.

V. OBJECTIVES

To resolve the immense damage to the ecosystem caused by air pollution by the excessive
use of gasoline, petrol, and alike; we want to present our study as a bandage over said
implication. Implying these issues, our study aims to create an alternative for fossil fuel-based
gasses, such as petrol and natural gas, using biologically-produced biogas out of earthworm
slime excretions. With the help of this study, the researchers can depreciate gas emissions
ejected at the atmosphere by a vast percentage.

Fumes produced by gasoline overrun cities and create major airborne diseases that are
proven hostile to life. These emissions are considerably of concern as inhabitants near a source
will sustain strokes, heart diseases, lung cancer, acute and chronic respiratory diseases.
Furthermore, impairment and tremors upon the ecological foundations are then brought by
further consumption of oil at an extended frequency — therefore shortening the tendency of
biological entities to flourish.

Since the composition of the biogas we construct are purely made of organic compounds, we
can expect it to be less energy consuming and safer to maneuver with. If, and in all means, our
research extends the reach of a capital, we can conduct a summit to upskill our clientele and
prepare them for an absolute scenario. Added to this, we as researchers can hereby register our
study as a complete replacement of oil reliance. Oil is an expensive and hard to derive material
— often causes a huge dilemma just by simply extracting a gallon. With our study, we can,
atleast to an extent, eliminate inadequate factors leading to a substance that powers this planet.

B. REVIEW OF RELATED LITERATURE

Anaerobic Digestion

According to Liebtrau, et. al (2017), Anaerobic Digestion (AD) is the microbial

conversion of organic materials or waste into energy-rich gas, biogas, which is used as a
renewable energy supplier. It is often applied in the treatment of sewer systems, solid waste, and
the disposal and conversion of agricultural residue. The biogas produced from AD mainly
consists of methane and carbon dioxide. This study also discusses that
in the context of biorefinery principles, Anaerobic Digestion is a treatment method for
reducing organic waste from other production processes as well.

Biofuels for a sustainable future

Based on a study conducted by Liu, et.al (2021), increasing energy consumption and
human dependence on fossil fuels that garnered greenhouse gasses and hence, climate change.
Major efforts have been taken to develop and test renewable fuel proxy. Given the abundance of
earthworm in nearly every environment, researchers have thought of wielding its properties to
create a product capable of reducing fuel dependency thus lessening environmental difficulty.
Through scientific methods, researchers hope to create an alternative that is cost-friendly,
efficient, and easily produced.

Ecological role of earthworm intestinal bacteria in terrestrial environments

Mingming Sun, et.al (2020) asserts that earthworm gut has a potential application that is
good for pollutant transformation including nitrification, methanogens (which produce methane
that is essential for biogas conversion), metal detoxification etc. Given this evidence, its
abundance of nutrients makes it ideal for colonization or anaerobic bacteria. Although limited
information about the earthworm's specific mechanism was gathered, it is highlighted in the
study the current problems together with the future prospect of its intestinal bacteria, which is
enough for the researchers to create an alternative for natural gas supply.

Humification and Microbial Activity of Earthworm Mucus in Vermicomposting

In a study published in 2018, Huang and Xia examined how earthworm mucus might aid
in the humification and decomposition of organic waste. For this, three vermicomposting
substrates were inoculated with "Eisenia fetida mucus" using cow dung (CD), fruit and
vegetable wastes (FVW), and sewage sludge (SS). A 20-day experiment showed that the mucus
could accelerate the humification and mineralization of organic components. In comparison to
substrates without mucus, treatments
incorporating mucus revealed an increase in dissolved carbon of 9.8% to 37.5%. The mucus
also significantly enhanced bacterial population and microbial activity, with FVW treatments
showing the largest increases. In addition, the mucus adversely impacted Firmicutes during
decomposition while favoring the growth of "Proteobacteria."

Biogas from Nitrogen-Rich Biomass as an Alternative to Animal Manure Co-Substrate in

Anaerobic Co-Digestion Processes
 Isabel et. al (2022) discovered in their study that nitrogen-rich biomass is an
appropriate substrate in biogas co-digestion instead of animal manure, which is commonly
used in the co-digestion process. If animal manure cannot be used to produce methane gas,
this biomass mixed with other substrates might replace it. The main component of the biogas
produced in AC-D and used as a fuel is methane. The study’s results showed that the most
elevated methane yield belongs to the
nitrogen-rich biomass, compared to the pig manure that was used in the
experimentation. A fertilizer of importance is the digestate produced in the AC-D
because it contains nitrogen and can replace mineral fertilizers, saving energy.

REFERENCES

Liebetrau, Jan, Sträuber, Heike, Kretzschmar, Jorg, Denysenko, Velina, Nelles, Michael.
2019. Anaerobic Digestion. Adv Biochem Eng Biotechnol. 2019;166:281-299. doi:
10.1007/10_2016_67. PMID: 28391363.

