A

Dissertation

On

Transforming Waste into Energy: Biogas Production from Fruit

Waste
Submitted to

Department of Microbiology

Sanjivani Arts, Commerce, and Science College, Kopargaon

Affiliated To
Savitribai Phule Pune University, Pune

In partial fulfillment of Master of Science

In
Microbiology
Submitted By
Mr. Tirse Adesh Uttam
Research Guide
Dr. S. B. Dahikar
Department of Microbiology
Sanjivani Arts, Commerce and Science
College, Kopargaon- 423603
(2023-2024)

Sanjivani Rural Education Society’s

I
 Sanjivani Arts, Commerce and Science
College, Kopargaon- 423603

CERTIFICATE

This is to certify that Mr. Tirse Adesh Uttam, has successfully completed the project

titled “Transforming Waste into Energy: Biogas Production from Fruit Waste”

in the academic year 2023-24 for the partial fulfillment of degree, Masters of science

in Microbiology, Department of Microbiology, Sanjivani College of Arts, Commerce

and Science, Kopargaon under the guidance of Dr. S. B. Dahikar and Mr. S. V.

Bhakare. This is candidates own work.

Co-Guide Research Guide Head of Department

Principal

DECLARATION
This is to declare that I, Mr. Tirse Adesh Uttam student of master of Science in
Microbiology (Academic Year 2023-24), Sanjivani Arts, Commerce and Science
College Kopargaon, have given original data and information to the best of my
knowledge in the project report titled “Transforming Waste into Energy: Biogas
Production from Fruit Waste” under the guidance of Dr. S. B. Dahikar.

This work has not been submitted to any other University or Institute for the award of
a degree or diploma. I have prepared this report independently and properly
acknowledge the material collected from secondary sources wherever requires. I
solely own the responsibility for the originality of the entire content.

I assure that I will not share any of this content and information with any other
organization or publish this work without the permission of the research guide.

Place : Kopargaon Mr. Tirse Adesh

Uttam

Date : M.Sc.
Microbiology

III
 ACKNOWLEDGEMENT

I would like to express my sincere gratitude and appreciation to my guide Dr. S. B.

Dahikar for his priceless guidance and valuable support in shaping my research work
and my scientific career. He has always set the best examples for me to include for
skills necessary to confidently face new challenges and solve problems independently.

I am also thankful to my principal Dr. S. B. Dahikar and HOD of Microbiology

Department Dr. S. A. Bhutada for allowing me to utilize every facility of the
laboratory, my colleagues, and non-teaching staff of our college for their readiness to
help throughout my research work.

I would also like to thank, Ms. R. D. Nagarkar , Mr. S. V. Bhakare, and every staff
member and lab assistant Mr. Minanath Shinde of the Sanjivani Arts, Commerce,
and Science College Koparagon for their laboratory assistance, cooperation, and
selfless help at every phase of our research work.

I would also like to expand my deepest gratitude to all those who have directly and
indirectly guided and help me with this research work.

Last, but not least, I am very grateful to my family for their continuous support in
completing our Project work and to my friends who boosted me up morally with their
continuous support. I sincerely acknowledge their contribution to my M.Sc. project
work and realize that words cannot adequately express this.

Mr. Tirse Adesh Uttam

 INDEX

Sr. No. Content Page No.

1 Title
2 Certificate
3 Declaration
4 Acknowledgement
5 List of Abbreviations
6 List of Tables
7 List of Figures
8 Abstract
9 Introduction
10 Review of Literature
11 Materials and Methods
12 Result and Discussion
13 Conclusion
14 Future Scope
15 References
16 Appendix

V
 LIST OF ABBREVIATIONS

Sr. No Abbreviation Full Form

1 Kg Kilogram
2 cm centimeter
3 gm gram
4 hrs hours
5 MJ/m3 Mega Joules per cubic
6 LPG Liquefied petroleum gas
7 pH Potential of hydrogen
8 AD Anaerobic digestion
9 VFAs Volatile fatty acids
10 TS Total solid
11 VS Volatile solid
12 LR Loading rate
13 DNSA 3,5-dinitrosalicyclic acid
14 BSA Bovine serum albumin
15 OD Optical density
16 mg milligram
17 ml milliliter
18 nm nanometer
19 µg microgram
20 HRT Hydraulic retention time
21 kWh/m3 kilowatt-hours per cubic
22 Oil/m3 Oil in cubic meter
23 Kg/m3 Kilograms per cubic meter
 LIST OF TABLES

Sr. No. Table name Page No.

1 Biogas Composition
2 General Features of Biogas
3 Composition of Fluid thioglycollate media
4 List of chemical reagents used in
Experiment.
5 List of materials used in Experiment
6 DNSA procedure table
7 Procedure table of Folin Lowry method
8 Test plant biogas production in gm
9 Control plant biogas production in gm
10 Result of TS (%) and VS (%)
11 DNSA result
12 Folin Lowry result

VII
 LIST OF FIGURES

Sr. No. Figure name Page No.

1 Biogas: Converting waste into Energy.
2 Anaerobic digestion degradation steps
3 Fruit waste used for the present study
4 Construction of 20 lit anaerobic digester
5 Inoculum (A) Before incubation (B) After
incubation
6 Slurry preparation
7 Digester of 200ml (A) Day 1 (B) Day 8
8 Biogas production (A) Day 1 (B) Day8
9 Comparative study of biogas production in
Test plant and Control plant.
 Transforming Waste into Energy: Biogas Production from Fruit
Waste
Tirse A. U, Dahikar S. B., and Bhakare S.V.
Department of Microbiology,
Sanjivani Arts Commerce and Science College, Kopargaon- 423603
tirseadesh@gmail.com

Abstract

This study investigates the generation of biogas through the anaerobic digestion of
fruit waste mixed with an inoculum. The examination involved evaluating the total
solids, volatile solid, moisture content and ash content of the waste materials. The
impact of utilizing various fruit wastes, such as banana peel, mango, water melon,
guava, pumpkin, papaya, orange peel and chickoo were used in a 20 lit digesters.
Microorganisms, particularly those from the Methanogenesis family play vital role in
transforming this waste into biogas, where methane serves as the main component.
Initially, a defined amount of fruit waste, sourced from the market, was acquired.
Later, in the laboratory, an inoculum was prepared and mixed with the fruit waste in
an anerobic digestor. Throughout daily observations, there was a progressive rise in
gas production until, on the 8 th day, the generation of biogas come to a halt.
Evaluation of the generated biogas was monitored daily by weighing the gas stored in
a vechile tire tube in grams over a period of 8 days. The research showed alternative
energy sources, such as biogas, serves as a sustainable or renewable energy source
and finds applications across various purposes. It should be intensified to address
ecological threats like environmental pollution, deforestation, desertification, erosion.
Keywords: Anaerobic digestion, Fruit Waste, Biogas, Methane

IX
 CHAPTER 1
INTRODUCTION
 INTRODUCTION
In our rapidly advancing world, the demand for energy consumption is increasing
rapidly every day (Leta Deressa et al., 2015 ). The scarcity of petroleum and coal
poses a global threat to fuel supply, prompting research in various areas to explore
alternative energy sources, such as renewable energy resourses (Suyog, V.,
2011 ).Using renewable and sustainable energy sources is the best way to meet a
country’s energy needs (Donald, L., 1998). It is highly preferable for the development
of renewable energy to have no negative impacts on the environment. Certainly
generating renewable energy from easily accessible local materials is highly
beneficial and lowers production cost (Lohri, C., 2009). Renewable energy resources
include solar energy, wind energy, various thermal and hydro sources of energy, as
well as biogas. Biogas stands apart from other renewable energies due to its unique
ability to utilize, manage, and harvest organic wastes while simultaneously generating
fertilizers and water for agricultural irrigation. Biogas is not constrained by
geographical limitations, and it doesn’t demand sophisticated technology for energy
production. Moreover, it is straightforward to use and implement (Suyog, V., 2011 ).

