Bioenergy - Anaerobic digestion of

cattle manure and methane

production
Submitted by: Jakob Zidi
School of science and engineering
Department of Biotechnology, Chemistry and Environmental Engineering
Section for Sustainable Biotechnology
Aalborg University (Copenhagen)
Date 18.12.2014

Abstract
This study investigates the potential of producing bio-energy from organic waste
materials, and more specifically the production of methane from cattle manure. It
involves an experiment with a continuous stirred-tank reactor(CSTR), where the
manure is used as substrate for the methane production. Moreover, this study explore
the processes taken place during the anaerobic digestion. The maximum methane
yield obtained from the AD process was reached after 19 days occupying 66,55% of
the biogas produced in an amount of 363,54mL/gVS. Besides measuring the
biogas/methane production an analyze of ammonia and volatile fatty acids where
performed to get insight in possible process inhibition causes. But the results of the
measurements indicated a successful anaerobic digestion without any significant
inhibition. Furthermore, were measurements of the total solids(TS), the volatile
solids(VS) and the chemical oxygen demand(COD) performed to receive knowledge of
the efficiency of the conversion of the organic matter. Even though conflicting
measurement of organic matter conversion was shown in the TS/VS removal and the
COD removal, the total output of biogas production turned out to be successful and
increasing the whole period, except for when the initial manure was changed, which
resulted in a drop in the biogas/methane yield. Ultimately, the AD process was
considered successful and stable with a successful production of biogas and methane.

Contents
Abstract.......................................................................................................................... 1
Table of figures............................................................................................................... 3
Table of tables................................................................................................................ 4
1. Introduction:............................................................................................................... 5
1.1 Historical background of anaerobic digestion:.......................................................6
2. Overview of anaerobic digestion:...............................................................................6
2.1 First step (Hydrolysis):........................................................................................... 7
2.2 Second step (Acidogenesis)..................................................................................8
2.3 Third step(Acetogenesis)....................................................................................... 8
2.4 Fourth step (Methanogenesis)...............................................................................8
2.5 Factors that affect anaerobic digestion process..................................................10
2.5.1 Temperature.................................................................................................. 11
2.5.2 pH value and optimal intervals.....................................................................11
2.5.3 Volatile fatty acid(VFA) accumulation............................................................12
2.5.4 Ammonia....................................................................................................... 12
2.5.5 Other factors (Sulphide, macro and micro nutrients (trace elements), Light
metals, Heavy metals)........................................................................................... 12
2.6 Optional parameters........................................................................................... 13
2.6.1 Particle size................................................................................................... 13
2.6.2 Water content............................................................................................... 13
2.6.3 Reactor type.................................................................................................. 13
2.6.4 Organic loading rate (OLR)............................................................................13
2.6.5 Hydraulic retention time (HRT)......................................................................14
2.7 Biogas................................................................................................................. 14
2.7.1 Definition of biogas....................................................................................... 14
2.7.2 Sources of biogas:......................................................................................... 14
2.7.3 Uses of biogas:.............................................................................................. 15
3. The study................................................................................................................. 15
3.1 Objective of this study......................................................................................... 15

3.2 Structure of this report........................................................................................ 16

4. Materials and Method............................................................................................... 16
4.1 Substrate............................................................................................................. 16
4.2 Reactor set-up (mixing and time settings):.........................................................16
4.3 Calibration of pump and gas-meter.....................................................................17
4.3.1 Calibration of peristaltic pump......................................................................17
4.3.2 Calibration of Gas-meter...............................................................................18
4.2.3 Continuous Experiment:................................................................................18
4.3 Analytic methods................................................................................................ 19
4.3.1 Determination of TS/VS and measurement of pH.............................................19
4.3.2 Ammonium.................................................................................................... 20
4.3.3 Chemical oxygen demand (COD)..................................................................20
4.3.4 Volatile fatty acids(VFA)................................................................................20
4.3.5 Biogas measurement.................................................................................... 21
5. Results and discussion............................................................................................. 21
5.1 Continuous reactor (CSTR).................................................................................. 21
5.2 Biogas and Methane yields..................................................................................21
5.3 Methane content................................................................................................. 22
5.4 pH........................................................................................................................ 23
5.5 TS/VS measurements.......................................................................................... 24
5.6 Chemical oxygen demand (COD)........................................................................25
5.7 Ammonia production........................................................................................... 26
5.8 Volatile fatty acids (VFA's)...................................................................................26
6. Conclusion................................................................................................................ 31
7. Recommendations for further work..........................................................................31
8. References................................................................................................................ 32