Liu, Yuzhong, Cruz-Morales, Pablo, Zargar, Amir, Belcher, Michael S., Pang, Bo,
Englund, Elias, Dan, Qingyun, Yin, Kevin, & Keasling, Jay D. 2021. Biofuels for a
Sustainable Future. Cell, 184(6), 1636–1647. https://doi.org/10.1016/j.cell.2021.01.052

Sun, Mingming, Chao, Huizhen, Zheng, Xiaoxuan, Deng, Shaopo, Ye, Mao, & Hu, Feng.
2020. Ecological role of earthworm intestinal bacteria in terrestrial environments: A
review. The Science of the total environment. 740, 140008.
https://doi.org/10.1016/j.scitotenv.2020.140008

Huang, Kui, & Xia, Hui. 2018. Role of earthworms' mucus in vermicomposting system:
Biodegradation tests based on humification and microbial activity. The Science of the
total environment. 610-611, 703–708.

https://doi.org/10.1016/j.scitotenv.2017.08.104

Isabel, Parralejo A.A., Luis, Royano B., Juan, Cabanillas P., Jerónimo, Gonzales C. 2022.
Biogas from Nitrogen-Rich Biomass as an Alternative to Animal Manure
Co-Substrate in Anaerobic Co-Digestion Processes. Energies 2022, 15, 5978.

https://doi.org/ 10.3390/en15165978

C. METHODOLOGY

I. Gathering of the materials

 Earthworms will be gathered from moist soil in Villar Sipag in Las Pinas City, and
hand gloves and a large container will be used to gather them. The hand gloves, large
container, and the shovel will all be bought in nearby hardware stores.

II. Extraction of earthworm slime

With (5) lbs or (2) kgs of earthworms at hand, they can excrete mucus at a rate of
0.2-0.5% per day. The worms will then be brought to a 10-gallon glass container to let
them secrete needed mucus — researchers will then fixate for 5 days. Temperature is
carefully adjusted to 10–35°C. After the due date is over, the researchers will
successfully harvest 2000-5000 grams of slime. The earthworm will then be dispersed
at a small patch of land near one of the researcher’s homes to be used again at a later
date. Furthermore, the container used well be cleansed with disinfectants and water to
avoid any transmission of pathogen earthworms may carry.

III. Isolating nitrogen from earthworm slime

Using denitrification, the method of converting nitrate and nitrite to nitrogen gas, the
researchers can add extracted slime on the container in preparation for the isolation
process. According to Serniak L. (2017), earthworm slime has high concentrations of
glucose, acetate, lactate, and organic acids that have a great probability fuel
denitrification The container will then be de-oxygenated to promote anaerobic
denitrifiers. Organic acids will supplement denitrifiers to convert the latter into nitrogen
which are needed in anaerobic digestion.

IV. Transportation of enhanced slime to a biogas refinery

After the synthesization process of the earthworm slime, it will be then contained in
a barrel and will be shipped to a local biogas refinery in the country for conversion.

V. Anaerobic digestion process

Though the researchers will not facilitate the entire conversion itself, the process
involves inputting the enhanced waste material into the anaerobic digester. The slime
will be mixed with water as the bio digester will also be filled with water to create an
air lock or oxygen-free environment. According to
Homebiogas (n.d), once it is put in the chamber, the bacteria will break down the organic
materials left in the slime and as the matter flows in the tank, biogas will start
accumulating in the upper part of the digester. Soon, the biogas will be transported into a
separate container using a hose.
VI. Produced biogas methane content analysis
After the substrate has been converted into biogas, the researchers will measure the
amount of methane (CH4) the biogas carries to determine whether the earthworm slime
is an effective and suitable substrate for conversion into biogas. Using biogas yield
analysis and chemical composition methodology, the researchers will compare existing
biogas produced from animal waste,
wastewater biosolids, and the likes to see where the earthworm slime biogas is lacking.

VII. Risk and safety

The researchers will be handling earthworm slime, which is an example of animal
waste, meaning they will have to work in a BSL-2 facility for safety. Laboratory attire
and equipment will be used for precaution. All practices and procedures will be done
with caution as well and there will be a qualified scientist to guide the researchers along
the experimentation process and methane content analysis.

E. WORKPLAN AND TARGET DELIVERABLES

I. Expected Output
The researchers expect that the earthworm slime will be a compatible and
suitable substrate in the conversion or production of biogas. It is also anticipated
that nitrogen in slime can be used as a main factor for anaerobic digestion —
employing the final step of the nitrogen cycle, denitrification.
II. Potential Impact
The possible significant impacts of the expected output are the
increase of usage of biogas and the promotion of environmental-friendly fuel.
The researchers anticipate that the study can replenish demounted oil supply the
current market fails to retrieve as well, and provide cheaper options of fuel for
the public.

III. Workplan and Timetable

Objectives Target Location Month
Deliverables
Gathering of Shovels, Local grocery stores November
equipment buckets, and or hardware stores
hand gloves
will be bought
in nearby
department
stores or
hardware
stores.
Gathering of The Villar Sipag, Las November
earthworms researchers Pinas City
will use a
shovel to
search for
earthworms
and all
gathered
worms will be
put in a
bucket,
weighing about
5 lb or 2 kg.

Preparation of Gathered BSL-2 Facility November

earthworms for worms will be
extraction gently cleaned
and will be
covered in thin
film. Then it
will be stored at
a cool
temperature for
3 days to 1
week for
nitrogen
separation.
 The extracted
Nitrogen extraction slime will then BSL-2 Facility December
process be in a machine
that separates
the slime and
its components.

Precautionary After the December

measures nitrogen
isolation, the
researchers
will be
performing the
anaerobic
digestion
process with
proper attire
and safety
measures.
Conducting the The Local biogas refinery
biogas conversion researchers
will transport
the barrel of
slime to a
nearby biogas
refinery with a
biogas digester
and authorized
personnel will
start the biogas
conversion.

Produced biogas The chemical Local Biogas

methane analysis composition of Refinery
the biogas
 made from
earthworm
slime will be
analyzed
evaluating its
yield using a
method
suggested by
Pham et. al
(2013)
involving a
“1,000 mL
syringe and
injecting into a
butyl bung then
drawing the
plunger out
until the
pressure in the
headspace
dropped to
ambient
pressure”.
Equipment Machines and December
disposal tools will be
cleaned and
other wastes
will be
disposed of
properly. The
researchers will
also clean
themselves to
avoid
spreading of
any bacterias
or germs.