Dr. B.R.

Ambedkar National Institute of Technology, Jalandhar

Industrial Practice Training (BTX-300) Report

On
Anaerobic Digestion of Water Hyacinth under Thermophilic Conditions for
the enhanced production of Biogas
Submitted by
Ritika (19111036)
Final year student
Department of Biotechnology

Under the Guidance of

Dr. Sachin Kumar
Scientist C
Biochemical Conversion Division
Sardar Swaran Singh National Institute of Bioenergy

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DECLARATION

I hereby declare that the work which is being presented in this project report entitled “Anaerobic
Digestion of Water Hyacinth under Thermophilic Conditions for the enhanced production of
Biogas” is being submitted by me in the partial fulfillment of the requirement for the completion
of the curriculum of 7th Semester in Bachelor of Technology in Biotechnology at Dr. BR
Ambedkar National Institute of Technology, Jalandhar is an authentic record of my work carried
out under the guidance of Dr. Sachin Kumar, Scientist C, Biochemical Conversion Div., Sardar
Swaran Singh National Institute of Bioenergy. The matters embodied in this report have not been
submitted by me for the award of any other degree or diploma.

Date:- Ritika
(19111036)

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CERTIFICATE

This is to certify that the project work entitled “Anaerobic Digestion of Water Hyacinth under
Thermophilic Conditions for the enhanced production of Biogas”, is a bonafide record of work
submitted by Ritika (19111036), Final year student of the Department of Biotechnology, Dr. BR
Ambedkar National Institute of Technology, Jalandhar, under the guidance of Dr. Sachin Kumar,
Scientist C, Biochemical Conversion Div., Sardar Swaran Singh National Institute of Bioenergy,
during the session 2022-23.

Place: Jalandhar Guide

Date: Dr. Sachin Kumar
Scientist C
Biochemical Conversion Division
Sardar Swaran Singh National Institute of Bioenergy

HOD
Dr. Nitai Basak
Department of Biotechnology
Dr. B. R. Ambedkar National Institute of Technology

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 ACKNOWLEDGEMENT

I wish to offer my sincerest thanks Dr. G. Sridhar, Director, Sardar Swaran Singh National
Institute of Bioenergy, Jalandhar- Kapurthala Road; for his kind permission to carry out my
thesis in the Research and Development Laboratory 2 of Sardar Swaran Singh National Institute
of Bioenergy. My heartfelt gratitude goes to my supervisor, Dr. Sachin Kumar, Scientist C,
Biochemical Conversion Division, Sardar Swaran Singh National Institute of Bioenergy,
Jalandhar for his constant inspiration, constructive criticism, supervision, and valuable
suggestions without which this thesis would not have been completed. I acknowledge my esteem
to Miss. Gagan Preet Kaur (PhD. Scholar), Miss. Nisha Yadav (PhD. Scholar), and Aashirwad
(B. tech Student, UPES, Dehradun), Sardar Swaran Singh National Institute of Bioenergy for
their keen interest, constant guidance, and encouragement of my work. I would like to thank all
the teaching assistants along with the library attendants who cooperated with me and assisted me
throughout the study. My deepest appreciation goes to my thesis partner Manvi, for her help and
pleasant company who supported me immensely during my time of crisis. I am also thankful to
all my friends for their interest, constant support, and positivity towards my work. Finally, I owe
my earnest gratitude and appreciation to my parents and grandparents, who shouldered all my
responsibilities for such a prolonged period, with smiling faces who believed in me and allowed
me to carry out my work undisturbed.

Ritika (19111036)

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 TABLE OF CONTENTS
S.No. Contents Page No.
1. Abstract
2. Introduction
3. Literature Review
4. Materials And Methods
5. Results And Discussion
6. Conclusion
7. References

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ABSTRACT

In the current scenario, research into the creation of renewable energy from organic resources
and waste has benefited from the excessive use of conventional fuels and the influence of
greenhouse levels in the atmosphere. The majority of the energy used worldwide is produced
from fossil fuels due to the enormous demand for energy. Anaerobic digestion (AD), which
combines the creation of biofuel and environmentally friendly waste management, is referred to
as an alternative and effective technique in recent studies. The growing interest in producing
biogas from biowaste using complicated anaerobic digestion (AD) has created new opportunities
for improving and modernizing the processes used to produce biogas. To enhance the output and
quality of biogas, the biogas industry is utilizing a variety of technology advancements. The
aquatic plant known as the water hyacinth (Eichhornia crassipes) is known for its ability to grow
very quickly, freely float over water, and completely cover the water's surface. Because of their
high fermentation content, cellulose, nitrogen, and other vital elements, water hyacinth leaves
can be used as a biogas fuel. The aim of this study is to study the anaerobic digestion of water
hyacinth under the thermophilic conditions for enhanced biogas production.