Table of figures

Figure
Figure
Figure
Figure

1:
2:
3:
4:

Anaerobic digestion biochemical conversion pathways (W. Parawira, 2004). . .7

Calibration graph for pump(water)................................................................17
Calibration graph for pump(manure).............................................................18
Picture of the Continous stirred tank reactor system.....................................19

Figure 5: Gas chromatograph (GC) for measurement of biogas production and methane
composition.................................................................................................................. 21
Figure 6: Development of biogas and methane gas during the period of the AD process
..................................................................................................................................... 22
Figure 7: Yield of the methane production during the AD process................................23
Figure 8: Measurement of pH during the period of the AD process..............................23
Figure 9: Development in Total solids(TS) in the influent and effluent during the AD
process......................................................................................................................... 24
Figure 10: Development in Volatile solids(VS) in the influent and effluent during the AD
process......................................................................................................................... 24
Figure 11: Development of the Chemical oxygen demand(COD) during the AD process
..................................................................................................................................... 25
Figure 12: Measurements of the Ammonia accumulation during the AD process.........26
Figure 13: Development of Acetate during the period of the AD process.....................27
Figure 14: Development of Propionate during the period of the AD process................28
Figure 15: Development of Iso-butyrate during the period of the AD process..............28
Figure 16: Development of Butyrate during the period of the AD process...................29
Figure 17: Development of Iso-valerate during the period of the AD process..............29
Figure 18: Development of Valerate during the period of the AD process....................30
Figure 19: Development of VFA during the period of the AD process...........................30
Table of tables

Table 1: Acetogenic, methanogenic and syntrophic reaction: Free energy, pH 7, 1 at, all
reactants and products at 1M concentration (W. Parawira, 2004)................................10
Table 2:Comparison of mesophilic and thermophilic temperatures in the AD
process(W.Parawira, 2004)........................................................................................... 11
Table 3: Composition of biogas (M. Balat & H. Balat, 2009)..........................................14
Table 4: Overview of production of manure in EU-27 (M. Balat & H. Balat, 2009).........15
Table 5: Calibration for water....................................................................................... 17
Table 6: Calibration for manure.................................................................................... 18
Table 7: Observations and measurements of the reactor.............................................21