In the United States, there has been significant interest in anaerobic digestion as a
method for producing sustainable fuel source and fertilizer (Garba and Sambo, 1995).
Biogas is a combustible gas formed through the fermentation of organic materials in
an oxygen-free environment (N. S. Usman et al., 2009). The biogas is also knowm by
several other names, including dung gas, marsh gas, gobar gas, sewage gas, and
swamp gas (Dangoggo and Fernande, 1986).

Deforesttion poses a significant challenge in developing nations such as India, where

a substantial portion relies on charcoal and fuelwood for energy, leading to the
necessity of cutting down forest. Utilizing dung and firewood for energy adversely
impacts public health, as the smoke emitted contributes to air pollution. A sustainable
and eco-friendly alternative for energy is imperative. (Suyog, V., 2011 ). Before
disposal, fruit waste can undergo chemical and biological treatment to produce
valuable by-product (T. G. Narayani and P. Gomathi Priya, 2012). Fruit waste
contains high calorific and nutritive value for microbes, contributing to a significant
increase in methane production efficiency, as previously mentioned. It means
increasing efficiency and reactor size results in reduced biogas production costs.

XI
Improper disposal of fruit waste in landfills in many cities poses public health risk,
contributing to diseases such as malaria, cholera, and typhoid. Additionally, it releases
foul odors and methane, a significant greenhouse gas that contributes to the issue of
gobal warming (Suyog, V., 2011 ).

Biogas technology offers a dual benefits: the biogas production can fulfil energy
needs while the remaining organic residue serves as valuable fertilizer. This
renewable energy source is derived from the breakdown of animal and plant wastes,
consisting primarily of Methane (CH4) and Carbon dioxide (CO2), along with minor
impurities such as Hydrogen (H), Hydrogen sulfide (H2S), and Nitrogen (N) (Heb, F.,
2009). The generation of methane in anaerobic digestion is closely connected to or
dependent on the quantity and type of organic matter introduced into the system.
Therefore, fruit waste and inoculum can undergo anaerobic digestion for energy
production using various methods. Presently, key approaches include single-phase
digestion, two-phase digestion, dry fermentation, and co-digestion (Chanakya, H. N.
et al., 2006). An fascinating approach to enhance the efficiency of anaerobic digestion
of waste is co-digestion, which involves adding a co-substrate. This method can boost
biogas yield by creating a positive synergistic effect in the digestion medium and
supplying nutrients that may be lacking for microorganisms (Rungvichaniwat. J.,
2003). Therefore, employing co-digestion of fruit wastes and inoculum present an
alternative method to enhance biogas technology.

What is the reason for choosing this plant?

Proper disposal of fruit waste from markets or juice shops can be achieved in an eco-
friendly manner. When calculating the cost-effectiveness of waste disposal, it’s
important to consider more than just monetary aspects. Managing waste disposal
properly helps maintain hygiene by preventing food dumping in inappropriate places.
This contributes to the value of biogas plants, which utilize natural processes
involving microorganisms to converet fruit waste into useful resources.

Anaerobic treatment is utilized for processing by-product and wastes from food
processing and agriculture based industries (Rajesh Banu. J. et al., 2007; Ogunnusi et
al., 2009). The anaerobic fermentation of manure for biogas production does not
decrease it’s value as a fertilizer supplement, as treated sludge retains it’s available
nitrogen and other beneficial substances (Beatriz. M et al., 2010).
Anaerobic digestion shows promise for treating fruit waste. While it’s common in
rural areas of developing countries to use anaerobic digestion for animal dung, there’s
limited information on it’s technical and operational feasibility for testing organic
solid waste in those region. Numerous factor affects design and performance of
anaerobic digestion, including feedstock characteristics, digester design, and real time
operational conditions. Understanding the physical and chemical characteristics of
organic waste is crucial for designing and operating digester. The factors directly
impact biogas production and process stability during anaerobic digestion. Key
characteristics include moisture content, nutrient content, particle size, and
biodegradability (Suyog, V., 2011).

Recent advancements in technology have led to various modification and

improvements aimed at reducing the costs associated with biogas production. These
include methods to accelerate bacterial fermentation, reduce reactor size, use starchy
or sugary materials for production, modify feed materials for fermentation, improve
effluent management, and create compact equipment suitable for small-scale gas
production, such as in backyards.

1.1 BIOGAS

BIOGAS, a renewable energy source, is generated from various raw materials

including agricultural waste, manure, municipal waste, plant material, sewage, green-
waste, wastewater, and food waste (NNFCC, 2011). Biogas is generated through
anaerobic digestion, a process that involves anaerobic organisms or methanogens,
within an enclosed system such as anaerobic digester, biodigester, or bioreactor
(Insyde, 2018). It can be employed in a gas engine to transform the gas's energy into
both electricity and heat. The composition of biogas fluctuates based on the substrate
composition and the conditions within the anaerobic reactor, including temperature,
pH, and substrate concentration (Hafner et al., 2017).

1.2 CHARACTERISTICS OF BIOGAS

 Biogas is primarily composed of Methane (CH4) and Carbon Dioxide (CO2),

with small amount of other gases such as Hydrogen sulfide (H2S), Nitrogen
(N), and Water vapor (H2O). The composition of biogas is influenced by the
feed material used in it’s production.

XIII
  Biogas is a versatile fuel that can replace traditional sources such as firewood,
cow dung cake, petrol, LPG, diesel, and electricity, depending on the specific
task and local supply conditions and limitations.
 Biogas, which is roughly 20% lighter than air, has an ignition temperature
ranging from 650-750oC.
 Biogas burns with a blue flame with reddish yellow mixture, similar to LPG
gas and is both odorless and colorless.
 The caloric value of biogas is typically around 20 Mega Joules per cubic
(MJ/m3), and it generally burns with an efficiency of 60% in a conventional
biogas stove.
 Biogas digester systems produce a residue of organic waste after anaerobic
digestion that is rich in nutrients, particularly in the form of ammonia, making
it superior to normal organic fertilizer. This residue can be used effectively as
manure.

Constituents Formula % composition

Methane CH4 55-65%

Carbon Dioxide CO2 35-45%

Water H2O 2-7%

Hydrogen sulphide H2S 0-2%

Ammonia NH3 0-0.05%

Nitrogen N 0-2%

Oxygen O2 0-2%

Hydrogen H 0-1%

Table 1: Biogas Composition (Prakash et al., 2005, Schnurer and Jarvis, 2010)
1.3 PROPERTIES OF BIOGAS

 Biogas generally has a natural pH, approximately 7,which makes it compatible

with many applications.
 Biogas volume changes with variations in temperature and pressure.
 The calorific value of biogas fluctuates with alterations in temperature,
pressure and water vapor content.
 The amount of water vapor in biogas varies with changes in temperature and
pressure.
 It is safe to handle and does not emit any unpleasant smell.

Energy Content 6-6.5kWh/m3

Fuel Equivalent 0.6-0.65 l oil/m3 biogas

Explosive Limits 6-12 % biogas in air

Ignition Temperature 650-750oC

Critical Pressure 75-89 bar

Critical Temperature -82.5oC

Normal Density 1.2kg/m3

Smell Bad eggs

Calorific value without CO2 26 MJ/m3

Calorific value with CO2 18.7 MJ/m3

Viscosity 1.297×10-5kg/sec/m

Specific gravity 0.94

Table 2: General features of Biogas ( Deublein and Steinhauser, 2008)

XV
 1.4 FACTORS AFFECTING YIELD AND PRODUCTION OF
BIOGAS

Numerous factor influence the fermentation process of organic substance under

anaerobic conditions are,

 The type and composition of the feedstock (organic material) used in the
digester have a significant impact on biogas.
 The biogas production is affected by temperature inside the digester. The
temperature affects the activity of the microorganisms responsible for
anaerobic digestion.
 The hydraulic retention time, or the amount of time that organic matter spends
in the digester, is important for biogas production. Longer retention time allow
for complete digestion of the feedstock and higher biogas production.
 The acidity and alkalinity (pH value) of the substrate are important
consideration for biogas production.
 The rate of material flow and it’s dilution are also important factors in biogas
production.