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 1. INTRODUCTION

Currently, modern culture considers energy to be a basic necessity. The community used a
variety of technology to promote convenience in daily life, which raised the requirement for
energy use in addition to population growth and rising consumption. The world's present energy
supply is heavily dependent on fossil fuels (crude oil, lignite, hard coal, natural gas). These are
the fossil remains of extinct plants and animals that have spent hundreds of millions of years
under pressure and heat in the Earth's crust. Fossil fuels are nonrenewable resources because
their reserves are being used up considerably more quickly than they are being replenished. The
current situation has led to research efforts into the generation of alternative fuels from bio
resources due to the unusual use of conventional fuels and the impact of greenhouse emissions
on the environment. Global energy consumption has rapidly expanded, and now, fossil fuels
supply around 88 percent of this need. Carbon dioxide (CO 2) is the primary greenhouse gas
(GHG) that is contributing to the atmosphere's rapid increase in GHG emissions. Advanced
solutions to handle bio-waste and simultaneously create renewable energy are required to fix
such environmental challenges. Researchers have recently become quite interested in using
biomass as a source of renewable energy, especially because the technology is safe for the
environment.
Biogas is the byproduct of methanogenic bacteria's anaerobic digestion or breakdown (in an
oxygen-free environment). Because the methane (CH4) in biogas has a heating value of 50
MJ/kg, it has a lot of potential as a new energy source. Since there is only one carbon in each
chain of methane (CH4), its combustion can be more environmentally benign than that of fuels
with extensive carbon chains. The reduced CO 2 created during the combustion of short carbon
chain fuels is the root of this issue. The tendency to convert waste into a useful resource by using
it as a substrate for AD is one of the primary benefits of producing biogas.

1.1 Biochemistry of Anaerobic fermentation

Three phases of anaerobic fermentation include the hydrolysis phase, acid phase and methane
forming phase.
During hydrolysis phase, large molecules are broken down into smaller sizes by enzymes which
in turn are decomposable by bacteria. The monomeric components that are liberated during the
hydrolysis phase serve as the acid forming phase's substrate. Complex organic compounds are
broken down into simpler acids like volatile fatty acids during the periodic acid phase. One
typical by-product of the breakdown of proteins, starches, and fats is acetic acid. Methane
bacteria can produce methane and carbon dioxide or by converting carbon dioxide to methane
utilizing hydrogen gas or format made by other bacteria. Methane bacteria are totally anaerobic.
Methanogenic bacteria react to different parts of the substrate in different ways. Methane is
created when methanogenic bacteria interact with acetic acid, methanol, carbon dioxide, and
hydrogen. Methanogenic bacteria's activity is slowed down by oxygen. However, to increase the

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overall process performance and to lower the associated cost, biological hydrolysis, which is the
rate-limiting step for anaerobic degradation, must be addressed.
1.2 Properties and composition of biogas

About 60 to 70 percent of biogas is made up of methane, 30 to 40 percent is carbon

dioxide, and traces of ammonia, hydrogen sulfide, mercaptans, and other toxic gases are
also present. Biogas has an ignition temperature between 650 and 750֯ C and is
approximately 20% lighter than air. The second significant component, carbon dioxide,
which is not combustible, contributes to make biogas less combustible. One of its main
contaminants, hydrogen sulphide, has a smell identical to that of rotten eggs, making it
simple to notice when biogas is leaking whenever it does. Biogas has blue flame. Calorific
value of biogas varies within 500 to 700 BTU per cubic foot.
Due to its distinct qualities, biogas has various advantages. Cooking, lighting, and heating
are only a few of the diverse end uses for biogas, which also has industrial applications for
combined heat and power (CHP) generation, transportation fuel (after being improved to
biomethane), and other uses when enhanced to natural gas quality. Large-scale plants are
built in several developing nations primarily for the generation of electricity using fuel
cells or CHP engines.

1.3 Important aspects and safety aspects

The acidity of the digester can be checked by dipping the strip of litmus paper. Higher
acidity indicates the imbalance in various bacteria. The number, size, and kind of animals
providing feed, the dilution of the slurry, and the retention period are some of the variables
that affect the capacity of the digester in a biogas plant. Higher the retention time larger the
size of digester is required.
Proper mixing of the digester contents provides-
 Intimate contact between micro-organisms and the substrate.
 Maintaining the uniform temperature.
 The distribution of bacteria and volatile solids throughout slurry.
 Prevent scum or crust from forming on the slurry's surface, which would obstruct the
release of biogas and minimizes sludge formation.
 Prevents accumulation.
Biogas must be handled carefully because it is an explosive gas. The most important
moment to follow safety precautions is while digester and gas line repairs are taking place.
You can check for any leaks by running soapy water over gas taps, gas cocks, joints, and
fittings to see if any bubbles appear. Appliances that are prone to fire should not come into
touch with gas leaks.