1. Introduction:
One of the major issues both scientifically and politically during the past decade has
been the increasing temperatures due to the increasing emissions of greenhouse
gasses (GHG)(S.H. Schneider, 1989). Global warming has forced the world to pay
attention to the industrial emissions and different kind of restrictions has been made
to lower to damage. In recent years several countries have made agreement to lower
their emissions of carbon dioxide (CO2) to minimize the damage. To achieve this goal
rules been made about amounts of CO2 emission allowed. Power plants and other
companies receive or buy CO2 emission allowances in order to emit large amounts of
CO2. In Denmark the Danish energy agency manages the EU Emissions Trading
Scheme(Dansk Energi | Rekordlave CO2-kvotepriser ovenp EU afstemning, n.d. ;
Dansk Energi | Rekordlave CO2-kvotepriser ovenp EU afstemning. (n.d.)). Even
though it is commonly thought that CO2 is the most effective greenhouse gas research
has shown, that methane(CH4) is significant more effective, and have an absorption of
infrared radiation, which is 20 to 30 times more effective per molecule in respect to
CO2 (S.H. Schneider, 1989).
Today, the modern society produces a large amount of wastes, that potential can be a
tremendous threat to the environment, but also to health of humans, animals and
other living organisms. As a result of attempting to minimize the risk of this threat a
lot of different waste treatments and disposals methods have been developed, and are
used to prevent and control this problem. The selection of method must always take in
consideration, which one will give maximum safety and minimum environmental
impact. Furthermore the method should, as far as possible, be based on attempting to
recycle the end products of the process. One of the today's trends is to reduce the
stream of wastes going to landfill by recycling the organic material and plant material.
One way of achieving this goal is by anaerobic digestion (AD), which not only will
reduce the energy consumption, but may also produce an access of energy.(B.K.
Ahring, 2003)
Anaerobic digestion has in many years been implemented as a mean of stabilizing the
produced sewage sludge. However, in recent years the AD technology has been
developed and expanded to emphasize the treatment and energy recovery from a
wide range of different wastes. The major areas includes animal wastes, organic

household wastes and industrial wastewaters and industrial organic materials (B.K.
Ahring, 2003).
In the present time large amounts of animal manure and slurries are produced by the
animal breeding sector, which represents a constantly pollution risk, that potentially
could have a negative impact on the health of the environment if is not managed
correctly. To prevent emissions of GHG and accumulation of too high amounts of
nutrients and organic matter into the natural environment produced by the industries,
an optimal solution of recycling is of crucial importance. (J.B. Holm-Nielsen et al.,
2009) In Denmark, as a result of a high livestock population, about 25 million tonnes
of livestock waste is produced each year. Currently only about 5% of the waste is
treated by the 83 biogas plants in Denmark. It is the goal of the Danish government to
increase this number to about 40% by the year 2020, as a mean of decreasing the CO 2
emissions(B.M. Salces et al., 2014).
One of the major product from anaerobic digestion is the production of biogas and
fertilizers. Biogas can be almost being produced from everything from biological feedstocks, including agricultural wastes and different organic wastes from all over society.
(J.B. Holm-Nielsen et al., 2009)

1.1 Historical background of anaerobic digestion:

Anaerobic digestion has been used for many decades and is one of the oldest
technologies, which has been highly advanced during the years. The use of microbial
degradation was first utilized by the Egyptians about 5000 years ago where the
acidified bread with leaven. And likewise the Sumerians and Babylonians gardened
juices into alcohols by the process of fermentation. Later biogas was used by the
Assyrians for heating of water in their baths 10th century BC, and later in the Persian
regime in the 16th century. In the 17th century the anaerobic digestion technology
advanced and scientific research was established by J.B.V. Helmont that found, that
yielded flammable gasses from decaying organic materials. In 1808, a scientist named
Sir Humphry Davy demonstrated the production of methane by AD of cattle manure.
The industrialization of the AD process started in 1859 with the first digestion plant in
Bombay, India. In the end of 19th century the AD process made it to Europe in England
where biogas was produced from a sewage treatment facility, which were used to fuel
lamps in Exeter, Devon, a city in south England. Further discoveries was found, due to
the developments within the field of microbiology. In 1930, research had let Buswell
and others to identify anaerobic microorganisms as well as conditions for promotion of
methane production. Since then many different low-cost project has been deployed as
foreign aid project in developing countries. The research within this area has also
developed a lot and come far in the understanding of the processes taken place in the
anaerobic digestion system, which has let to establishment of many AD plant all over
the globe(S. Verma, 2002).