1.5 ADVANTAGES OF BIOGAS TECHNOLOGY

 Production of renewable and sustainable energy from organic waste.

 Conversion of organic waste into a premium-quality fertilizer.
 Enhancement of waste management techniques.
 Possibility of decentralized energy generation in rural regions.
 Enhancement of hygienic conditions by reducing pathogens.
 Environmental benefits including soil, water, and air protection.
 Micro-economic advantages from energy and fertilizer alternatives.
 Macro-economic benefits from decentralized energy production and

environmental conservation
 Figure 1 : Biogas: Converting waste into Energy.

1.6 PRODUCTION PROCESS OF BIOGAS

In a usual biogas system, you’ll find the following components:

1. Collection system for organic waste.

2. Anaerobic digester for the breakdown of organic material.
3. Storage system for effluent or digestate.
4. Infrastructure for handling the biogas production.
5. Utilization system for using the biogas.

Biogas is considered a sustainable energy source. Biogas produced through anaerobic

digestion, a process in which microorganisms decompose organic matter in the
absence of oxygen. Methanogens, which are methane producing bacteria, are the final
step in a series of microorganisms that breakdown organic matter and recycle the
decomposition product back in the environment (Suyog, V. et al., 2011)

.1.7 PRINCIPLES FOR PRODUCTION OF BIOGAS

XVII
There are two primary forms of digestion:

1. Aerobic digestion
2. Anaerobic digestion

Aerobic digestion is a microbial process that take place in the presence of oxygen,
where organic material undergoes oxidation, resulting in the production of nitrate,
phosphate, and carbon dioxide (Martin, J. et al., 2015). In aerobic digester, microbial
growth tends to be high, allowing for shorter retention times during biogas
production (Ohimain and Izah, 2017).

Anaerobic digestion is a series of processes in which microorganisms decompose

biodegradable material without presence of oxygen (NNFCC, 2011). Anaerobic
digestion (AD) is a process utilized in both industrial and domestic settings for waste
management or fuel production. This fermentation process is commonly employed in
the industrial protection of food and beverages, as well as in home fermentation
practices.

1.8 ANAEROBIC DIGESTION

Anaerobic digestion (AD) is a natural process that breaks down organic materials into
gases and liquids using anaerobic microorganisms. The gas produced, known as
biogas, can be burned at normal room temperature and pressure. The residue left after
bacterial action is called "slurry." (Ravindra , B. et al., 2023)

Biogas production is typically comprised of several key stages.

1. Hydrolysis
2. Acidification
3. Acetogenesis
4. Methanogenesis

1.8.1 HYDROLYSIS:

In the initial step, complex organic compounds like lipids, proteins, and
polysaccharides undergo hydrolysis, or liquefaction, converting them into soluble
monomers or oligomers such as amino acids, long-chain fatty acids, sugars, and
glycerol. This hydrolysis process is facilitated by hydrolytic or fermentative bacteria,
which release extracellular enzymes. Subsequently, acidogenic bacteria ferment the
simple soluble compounds into a mixture of carbon dioxide (CO2), hydrogen (H2),
alcohol, and low molecular weight volatile fatty acids (VFAs) like propionic and
butyric acids, in a process known as acidogenesis (K. C. Surendra et al., 2014).

1.8.2 ACIDIFICATION:

During the acidification step, fermentative microorganisms utilize sugars, fatty acids,
and amino acids from hydrolysis to produce organic acids such as acetic, propionic,
butyric, and fatty acids, as well as hydrogen and carbon dioxide. Amino acids can also
be used as energy and carbon sources by strict or facultative fermentative anaerobic
bacteria. This step is the fastest reaction in the anaerobic digestion of complex organic
matter. An increased concentration of hydrogen leads to the accumulation of electron
sinks like lactate, ethanol, propionate, butyrate, and higher volatile fatty acids. Acetic
and butyric acids are crucial precursors for methane formation. Therefore, the
concentration and proportion of individual volatile fatty acids produced in the
acidogenic stage are important factors influencing the overall performance of the
system (W. Parawira, 2004).

1.8.3 ACETOGENESIS:

In acetogenesis, acetogenic bacteria anaerobically oxidize alcohols and volatile fatty

acids, producing acetate, hydrogen sulfide (H2S), and carbon dioxide (CO2). Acetate
can also be produced from hydrogen (H2) and CO2 by hydrogen-oxidizing acetogenic
bacteria, called homoacetogens. In the final stage, acetotrophic and hydrogenotrophic
methanogens convert acetate, H2, and CO2 into a mixture of methane (CH4) and
CO2. Acetotrophic methanogens use acetate as a substrate in acetotrophic
methanogenesis (K. C. Surendra et al., 2014).

1.8.4 METHANOGENESIS:

Methane and carbon dioxide are primarily formed from hydrogen and carbon dioxide.
In the final stage of methanogenesis, methane is produced by two groups of
methanogenic bacteria. The first group converts acetate into methane and carbon
dioxide, while the second group uses hydrogen as an electron donor and carbon
dioxide as an acceptor to produce methane. The aceticlastic pathway accounts for
about 70% of methane production, with the hydrogen pathway being more energy-

XIX
yielding than the acetate pathway because it is not rate-limiting. Maintaining a low
hydrogen pressure in the system is crucial for the hydrogen pathway. An increase in
the partial pressure of hydrogen leads to the accumulation of volatile fatty acids and a
decrease in pH, which can cause the methanogenesis stage and the entire anaerobic
digestion process to fail. Methane-producing microorganisms are obligate anaerobes
and highly sensitive to environmental changes, although hydrogen-utilizing
methanogens have been found to be more resistant. The loading, efficiency, and
running stability of the methanogenesis phase are influenced by the terminal
fermentation products produced in the acidogenesis phase (W. Parawira, 2004).

Figure 2: Anaerobic digestion degradation steps (Ireen Maile et al., 2016).

 AIM AND OBJECTIVES

AIM:

Converting waste into energy, such as producing biogas from fruit waste, is an eco-
friendly practice that turns various types of waste into valuable resources. This
method involves the anaerobic digestion of organic materials like fruit waste to
produce biogas, a renewable energy source. It not only decreases landfill waste but
also creates a clean energy source for cooking, heating, and electricity generation.
Moreover, biogas production from fruit waste can lower greenhouse gas emissions
and promote a more sustainable, circular economy.

OBJECTIVES:

1. Enhancing biogas production through optimization techniques.

2. Utilization of fruit waste into useful energy sources helps reduce
environmental pollution and the need for landfill space.
3. Recycling nutrients involves repurposing essential elements and compounds
within ecosystem or biological systems.

XXI
 CHAPTER 2
REVIEW AND LITERATURE
 REVIEW OF LITERATURE

Solomon Lipus et al., (2015) studied on the production of biogas from a mixture of
fruit and vegetable wastes with cow manure in an anaerobic digester. It analyzed the
total solid, volatile solids, moisture content, and ash content of the wastes. The
feedstock included avocado, papaya, mango, tomato, banana peel, and cow manure,
and different digester volumes were used for biogas production. The researchers
tested the combustibility of the generated gas, and the anaerobic digestion process
required 55 days for complete digestion. They highlighted the sensitivity of anaerobic
digestion to pH changes, emphasizing the importance of maintaining a pH range of
6.7-7.4 for optimal performance. Temperature variations, both within the digester and
in the surrounding environment, were found to significantly impact the process. The
study suggested that adjusting these factors could facilitate co-digestion of fruit and
vegetable wastes with cow manure to produce biogas without the need for additional
nutrients or chemicals.

P. Gomathi Priya et al., (2012) conducted a study on biogas production from fruit
wastes at ambient temperature using a 500 ml anaerobic batch digester. They explored
co-digestion of fruit wastes with rice bran and cow dung. The biogas production from
different samples varied: Sample A (fruit waste) yielded 363 ml, sample B (fruit
waste 75% + cow dung 25%) yielded 405 ml, sample C (fruit waste 75% + rice bran
25%) yielded 315 ml, and sample D (fruit waste 50% + rice bran 25% + cow dung
25%) yielded 381 ml. Analysis through chromatography revealed that sample B had
the highest methane content (80%), while sample C had the highest carbon dioxide
content (50%). The study highlighted the significant impact of cow dung on the
digestion of fruit wastes, demonstrating the highest yield (405 mg) of biogas
production.