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 1.4 Factors affecting biogas yield

Effect of pH- The pH of the digester's contents has a significant impact on biogas
generation. It essentially serves as a pointer for a solution's acidity and alkalinity. Bacterial
activity is thought to be hindered by an environment that is both excessively acidic and too
alkaline. For improved gas output, a pH range of 7 to 8.5 is ideal. The amount of carbon
dioxide and volatile fatty acids produced in a digester as intermediate products during
fermentation mostly influences the pH of the digester's contents.
Effect of C:N ratio- Both carbon and nitrogen give the necessary nutrients for anaerobic
bacteria to function effectively. Nitrogen aids in constructing the cell structure of
microorganisms while carbon supplies the energy they require to survive. In general,
microorganisms use carbon 25 to 30 times more quickly than nitrogen during digestion,
which suggests that feedstocks should have a carbon content that is 25 to 30 times higher
than nitrogen.
Effect of temperature- The digester's content's temperature has a significant impact on the
biogas yield. Different temperature ranges have mesophilic and thermophilic bacteria at
their most active, producing the greatest amount of gas. It has been discovered that the
former are most active in the 35֯ to 40֯ C range and the latter in the 50֯ to 60 ֯C range.

1.5 Biogas yield from Water-Hyacinth

An aquatic plant known as a water hyacinth (WH) can grow very quickly and float freely
on the water's surface. This plant's ability for invasion has greatly damaged the ecosystem
in many places. It has demonstrated an incredibly rapid rate of growth and has formed a
dense, compact carpet that covers the entire body of water. This dense carpet blocks
sunlight from penetrating the water, which decreases the amount of oxygen in the water
and causes a number of issues, such as damage to aquatic plants and animals, irrigation
issues that prevent the transportation of water, and mosquito breeding grounds that
increase mosquito populations.
WH is a viable feedstock for the generation of biogas because it is locally cultivated, is
abundant in fermentable matter and vital nutrients like nitrogen. A helpful soil conditioner
and manure with no negative environmental consequences, digestate obtained during AD is
rich in macronutrients required for the proper growth of plants in addition to producing
bio-gas. 9 So, the use of WH as a renewable energy source to replace fossil fuels will offer
efficient control of this invasive plant on water bodies, and in the interim, the effluent after
AD might be used as an appropriate organic fertilizer for plant feeding. WH absorbs
certain metals and metabolites and hence can reduce pollution levels by 75-80 percent.

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 2. OBJECTIVE

This study is conducted with aim to: -

 Study of Anaerobic Digestion of Water Hyacinth under Thermophilic Conditions
 To check the volume of gas formed every day.
 To check the composition of biogas through Gas Chromatography.

3. LITERATURE REVIEW
3.1 Anaerobic Digestion
The process of breaking down organic matter by microbes without the use of oxygen is known as
anaerobic digestion (AD). Natural Gas is also produced by nature by the digestion of thousand
years of old plants by anaerobic bacteria. But the natural pressure and temperature converts the
methane into other gases such as ethane, propane, butane etc. That is why it is produced
manually by anaerobic digestion. The duration is usually fifteen days for the conversion of
organic matter into the biogas which is too short for the conversion of methane into other gases.
It is a Temperature dependent process. If the temperature is below 30℃, the process bounds to
slow down. The temperature range between 35-38℃ is the optimum range known as the
mesophilic range. Again, if it is between 40- 45℃ range, the process will slow down and at 55-
60℃ temperature range, which is also known as thermophilic range, it attains its peak.
In Anaerobic Digestion, Organic Matter is hydrolyzed by enzymes into simple sugars, Alcohols,
peptides and amino acids. These are then converted to volatile fatty acids, hydrogen, carbon di
oxide, water and some amount of methane. Finally, the Methane forming bacteria converts the
fatty acid into methane.
Anaerobic process can be divided into two phases namely the acid forming phase and the
methanogenesis for the production of the Biogas. Biogas is a combustible gas which contains,
60-65% methane, 35-40% carbon di oxide traces of hydrogen sulfide, ammonia and other
impurities. The hydrogen sulfide present in biogas is highly corrosive and hence it needs to be
removed. It can be removed by passing it through the steel wool and the 35%. CO2 can be
removed by passing through the lime water.
Anaerobic Digestion consist of three steps: -

1. Hydrolysis: - Conversion of large molecule into smaller ones by enzymes. These enzymes
are further decomposed by bacteria.
2. Acid Phase: - Complicated Molecules such as proteins, fats and carbohydrates are
decomposed by acid forming bacteria into organic acids, CO2, H2, ammonia and other
impurities. Bacteria can utilize only CO2, H2, HCOOH.