2. Overview of anaerobic digestion:

The evolutionary development in microbial degradation truly play a key role in the
recycling of the carbon on the plant. There exist many different communities of
microbes that has evolved to specifically degrade different organic material (S.B.
Leschine, 1995). The anaerobic digestion (AD) is one of the major sections of
degradation, which takes place in environments where free oxygen is not accessible.
One of the major valuable products of anaerobic digestion is the production of biogas,
which mainly consist of carbon dioxide and methane. This form of renewable energy
can be utilized for the production of heat and electrical energy. The AD process occurs
naturally in anaerobic ecosystems such as sediments, paddy fields, water logged soils
and in the rumen(B.K. Ahring, 2003). For the process of AD sufficient nutrients in
relation to the amount of carbon are required to give an efficient digestion of the
organic material. The environment of which the AD process is taken place should have
a pH value close to neutral and the level of the inhibitory compounds should be kept
low. Furthermore competing electron acceptors, as nitrate, sulphate, oxidized iron and
manganese should not be present or at minimum be kept at low levels. The
temperature should also be stable to apply the optimal conditions for the
microbes(Geradi, 2003; Schrink, 1997). It is the enzymatic processes, that breaks
down the complex organic substances into the end products methane and carbon
dioxide. Other products are also generated in a minor quantities(less than 1% of total
gas volume), such as nitrogen, ammonia, hydrogen and hydrogen sulphide. In each
step of the AD process different microbes are responsible for the degradation,
including fermentative, acetogenic and methanogenic (acetotrophic and
hydrogenotrophic) microorganisms (Geradi, 2003).
Besides from the biogas, the AD process also produces a liquid effluent called the
digestate, which contains all the water, all the minerals and approximately half of the
carbon from the organic matter in the influent. The digestate is a nutrient rich
substance and can be used for many different purposes, but the most commonly
utilization of the digestate is for fertilizers, as feedstock for ethanol production and in
low-grade building material such as fiberboards. The water from the effluent of the AD
process can be recycled after treatment and used again. AD enhances the nutrient
availability and reduces the smell and pathogens of the substrate(Mata-Alvarez, 2003).
As mentioned earlier the AD is a process involving different groups of microorganisms
and the AD is therefore considered as a multi-step process. It consists of four different
reactions, hydrolysis, acidogenesis, acetogenesis and methanogenesis. Of the four
reactions only the first is extracellular hydrolysis) and the rest
intracellular(acidogenesis, acetogenesis and methanogenesis)(H.N. Gavala et al, 2003;
D.J. Batstone, 2002). The four steps are carried out by different groups of
microorganisms, the fermentative bacteria hydrolytic and acidogenic), the anaerobic
oxidizing bacteria(syntropic and acetogenic) and the methanogenic archaea(D.J.
Batstone,2002; Cheng H. and Wang L., 2013).

Figure 1: Anaerobic digestion biochemical conversion pathways (W. Parawira, 2004)

2.1 First step (Hydrolysis):

The hydrolytic step is a slow process, which will take a few hours before the
breakdown of the carbohydrates and furthermore a few days for the lipids and
proteins. The hydrolysis is the first step where complex organic material is broken
down into long-fatty acids, amino acids and simple sugars. The organic matter, such
as carbohydrates, proteins and fats, are converted by the extracellular enzymes
cellulase, amylase and protease or lipase. This step is performed by either anaerobic
bacteria or fungi. The carbohydrates are converted into sugar monomers, the proteins
into amino acids and the fats into long-fatty acids chains. The hydrolytic step is
considered as a rate-limiting step, because the large amount of lignocellulose in the
organic matter, which is hard to degrade due to the content of lignin. The final
products of this step is glucose, glycerol, long fatty-acids chains and amino acids,
which are used as substrates for the following step of the AD process(N. B. Basnet,
2014).