Sagagi, B. S. et al., (2009) in their study, they investigated biogas production from
fruit and vegetable waste materials and their impact on plants when used as fertilizer,
utilizing both digested and undigested sludge. The highest weekly individual
production rate was observed for the cow dung (control) slurry, averaging 1554 cm3,
followed by pineapple waste with 965 cm3 of biogas, then orange waste with 612
XXIII
cm3, and finally, pumpkin and spinach wastes with 373 cm3 and 269 cm3,
respectively. The findings suggested that the variation in biogas production depended
largely on the substrate's nature. All substrates were deemed suitable for biogas
production, and their spent slurries were identified as a viable source of plant
nutrients.

Dr. Anand Karve et al., (2003) studied on a compact biogas system that utilizes
starchy or sugary feedstock material. The analysis indicates that this new system is
800 times more efficient than conventional biogas plants.

Hilkiah Igoni et al., (2008) studied the impact of total solids concentration in
municipal solid waste on biogas production in an anaerobic continuous digester. The
concentration of total solids (TS) in the waste affects pH, temperature, and the
efficiency of microorganisms in the decomposition process. By investigating various
TS concentrations in an anaerobic continuously stirred tank reactor (CSTR), they
determined the conditions for optimal gas production. The findings revealed that as
the percentage of total solids (PTS) in municipal solid waste increased during
anaerobic digestion, there was a corresponding geometric increase in biogas
production. A statistical analysis established that the relationship between biogas
volume and total solids concentration follows a power function, indicating that
beyond a certain point, further increases in total solids concentration do not lead to
additional biogas production.

Prema Viswanath et al., (1991) studied on the anaerobic digestion of fruit and
vegetable processing wastes for biogas production. Different fruit and vegetable
wastes, such as mango, pineapple, tomato, jackfruit, banana, and orange, were fed
into a 60-liter digester, with each waste cycled every fifth day to operate the digester
as feed became available. The researchers analyzed the characteristics of the
anaerobically digested fluid and the digester's performance in terms of biogas
production at varying loading rates (LR) and hydraulic retention times (HRT). They
found that the maximum biogas yield of 0.6 m3/kg VS added was achieved at a 20-
day HRT and a 40 kg TS m-3day-1 loading rate. Hourly gas production was
monitored in the digesters operated at 16 and 24 days HRT, revealing that the major
yield (74.5%) of gas was produced within 12 hours of feeding at a 16-day HRT, while
at a 24-day HRT, only 59.93% of the total gas could be obtained in the same time
frame.

Suelen Pavi et al., (2017) conducted an evaluation of the anaerobic co-digestion of the
organic fraction of municipal solid waste (OFMSW) and fruit and vegetable waste
(FVW), focusing on biogas and methane yield, volatile solids (VS) removal rate, and
process stability. They carried out a batch experiment under mesophilic conditions
(35°C), varying the OFMSW/FVW ratios (VS basis) to 1/0, 1/1, 1/3, and 0/1. The
study revealed that co-digestion produced higher methane yields compared to mono-
digestion for both OFMSW and FVW. The optimal mixing ratio of OFMSW/FVW
was determined to be 1/3, resulting in an average cumulative biogas yield of 493.8 N
mL/g VS, methane yield of 396.6 N mL/g VS, and VS removal rate of 54.6%. When
compared to the mono-digestion of OFMSW and FVW, the average increase in
methane yield was 141% and 43.8%, respectively.

G. N. Tiwari et al., (2013) conducted a study on producing biogas using kitchen

waste, which results from the bacterial degradation of organic matter in the absence of
air. Biogas typically contains approximately 55‐65% methane and 30 ‐40% carbon
dioxide, with a high calorific value (around 4700 kcal or 20 MJ at around 55%
methane content). This gas can be effectively used for power generation through a
biogas-based power-generation system after dewatering and cleaning. Moreover, the
slurry produced in the process serves as valuable organic manure for farming,
sustaining soil fertility. The study evaluated the performance of different ratios of
kitchen waste in a metal-made portable floating type biogas plant with a volume
capacity of 0.018 m3, designed for outdoor climatic conditions in New Delhi, India.
Each biogas plant had a 30 kg slurry capacity in a batch system for all measurements.
Throughout the study period, the researchers measured temperature, solar radiation,
and relative humidity. They also analyzed the biogas constituents, pH, volume, and
rate of biogas production at different temperature levels on a daily basis. The study
compared the rate of biogas production from kitchen waste with other energy sources
used for cooking purposes, such as LPG, kerosene, and coal.

Ogur, E. O. and Mbatia, S. (2013) reported on biogas, produced through anaerobic

digestion of organic materials, offers a sustainable, reliable, and renewable energy
solution. Kitchen waste presents a significant potential for biogas production, with the

XXV
added benefit of waste treatment to reduce environmental impact and provide
nutrient-rich organic fertilizer. The primary goal of the study was to design an
anaerobic digester that could utilize food waste to generate biogas for Kabete
Technical Training Institute’s (KTTI) kitchen. Given that the institute primarily relies
on wood fuel, supplemented by expensive liquefied petroleum gas (LPG), the
substantial quantity of kitchen waste produced could sufficiently generate biogas for
kitchen use. This suggests that investing in this technology could be financially viable
for the institute.

Carlos Morales Polo et al., (2019) studied on the residual materials found in fruit and
vegetable markets, assessing their suitability for anaerobic digestion to produce
biogas as a renewable energy source. The findings indicate that these substrates are
highly suitable for anaerobic digestion due to their rich simple carbohydrate content
and high moisture and solids content (both total and volatile). These characteristics
make them easily solubilized and provide a significant amount of matter directly
accessible to the microorganisms responsible for anaerobic degradation. The process
proceeds smoothly, with only a slight release of acidic elements and without being
affected by the development of the buffer effect by ammonia. Furthermore, a two-
phase digestion phenomenon is observed, suggesting that despite the particulate
nature of the substrate, it effectively digests both the directly accessible and
inaccessible organic matter. In quantitative terms, 100 g of residue V produce 913.282
NmL of biogas, of which 289.333 NmL is methane. The disintegration constant is
0.200 days^-1, with 16.045% of the substrate degraded. As an innovative approach,
the study uses the hydrogen generated in the process as an indicator of stability and
process development. With the support of statistical analysis and mathematical
adjustments, the study provides a detailed characterization of the process and its
evolution, demonstrating that degradation is rapid and characterized by stable
hydrolysis.

Ireen Maile et al., (2016) studied on the significance of selecting operational

parameters—including temperature, pH, hydraulic retention time (HRT), organic
loading rate (OLR), inoculum to substrate ratio (ISR), particle size, and nutrient levels
with care to optimize biogas production from FVW. Additionally, the study
highlighted that factors like co-digestion, pre-treatment techniques, and digester
configuration play pivotal roles in enhancing methane yield from FVW.

Anika Ogemdi Chinwendu et al., (2019) studied on the determination of the potential
of biogas production from fruit wastes, such as mango, pawpaw, and watermelon, as
well as their combinations. It utilized locally fabricated fixed batch type anaerobic
digesters with a 45-day retention time. The substrates were processed into slurry
treatments by grinding and mixing with water in specific ratios, except for
watermelon, which had a higher moisture content and used a different ratio.
Monitoring of process pH, temperature, and viable anaerobic counts was conducted
using various tools. Biogas yield was quantified through liquid displacement, and
variations in pH, temperature, and anaerobic counts were observed over the retention
period. The total biogas volumes produced at the end of the digestion period varied
across substrates and combinations, with watermelon and melon displaying the
highest potential. This underscores the importance of these fruits in waste
management and Nigeria's energy transition vision.