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 3. Methanogenesis: - Methane forming bacteria converts fatty acids into methane.

There are four types of methanogenesis bacteria: -

1. Rod shaped sporulating cells: - Methanobacillus
2. Rod shaped non- sporulating bacteria: - Methanobacterium
3. Spherical cells in Sarcina Arrangement: - Methanosarcina
4. Spherical cells not in Sarcina Arrangement: - Methanococccus

Thermal stage Process temperatures Minimum retention time

Mesophilic 30℃-42℃ 30- 40 days
Thermophilic 43℃-55℃ 15-20 days
Table 1: Thermal stage and typical retention times

The size of the reactor depends upon the number, size and types of animal/plant providing feed,
slurry dilution and retention time.
Retention time is the most important factor as minimum 10 days retention time is necessary. The
longer the time period, the larger the reactor. Loading rate is the amount of volatile solids fed to
the reactor per day.

4. MATERIALS AND METHODS

4.1 Sample Preparation
Dried and Grinded Water Hyacinth was taken. The Water Hyacinth is characterized by high
content of the lignocellulosic biomass with a 10% average content of lignin, useful in both
thermochemical and biochemical processes.

4.2 Consortium Used

Water Hyacinth seed is used as the consortium.

4.3 Principle of the Experiment

100g of the feedstock is mixed with the 800 volumes of tap-water. This Experiment was
carried out under the thermophilic temperature. The increase in the gas bottle pressure results
in the production of the carbon di oxide (CO2) and methane (CH4). This gas travels through
the plastic tube and enter the second bottle which contains water. Then due to the diffusion,
the water replaced enters the third empty bottle. The volume of the water displaced is equal
to the gas formation. The water volume in the empty bottle is checked every day to get the
amount of the gas formed.

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5

1 2 3

Fig. 1 Schematic diagram of the experimental layout for biogas production

1. Bottle filled with Feedstock

2. Bottle filled with Water
3. Empty bottle
4. T- Shaped tube for the collection of biogas
5. Clamps to prevent the leakage

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 Figure 2. Feedstock is ready to be filled in first bottle of the setup

4.3.1 EXPERIMENTAL MATERIALS

The feedstock used in this study is water hyacinth. The water hyacinth is first dried and grinded
down. A 100 g of water hyacinth is mixed with 800 ml of water. The mixture is kept covered for
24 hours at room temperature.

4.3.2 EXPERIMENTAL SETUP

The experimental setup for the digested feedstock consists of three incubator bottles. Each bottle
is of the volume of two liters. First, the exact amount of 3.08 g of urea is added to the feedstock

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along with the consortium consisting of the water hyacinth seed. Then around 100 ml of water is
added to the feedstock and mixed it. This treated feedstock is filled in one of the bottles and the
mouth of the bottle is covered with a cork lid with two outlets. The cork is then sealed with the
lac for preventing any leakage. In the second bottle, the water is filled somewhat less from the
brim and covered with the two-outlet cork and sealed with lac. The third bottle is kept empty and
covered with two-outlet unfixed cork. The connection is made with plastic tubes from the first
bottle to the second bottle and from the second bottle to the third bottle. The whole setup is kept
inside the incubator at 52℃ which is a thermophilic temperature.

5. RESULTS AND DISCUSSION

Figure 2. shows the volume of water measured per day. The water measured per day is equal to
the gas formed inside the feedstock bottle per day.

Volume of Biogas Produced per Day

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Volume Of Water measured

10

0
0 2 4 6 8 10 12
Number of days

Figure. 3

5.1 Biogas Composition

Figure 3. shows methane concentration from the water hyacinth feedstock. The maximum
methane concentration from the water hyacinth was 71.1414%. This result that water hyacinth
promote favorable condition for methanation process.

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 Gas Chromatography of Biogas
80
70

Composition of Gas
60
50
40
30
20
10
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Number of days

methane Carbon di oxide Oxygen Nitrogen

Figure. 4

5.2 CONCLUSION
The study has shown that anaerobic digestion of water hyacinth is a viable and feasible process
for large biogas plants. On the other hand, the water hyacinth showed high biogas production. In
general, water hyacinth is potential feedstock for biogas production.

REFERENCES
Keche, Fetanu., et al., Anaerobic digestion of urea pretreated water hyacinth removed from Lake
Abaya; bio-methane potential, system stability, and substance conversion.
Adelodun., Temitope., et al., Biogas Generation from Co-Digestion Waste Systems: The Role of
Water Hyacinth.
M. Carlini, et al., Water hyacinth biomass: chemical and thermal pre-treatment for energetic utilization in
anaerobic digestion process.

Irina., Pushiri., et al., Invasive Water Hyacinth: Ecology, Impacts and Prospects for the Rural
Economy

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