2.2 Second step (Acidogenesis)

The following step in AD process is the acidogenesis, in which the substrate from the
hydrolytic step are used by anaerobic oxidizers and fermentative organism. This is
considered as the fastest step in the AD process. In this step product from the

hydrolysis are broken down into volatile fatty acids, alcohols, lactate, succinate and
hydrogen with by-products of carbon dioxide, ammonia and hydrogen sulphide. Mainly,
the anaerobic degradation pathway yields acetate, carbon dioxide and hydrogen, but
other fermentative intermediates are also produced. These products can be directly
as substrates for the methanogenic microorganisms(N. B. Basnet, 2014). When the
electron sinks appears in this step(see figure 1), it may accumulate lactate, ethanol,
propionate, butyrate and higher volatile fatty acids. This is a response from the
bacteria, due to increased hydrogen concentrations in the substrate. Many different
bacteria are involved in the two first steps of the AD process and therefore several
kinds of alcohols and organic acids usually are produced. The proportion and
concentration of the individual VFA's produced in the acidogenic step are determining
for the overall performance of the AD process. This is because acetic and butyric acids
are preferred precursors by the methanogenic bacteria for the methane production.
This step is also results in a higher energy yield for the microbes(W. Parawira, 2004).

2.3 Third step(Acetogenesis)

In this step the electron sinks produced in the acidogenesis are further converted by
the acetogenic bacteria. The acetogenic bacteria's conversion of the electron sinks
lactate, ethanol, propionate, butyrate and higher volatile fatty acids will result in the
formation of acetate, hydrogen and carbon dioxide. The acetogenic bacteria are very
slow growing and are sensitive to fluctuations in the organic loadings, and furthermore
they also are sensitive to environmental changes, which may result in long lagging
periods when they have to adjust to new environmental conditions. Also a low partial
pressure of hydrogen are required for the acetogenic bacteria to proceed with the
degradation. Because of that is a syntrophic association with the hydrogen consuming
methanogens necessary. The syntrophy refers to the independence of the hydrogen
consuming and hydrogen producing methanogenic microbes. The associations
between the acidogenic and acetogenic bacteria are required because such reactions
are thermodynamically unfavorable as indicated in table 1. The acetogenic bacteria
involves the butyrate- and valerate-degrading acetogenic bacteria for example
Syntrophomonas wolfei, propionate-degrading acetogenic bacteria, for example
Syntrophobacter wolinii and the homoacetogenic bacteria, that are responsiable for
converting the products from the previous step(acidogenisis) into acetic acid,
hydrogen and CO2(W. Parawira, 2004).

2.4 Fourth step (Methanogenesis)

In fourth step the methanogens breaks down the remaining acetic acid and hydrogen
into methane and carbon dioxide. It is only a limited number of substrates that are
able to be converted by the methanogens in the methanogenesis, such as, acetate,
H2, CO2, methanol and formate(N. B. Basnet, 2014). The CO2 and H2-consuming
methanogens reduces CO2 by using it as electron acceptor via formyl, methenyl and
methyl by an association with specific coenzymes, which finally result in the
production of methane(W. Parawira, 2004). It is the methylotropic methanogens, that
converts acetate and some other methyl group, which contain one carbon compound,
such as, methanol, into methane. Approximately 70% of the methane production
comes via the aceticlatic pathway, whereas about 30 % of the methane is produced