Daniele Cocco et al., (2013) studied on anaerobic digestion pilot plant that utilized
fruit and vegetable wastes as its sole substrate. The substrates were sourced from the
waste produced by the Fruit and Vegetable Wholesale Market of Sardinia, Italy.

The study spanned approximately 6 months and aimed to determine the optimal
operating parameters for the process, considering the varying availability of different
fruit and vegetable wastes throughout the year. The study found that the optimal daily
loading rate of wastes was 35 kg/d, with a corresponding hydraulic residence time of
27 days. The optimal organic loading rate ranged from 2.5 to 3.0 kgVS/m3d. On
average, the specific biogas production was about 0.78 Nm3/kgVS, with a specific
methane yield of about 0.43 Nm3/kgVS.

These experimental results were instrumental in the preliminary performance

evaluation of a full-scale anaerobic digestion power plant designed to treat all the fruit
and vegetable wastes produced by the Wholesale Market of Sardinia (9 t/d). The
estimated daily methane production of 290 Nm3/d would allow for a combined heat
and power unit with a power output of about 42 kW and an annual electrical

XXVII
production of around 300 MW h/year, representing approximately 25% of the
wholesale market's electrical consumption.

Lissens et al., (2004) studied on a biogas operation aimed to increase the total biogas
yield from 50% to 90% using several treatments, including a mesophilic laboratory-
scale continuously stirred tank reactor, an upflow biofilm reactor, a fiber liquefaction
reactor releasing the bacteria Fibrobacter succinogenes, and a system that adds water
during the process. These methods successfully led to significant increases in the total
yield. However, the study was conducted under highly controlled conditions, which
may limit its applicability under varying conditions. Nonetheless, Bouallagui et al.
(2004) determined that minor fluctuations in temperature do not severely impact
anaerobic digestion for biogas production.

Jantsch and Mattiasson (2004) studied how anaerobic digestion is a viable method for
treating wastewater and organic wastes, generating biogas as a valuable by-product.
However, due to challenges in start-up and operation, it is often overlooked. To
prevent instability issues and avoid acidification in anaerobic digesters, it is common
practice to maintain the organic load of the digester well below its maximum capacity.
Numerous factors influence biogas production efficiency, including environmental
conditions such as pH and temperature, the type and quality of substrate, mixing
efficiency, high organic loading, the formation of high volatile fatty acids, and
inadequate alkalinity.

Thomsen et al., (2004) discovered that raising the oxygen pressure during wet
oxidation of digested biowaste led to a significant increase in methane yield.
Typically, methane yield ranges from 50 to 60%, but this approach boosted it by 35 to
40%, highlighting its potential for enhancing methane recovery and offering
economic advantages.
 CHAPTER 3
MATERIALS AND METHODS

XXIX
 MATERIALS AND METHODS

MATERIALS

Media used for the present study is given below:

1. Fluid thioglycollate media

Ingredients Gms/litre

Tryptone 15.000

Yeast extract 5.000

Dextrose ( Glucose) 5.500

Sodium chloride 2.500

L-Cystine 0.500

Sodium thioglycollate 0.500

Resazurin sodium 0.001

Agar 0.750

Table 3: Composition of Fluid thioglycollate media

Chemical reagent used for present study is given below:

Sr. No Chemical Name

1 Sodium thioglycollate

2 DNSA Reagent (3,5-dinitrosalicyclic acid) (1 g of dinitrosalicylic acid +

200 mg of crystalline phenol + 50 mg of sodium sulphite in 100 ml of 1%
NaOH).

3 40% sodium potassium tartrate.

4 Working glucose standard 100 mg/ 100 ml (by dilute 10 ml of stock

solution to 100 ml)

5 Reagent I: 48 ml of A, 1 ml of B, 1 ml of C.

6 Reagent II- 1-part Folin-Phenol [2 N]: 1 part water

7 Reagent A-2% Na2CO3 in 0.1 N NaOH

8 Reagent B-1% NaK Tartrate in H2O

9 Reagent C-0.5% CuSO4.5 H2O in H2O

10 BSA Standard - 1 mg/ ml

11 0.5% Sodium hypochlorite

Table 4: List of chemical reagents used in experiment.

XXXI
The following materials were utilized in the construction of digesters.

Sr. No. Product name

1. Two 20 liter container (used for drinking water storage is used as

digester)

2. Gas tap

3. M-seal

4. Water level pipe ( for gas output)

5. Plastic cape (to seal container)

6. Funnel (for feed input)

7. Vehicle tire tube (for gas collection)

8. Balloon (for gas collection)

9. Plastic bottle of 200ml (as digester)

10. Seal tape

Table 5: List of materials used in Experiment.

METHODS

In this study, different kinds of fruit waste sourced from Kopargoan market and juice
shops were utilized as substrates for biogas production. Two 20-liter cylindrical
plastic anaerobic digesters and one 200 ml plastic bottle were employed for the
experiment.

The substrates used in this study were waste residues from fruits, including orange
peel, banana peel, mango, watermelon, chickoo, pumpkin, papaya, and guava. These
materials are considered fruit waste.

3.1 SAMPLING AND TREATMENT OF SAMPLES WERE

CONDUCTED AS FOLLOWS:

The fruit waste from the Kopargoan market and juice shops was collected by wearing
hand gloves, and plastic glasses, straws, and bags were separated from the fruit waste.
Each type of fruit waste was then separated and placed in individual polythene bags.

The gathered fruit waste was chopped into small pieces, approximately 2-5 cm in size.
These small pieces were then sun-dried separately for fifteen to twenty days,
depending on the type of fruit waste, as fruits like chickoo, papaya, watermelon, and
guava take more days to dry under the sun compared to other fruit waste.

Following sun drying, the fruit waste was mixed and ground using a mixer grinder
machine to obtain a powdered sample. This powdered fruit waste sample was then
stored in airtight polythene bags.

XXXIII
(A) Chickoo (B) Orange peel (C) Banana peel

(D) Papaya (E) Pumpkin (F) Watermelon

(G) Guava (H) Mango

(I) Dired mixed fruit waste (J) Fruit waste powder

 Figure 3: Fruit wastes used for the present study.

3.2 CONSTRUCTION OF THE ANAEROBIC DIGESTER FOR

BATCH SYSTEM

An experiment was conducted to generate anaerobic biogas from fruit wastes using a
setup consisting of two 20-liter plastic containers typically used for drinking water
storage. Each container had a diameter of 25 cm and a height of 48 cm. The digester
had two openings: one with a 5 cm diameter for feeding and another with a 1 cm
diameter connected to a vehicle tire tube through a water level pipe, serving as gas
storage. The digesters were painted black to prevent direct sunlight from reaching the
slurry, thereby minimzing the impact of sunlight on the digestion process. To ensure
airtightness, seal tape, m-seal, and stainless steel clamps (3/4” size) were used
throughout the digestion process.

Another set of digester was made from plastic bottle with diameters of 2 cm and 4 cm
at the mouth and bottom, respectively, and a height of 14 cm, resulting in a total
volume of 200 ml. These digester were connected to balloons through the mouth of
the bottle, serving as gas storage units.

Gas outlet

Feed inlet

20 Liter digeste

Water level pipe

Gas tap

Vechile tire tube

XXXV
 Figure 4: Construction of 20 lit anaerobic digester.

3.3 PREPARATION OF INOCULUM

To prepare the inoculum, 250 ml of distilled water was taken in a 500 ml conical
flask. Then, 7.43g of fluid thioglycollate media was added (Fluid thioglycollate
medium includes thioglycollate as a reducing agent, which lowers the oxygen tension
in the medium, creating a suitable environment for the growth of anaerobes) and
thoroughly mixed until it completely dissolved, resulting in 250ml of fluid
thioglycollate broth. This broth was autoclaved at 121°C under high-pressure
saturated steam for approximately 15 minutes. After autoclaving, the broth was
removed from the autoclave and allowed to cool to room temperature. Once cooled, 1
ml of fresh biogas slurry was inoculated into the broth under sterile conditions. The
inoculated broth was incubated at 50°C for 48 hours, as most methane-producing
methanogens are thermophilic.

(A) (B)

Figure 5: Inoculum (A) Before incubation (B) After incubation

3.4 PREPARATION OF SLURRY

3.4.1 Slurry preparation for Test plant anaerobic digester

The slurry was prepared by gradually adding 3 kg of ground fruit waste powder to 10
liters of distilled water in a bucket, stirring well to ensure thorough mixing. Following
this, 5g of sodium thioglycollate powder was introduced to the slurry to reduce
oxygen content and create anaerobic conditions in the digester. After incorporating
the sodium thioglycollate powder, 250 ml of inoculum was added to the bucket and
thoroughly mixed with the slurry. Once fully mixed, the slurry was transferred to the
anaerobic digester for biogas production.

3.4.2 Slurry preparation for Control plant anaerobic digester

The slurry was prepared by thoroughly mixing 3 kg of ground fruit waste powder with
10 liters of distilled water in a bucket. However, it was poured into the anaerobic
digester for biogas production without the addition of sodium thioglycollate and
inoculum.

Figure 6: Slurry preparation.

3.5 EXPERIMENTAL

The work is carried out in two stages at the laboratory scale: first in 200ml bottle and
then in 20-liter plastic containers.

In the first stage, a 200ml plastic bottle with dimensions of 4cm in diameter and 14cm
in height was utilized as an anaerobic digester for biogas production from fruit waste.
Approximately 150ml of well-mixed slurry, consisting of 100ml of distilled water, 1
ml of inoculum, 0.05 g of sodium thioglycollate, and 30 g of mixed fruit waste
powder, was poured through the bottle's mouth. Following the slurry pour, a black

XXXVII
balloon was affixed to the bottle's mouth to collect the biogas produced in the digester
over an 8-day period.

During the second stage of the experiment, two 20-liters plastic containers were
employed as anaerobic digesters for biogas production from fruit waste. One
container was designated as the test plant container, while the other was the control
plant container. The containers were sterilized using a 0.5% sodium hypochlorite
solution before being poured. The slurry prepared for the test was poured into the test
plant container, and the slurry for the control was poured into the control plant
container through the feed inlet using a funnel. To seal the digesters' inlets, plastic
caps were used, and both digesters were made airtight with M-seal and seal tape to
prevent gas leakage. For gas outlet, a 3/4" water level pipe was attached above the
plastic cap, with one end connected to the digester and the other to a vehicle tire tube
for biogas storage. Daily analysis of biogas production was conducted by weighing
the gas stored in vehicle tube in grams for 8 days.

The digesters were set up in the Microbiology laboratory of the Microbiology

department.

3.6 DATA COLLECTING AND ANALYTICAL METHODS

The physical characteristics, including total solids, volatile solids, moisture content,
and ash content, were assessed using a standard method for analyzing water and
wastewater (APHA, 1998). Samples of the slurry were collected at the beginning and
end of the digestion process for the determination of total solids (TS%), volatile solids
(VS%), ash content (%), and moisture content (%).

3.6.1 MOISTURE CONTENT (%)

The moisture content was determined as a percentage by taking 2 gm sample of fruit

waste on foil paper (w) and placing it in a hot air oven at 105 0 C for 24 hrs to dry. The
weight of the dried (d) sample was then measured. The percentage of moisture content
was calculated using the following equation.
 w−d
Moisture content (%) = ×100
w

3.6.2 ASH CONTENT (%)

2 grams of fruit waste powder were placed in a tarred china dish. The dish was then
heated in a muffle furnace at a temperature of 450°C. After reaching a red-hot state,
the dish was allowed to cool for two hours before the final weight was recorded. The
percentage of ash content was calculated using the following equation.

Ash content (%) = Final weight of sample / Initial weight of sample

×100

3.6.3 TOTAL SOLID (TS%)

Total solids refer to both the organic and inorganic components present in the
feedstock (Adams et al., 2010). Approximately 10 grams of the sample are
placed in a foil plate and dried until a constant weight is achieved at approximately
105 degrees Celsius in a Hot air oven.The percentage of total solids was calculated
using Equation below (Suyog,V., 2011).

Final weight
Total solids (%) ¿ ×100
Initial weight

3.6.4 VOLATILE SOLIDS (VS%)

Volatile solids, which indicate the organic matter content of the feedstock excluding
inorganic salts and ash, were also determined (APHA, 1998). A 3g sample of oven-
dried material was weighed (B) in an empty crucible (A) and heated to 550°C for 1
hour in a muffle furnace until a constant weight was achieved (C). The percentage of
volatile solids was calculated using Equation below (Fernandez et al., 2001).

C− A
Volatile solids (VS%)¿ ×100
B

3.6.5 DETERMINATION OF REDUCING SUGAR

XXXIX
The reducing sugar content was assessed using the DNSA (3,5-dinitrosalicylic acid)
method as outlined by Shrestha in 2002. The DNS reagent was prepared by dissolving
1 g of DNSA, 200 mg of crystalline phenol, and 50 mg of sodium sulfite in 100 ml of
1% NaOH. Additionally, a 40% solution of sodium potassium tartrate was prepared.
A glucose standard solution of 100 mg/100 ml was created by diluting 10 ml of the
stock solution to 100 ml. The procedure involved allocating 12 clean, dry test tubes
for each sample. Successive volumes of the glucose standard (ranging from 0.1 ml to
1 ml) were pipetted into a series of test tubes, along with corresponding volumes of
the sample (ranging from 0.9 ml to 0 ml), ensuring a total volume of 1 ml in each. A
test tube containing 1 ml of distilled water was used as the blank. Next, 1 ml of the
DNS reagent was added to all test tubes, which were then placed in a boiling water
bath for 5 minutes. After cooling to room temperature, absorbance readings at 540 nm
were recorded. A concentration-absorbance graph was plotted using the glucose
standard, and the concentration of the unknown sample was estimated from this
graph.

Table 6: DNSA procedure table

Stock D/W Conc.(µg/ml) DNSA D/W(ml) O.D at

solution(ml) (ml) reagent 540nm

0.1 0.9 20 1 8 0.41

0.2 0.8 40 1 8 0.22

0.3 0.7 60 1 8 0.32

0.4 0.6 80 1 8 0.41

0.5 0.5 100 1 Boiling 8 0.45

water
0.6 0.4 120 1 bath for 8 0.47
5
0.7 0.30 140 1 minutes 8 0.70

0.8 0.2 160 1 8 0.65

0.9 0.1 180 1 8 0.79

1 00 200 1 8 0.8
Mix the contents thoroughly and cool by immersing the test tubes in cold water. After
cooling, take absorbance readings at 540 nm. Plot the standard curve using the
absorbance values obtained from the glucose standard. Calculate the amount of
reducing sugar in the sample by comparing its absorbance to the standard curve.

3.6.6 DETERMINATION OF PROTEIN

To determine the protein content in the provided sample using the Folin-Lowry
method. 0.2 ml of BSA working standard was distributed into five test tubes, each
containing 1 ml of distilled water to make up the volume. The tube with only 1 ml of
distilled water served as the blank. Afterwards, 4.5 ml of Reagent I was added to each
tube, followed by incubation for 10 minutes. Subsequently, 0.5 ml of Reagent II was
added to each tube, and the mixture was incubated for an additional 30 minutes.
Absorbance readings were taken at 660 nm, and a standard graph was constructed
using these values. This graph was then used to estimate the amount of protein present
in the given sample.

Table 7: Procedure table of Folin Lowry’s method.

Stock D/W Conc. Volume Volume O.D at

soluti (ml) (µg/ of of 660 nm
on ml) reagent reagent
(ml) D(ml)
(ml)

0.1 0.9 20 5 0.5 0.17

0.2 0.8 40 5 0.5 Incubation 0.29

Incubation at room
0.3 0.7 60 5 0.5 0.35
at room temperatu
0.4 0.6 80 5 temperatu 0.5 re for 30 0.40

re for 5 minutes in
0.5 0.5 100 5 0.5 0.51
min.

XLI
0.6 0.4 120 5 0.5 0.55

0.7 0.3 140 5 0.5 0.43

0.8 0.2 160 5 0.5 0.53

dark
0.9 0.1 180 5 0.5 0.60

1.0 00 200 5 0.5 0.64

CHAPTER 4
RESULT AND DISCUSSION
XLIII
 RESULT AND DISCUSSION

In an experiment using fruit waste, it was found that, on average, 3 kilograms of fruit
waste material can produce approximately 600 grams of biogas. This biogas consists
mainly of methane and carbon dioxide and can serve as a sustainable energy source
for cooking, heating, and generating electricity. Furthermore, the remaining material
after biogas production, called digestate, can be utilized as a nutrient-rich fertilizer.

4.1 BIOGAS PRODUCTION IN 200 ML DIGESTER

(A) Day 1 (B) Day 8

Figure 7: Digester of 200ml (A) Day 1 (B) Day 8

In this study, biogas production was carried out in a 200 ml digester over a period of 8
days. Although gas production was observed, but it was not quantified. This study
was conducted as a trial to produce biogas from fruit waste. Further this study was
carried out using two 20-liter digesters
4.2 BIOGAS PRODUCTION IN 20 LITER DIGESTERS

(A)

(B)

Figure 8: Biogas production (A) Day 1 (B) Day 8

In this study, it was observed that the fruit waste test plant produced a greater amount
of gas compared to the control plant . This suggests that the test plant, with the
addition of inoculum and sodium thioglycollate, produced more gas than the control
plant, which did not use inoculum and sodium thioglycollate. Therefore, the use of
inoculum and sodium thioglycollate in fruit waste appears to offer a more efficient
method for biogas production. The biogas generated was stored in a vehicle tire tube
and weighed daily on a weighing balance for up to 8 days.

XLV
4.3 BIOGAS PRODUCTION OF TEST PLANT AND CONTROL PLANT

Table 8: Test plant biogas production in gm.

Days Initial weight of vechile tube Initial weight of tube – Final

(gm) weight of tube(gm)

1 350 25

2 350 48

3 350 60

4 350 83

5 350 110

6 350 102

7 350 97

8 350 82

Table 9: Control plant biogas production in gm.

Days Initial weight of vechile tube Initial weight of tube – Final

(gm) weight of tube (gm)

1 350 00

2 350 10

3 350 18

4 350 35

5 350 50

6 350 51

7 350 43

8 350 37
The study involved daily monitoring of biogas production in a test plant and control
plant over an 8-day period, achieved by weighing vehicle tire tubes used for gas
storage. Daily gas production in grams was determined by subtracting the initial tube
weight from the final tube weight.

4.4 COMPARISION OF TEST PLANT AND CONTROL PLANT

Comparision
120 of Test plant and Control plant
100
weight in garm

80

60

40

20

0
1 2 3 4 5 6 7 8

weight of test weight of control

Figure 9: Comparative study of biogas production in Test plant and Control

plant.

Graph analysis : The graph illustrates that gas production initially rises until day 5, but
subsequently decreases as acid concentration increases in both digesters, leading to a
pH below 7 after 6-7 days. This suggests a significant impact of acid concentration on
biogas production.

4.4 MOISTURE CONTENT (%)

The Moisture content (%) of fruit waste powder,

w−d
Moisture content (%) =
w
×100

= (2- 1.75/ 2) ×100

Moisture content (%) = 12.8 %

XLVII
4.3 ASH CONTENT (%)

The Ash content (%) of fruit waste powder,

Ash content (%) = (Final weight of sample / Initial weight of sample) ×100

= (0.24 / 2) ×100

Ash content (%) = 12 %

4.4 TOTAL SOLID (TS%) AND VOLATILE SOLID (VS%)

Component Test plant Control plant

Day 1 Day 8 Day 1 Day 8

TS % 22.3% 18.6% 23% 21%

VS % 7.14% 4.6% 10.7% 9.3%

Table 10: Result of Total Solid (%) and Volatile Solid (%)

In this study, the anaerobic digesters were operational from day 1 to day 8. The
reduction efficiencies of total solids (TS%) and volatile solids (VS%) for fruit waste
were 18.6% and 4.6%, respectively, in the test plant, and 21% and 9.3%, respectively,
in the control plant. These results suggest that the fruit waste was effectively
converted to biogas, indicating the need to add inoculum and sodium thioglycollate to
the digester.

4.5 DETERMINATION OF REDUCING SUGAR

The levels of reducing sugars in fruit waste samples were assessed using the DNSA
(3,5-Dinitrosalicylic Acid) method. Absorbance measurements were taken at 540 nm,
and the results were quantified as reducing sugar concentrations in micrograms per
milliliter (µg/mL). These values, which ranged from 0.4 to 0.9, suggested differing
amounts of reducing sugars present in the samples.
Table 11 : DNSA result table

OD of Blank 00

OD of Test 0.6

These results imply that the fruit waste samples analyzed have a concentration of
reducing sugars of 180 µg/mL.

4.6 DETERMINATION OF PROTEIN

Table 12: Folin Lowry result table

OD of Blank 00

OD of Test 0.9

Based on the standard graph, the protein concentration was determined to be 142
µg/mL when estimated using the Folin-Lowry method.

XLIX
CHAPTER 5
CONCLUSION
 CONCLUSION

1. The results suggest that addition of inoculum and reducing agent like sodium
thioglycollate into fruit waste powder supplemented media enhances gas production
significantly compared to control. This indicates that employing these additives can
lead to a more efficient biogas production process.

2. The gas production initially increases until day 5 but then declines as acid
concentration rises in both digesters, resulting in a pH below 7 by day 6-7. From this
it can be conclude that there is a significant influence of increase in acid
concentration on overall biogas production.

3. The anaerobic digesters operated from day 1 to day 8, demonstrating reduction

efficiencies of total solids (TS%) for fruit waste at day 1 and day 8 were 22.3% and
18.6%, and volatile solid (VS%) for fruit waste at day 1 and day 8 were 7.14% and
4.6%, respectively, in the test plant, and total solids (TS%) for fruit waste at day 1
and day 8 were 23% and 21%, and volatile solid (VS%) for fruit waste at day 1 and
day 8 were 10.7% and 9.3%, respectively, in the control plant. These findings indicate
effective conversion of fruit waste into biogas, highlighting the necessity of adding
inoculum and sodium thioglycollate to the digester.

LI
 CHAPTER 6
FUTURE SCOPE
 FUTURE SCOPE

Fruit waste biogas holds significant promise for sustainable waste management and
renewable energy production. In response to the global imperative to reduce reliance
on fossil fuels, this technology offers a compelling environmental advantage. By
utilizing anaerobic digestion, fruit waste, including peels, pulp, and seeds, can be
broken down by microorganisms to produce biogas—a renewable energy source
comprising methane and carbon dioxide. This biogas can serve as a sustainable
energy solution for electricity generation, heating, and cooking, effectively curbing
greenhouse gas emissions. Furthermore, the nutrient-rich digestate remaining after the
process can be repurposed as organic fertilizer, completing the nutrient cycle and
enhancing soil health. With the growing focus on sustainability and circular economy
principles, fruit waste biogas is composed to play a essential role in addressing waste
management challenges while advancing renewable energy production. The future
scope of fruit waste biogas extends beyond its environmental benefits. It also holds
economic potential. As governments and industries increasingly prioritize renewable
energy, investments in biogas production facilities using fruit waste could increase.
This could create job opportunities, especially in rural areas where fruit cultivation
and processing are prominent. Additionally, biogas production from fruit waste can
help reduce the burden on landfills and incineration facilities, addressing waste
management challenges. Moreover, the byproducts of biogas production, such as
biofertilizers, can contribute to sustainable agriculture practices, further enhancing the
overall environmental impact. Overall, the future of fruit waste biogas appears
promising, offering a sustainable and circular solution to waste management,
renewable energy production, and agricultural sustainability.

LIII
CHAPTER 7
REFERENCES
 REFERENCES

Sagagi, B., Garba, B., & Usman, N. (2009). Studies on biogas production
from fruits and vegetable waste. Bayero Journal of Pure and Applied
Sciences, 2(1), 115-118.
Deressa, L., Libsu, S., Chavan, R. B., Manaye, D., & Dabassa, A. (2015).
Production of biogas from fruit and vegetable wastes mixed with
different wastes. Environment and Ecology Research, 3(3), 65-71.
Viswanath, P., Devi, S. S., & Nand, K. (1992). Anaerobic digestion of fruit
and vegetable processing wastes for biogas production. Bioresource
technology, 40(1), 43-48.
Donald, L. Biomass for Renewable Energy, Fuels and Chemicals; Academic
Press. 1998; p 445-491.
Lohri, C. (2009) Research on anaerobic digestion of organic solid waste at
household level in Dares Salaam, Tanzania; Bachelor Thesis at
ZHAW (Zurich University of Applied Sciences) in collaboration with
Eawag (Swiss Federal Institute of Aquatic Science and Technology).
Heb, F.(2009) Decentralised Anaerobic Digestion of Market Waste – A Case
Study in Thiruvananthapuram, India. Sandec Report.
Chanakya, H.N.; Ramachandra, T.V.; Vijagachamundeeswari, M. (2006)
Anaerobic digestion and reuse of digested products of selected
products of urban solid waste.
Narayani, T. G., & Priya, P. G. (2012). Biogas production through mixed fruit
wastes biodegradation.
Rungvichaniwat J. Msc Thesis. Mahidol University, 2003.
APHA.(1998) Standard Methods for the Examination of Water and
Wastewater. 20th edition. Washington, D.C, USA.
Vij, S. (2011). Biogas production from kitchen waste. a report of Bachelor
degree, Department of Biotechnology and Medical Engineering, NIT,
Raurkela.
Karve, A. D. (2007). Compact biogas plant, a low cost digester for biogas
from waste starch.
Igoni, A. H., Abowei, M. F. N., Ayotamuno, M. J., & Eze, C. L. (2008). Effect
of total solids concentration of municipal solid waste on the biogas
produced in an anaerobic continuous digester. Agricultural
Engineering International: CIGR Journal.

LV
Pavi, S., Kramer, L. E., Gomes, L. P., & Miranda, L. A. S. (2017). Biogas
production from co-digestion of organic fraction of municipal solid
waste and fruit and vegetable waste. Bioresource technology, 228,
362-367.
Jarvis, G. N., Strömpl, C., Burgess, D. M., Skillman, L. C., Moore, E. R., &
Joblin, K. N. (2000). Isolation and identification of ruminal
methanogens from grazing cattle. Current microbiology, 40, 327-332.
Ogur, E. O., & Mbatia, S. (2013). Conversion of kitchen waste into biogas.
The International Journal of Engineering and Science (IJES), 2(11),
70-76.
Morales-Polo, C., Cledera-Castro, M. D. M., & Moratilla Soria, B. Y. (2019).
Biogas production from vegetable and fruit markets waste—
Compositional and batch characterizations. Sustainability, 11(23),
6790.
Maile, I., Muzenda, E., & Mbohwa, C. (2016). Optimization of biogas
production through anaerobic digestion of fruit and vegetable waste: a
review.
Chinwendu, A. O., Catherine, A. O. B., Bassey, A. E., & Okon, E. U. (2019).
The potential of biogas production from fruit wastes (Watermelon,
Mango and Pawpaw). World Journal of Advanced Research and
Reviews, 1(3), 052-065.
Scano, E. A., Asquer, C., Pistis, A., Ortu, L., Demontis, V., & Cocco, D.
(2014). Biogas from anaerobic digestion of fruit and vegetable wastes:
Experimental results on pilot-scale and preliminary performance
evaluation of a full-scale power plant. Energy conversion and
management, 77, 22-30.
Otun, T. F., Ojo, O. M., Ajibade, F. O., & Babatola, J. O. (2015). Evaluation
of biogas production from the digestion and codigestion of animal
waste, food waste and fruit waste. International Journal of Energy and
Environmental Research, 3(3), 12-24.
Viswanath, P., Devi, S. S., & Nand, K. (1992). Anaerobic digestion of fruit
and vegetable processing wastes for biogas production. Bioresource
technology, 40(1), 43-48.
Pavi, S., Kramer, L. E., Gomes, L. P., & Miranda, L. A. S. (2017). Biogas
production from co-digestion of organic fraction of municipal solid
waste and fruit and vegetable waste. Bioresource technology, 228,
362-367.
Lattieff, F. A. (2016). A study of biogas production from date palm fruit
wastes. Journal of cleaner production, 139, 1191-1195.
Manthia, F., Amalin, N., Matin, H. H. A., & Sumardiono, S. (2018).
Production of biogas from organic fruit waste in anaerobic digester
using ruminant as the inoculum. In MATEC Web of Conferences (Vol.
156, p. 03053). EDP Sciences.
Knol, W., Van Der Most, M. M., & De Waart, J. (1978). Biogas production by
anaerobic digestion of fruit and vegetable waste. A preliminary study.
Journal of the Science of Food and Agriculture, 29(9), 822-830.
Bouallagui, H., Cheikh, R. B., Marouani, L., & Hamdi, M. (2003). Mesophilic
biogas production from fruit and vegetable waste in a tubular digester.
Bioresource technology, 86(1), 85-89.
Neto, J. G., Ozorio, L. V., de Abreu, T. C. C., Dos Santos, B. F., & Pradelle,
F. (2021). Modeling of biogas production from food, fruits and
vegetables wastes using artificial neural network (ANN). Fuel, 285,
119081.
dos Santos, L. A., Valença, R. B., da Silva, L. C. S., de Barros Holanda, S. H.,
da Silva, A. F. V., Jucá, J. F. T., & Santos, A. F. M. S. (2020).
Methane generation potential through anaerobic digestion of fruit
waste. Journal of Cleaner Production, 256, 120389.

LVII
CHAPTER 8
APPENDIX
 PARTICIPATION AND ABSTRACT SUBMISSION IN

INTERNATIONAL CONFERENCE

International Conference on Innovations and Biotechnology Research for Sustainable

Bioresources and Bioeconomy: Challenges and Practices"(IBRSBB-2024) Organized
by Sanjivani Art's Commerce and Science college Kopargaon on 15 & 16
February ,2024 in Association with Microbiologists Society (India) (MBS), Ural
Federal University (Russia), Swedish South Asian Network for Fermented Foods
(Anand, Gujrat), Institute of Fundamental and Applied Reserved, National Research
University (TIIAME), (Tashkent, Uzbekistan).

Transforming Waste into Energy: Biogas Production from Fruit

Waste
Tirse A. U, Dahikar S. B., and Bhakare S.V.
Department of Microbiology,
Sanjivani Arts Commerce and Science College, Kopargaon- 423603
tirseadesh@gmail.com

Abstract
This study investigates the generation of biogas through the anaerobic digestion of
fruit waste mixed with an inoculum. The examination involved evaluating the total
solids, volatile solid, moisture content and ash content of the waste materials. The
impact of utilizing various fruit wastes, such as banana peel, pineapple, tomato,
pumpkin, sweet lime peel and chickoo were used in a 20 lit digestor. Microorganisms,
particularly those from the Methanogenesis family play vital role in transforming this
waste into biogas, where methane serves as the main component. Initially, a defined
amount of fruit waste, sourced from the market, was acquired. Later, in the laboratory,
Methanogens spp. were isolated and their inoculum was mixed with the fruit waste in
an anerobic digestor. Throughout daily observations, there was a progressive rise in
gas production until, on the 15th day, the generation of biogas come to a halt.
Evaluation of the generated biogas was performed by chromatography analysis. The
research showed alternative energy sources, such as biogas, serves as a sustainable or
renewable energy source and finds applications across various purposes. It should be
intensified to address ecological threats like environmental pollution, deforestation,
desertification, erosion.

LIX
Keywords: Anaerobic digestion, Fruit Waste, Biogas, Methane