from the conversion of carbon dioxide and hydrogen(N.H. Basnet, 2014). Only few
species are capable of using the aceticlatic pathway to produce methane, whereas all
known methanogenic species are able to produce methane from carbon dioxide and
free hydrogen(W. Parawira, 2004). In the AD process hydrogen consuming
methanogens are among the fastest growing microbes and it is therefore likely that an
accumulation of hydrogen could occur. The minimum doubling time for the hydrogenic
microbes are 6 hours, while the aceticlatic microbes, which are among the slowest
growing, have a doubling time of 2.6 days. Even though hydrogen accumulation are
likely to occur, it is important for AD process, that the hydrogen pressure are kept low
to prevent overload or toxic inhibition of the microorganisms in the process(N.H.
Basnet, 2014). If the process becomes unstable for example, when the hydrogen
partial pressure increases, it would lead to an accumulation of VFA's, which will mean
the pH will decrease and inhibit the pH-sensitive microorganisms, which will result in
leading to failure of the methanogenic phase and thereby the whole AD process(W.
Parawira, 2004). Furthermore, the aceticlatic microorganisms has shown to be more
sensitive to changes in environmental conditions, whereas the hydrogen consuming
microorganisms has shown to be more resistant. Therefore the methanogenesis from
acetate is rate limiting in most cases(N.H. Basnet, 2014). The methanogens
microorganisms belong to the Archaea, which is a unique group of phylogenetically
different organism, that differ from the main group of microbes in the AD process,
which all are from the prokaryotic domain. Archaea are obligate anaerobes, that
means oxygen is toxic to these species. Archaea shares properties with both the
prokaryotes and eukaryotes microorganisms. They possess a prokaryotic cell structure
and organization, but share features with the eukaryotes like the presence of introns in
their genome, homologous sequences in tRNA and rRNA and a similar RNA polymerase
subunit organization. The methanogenic microbes include, the acetotrophic
methanogens, the hydrogenotrophic methanogens, and methylotrophs which convert
methyl compounds like methanol and methylamines. Of the many known
methanogenic species, only two,
Methanosarcina and Methanosaeta, are known to be able to grow from aceticlastic
reactions. Some of the acetate using methanogens are Methanosarcina barkeri and
Methanosaeta soenhngenii. Species of the Methanosaeta genera are known to be very
slow growing with doubling times of 4 to 9 days.

Table 1: Acetogenic, methanogenic and syntrophic reaction: Free energy, pH 7, 1 at, all
reactants and products at 1M concentration (W. Parawira, 2004)

2.5 Factors that affect anaerobic digestion process

During the anaerobic digestion process various problems may occur, but mainly the
complications lies with the methane yield and process instability. A number of factors
can result in a decreasing biogas production due to different factors, such as
temperature, pH, nutrient supply, organic loading rate (OLR) and hydraulic retention
time(HRT). Moreover factors of toxic and/or inhibitory nature can occur with to high
accumulation of different substrates such as, volatile fatty acids (VFA), ammonia,
sulfide, metal ions and to high levels of proteins in the waste. All these factors are the
main causes of process instability and in worst case process failure. Normally the main
problem is to maintain the correct balance between the acid forming and methane
forming microbes. The nature of these two groups of microorganisms differ in many
ways and works optimally in different environmental conditions, and further they also
have different nutrient needs, growth kinetics and physiologies. An imbalance in one
step of the AD process will affect the whole process and cause a reduction of the
desired output. The two first steps of the AD process (hydrolysis and acidogenesis) are
closely related to each other, as are the two last steps (acetogenesis and
methanogenesis). When degradation of the first step (the hydrolysis) runs to fast it
would result in an accumulation of acid, which would cause the pH to drop and thereby
inhibit the methanogenetic microorganisms. An accumulation of CO 2 would also result
in a drop in the pH, because when CO 2 reacts with the water is get dissolved and form
carbonic acids, which also would have a negative effect on the methanogenic
microorganisms. Naturally if the acid are accumulated in the acidogenesis, it would
also lead to a pH drop and affect the process. From this it is known, that indication of

process instability would be caused by increasing concentration of VFA's, pH drop and

a disruption of the steady state (N.H Basnet, 2014).
2.5.1 Temperature
The AD process is highly influenced by the temperature and the microorganisms are
divided into three different classification; the psychrophilic microorganisms (0-20 C),
the mesophilic microorganisms(20-42 C) and the thermophilic microorganisms(42-75
C)(W. Parawira, 2004). The temperature is directly connected to the hydraulic
retention time (HRT)(N.H. Basnet, 2014). The outcome of the AD process differs with
the temperature and different advantages and disadvantages apply to each of the
three temperature intervals. The psychrophilic temperature is normally not used due
to the slow nature of microbes growing and metabolisms. In the industry, mesophilic
and thermophilic temperature levels are normally applied, since this will result in the
most profitable outcome. The mesophilic conditions is currently the most used and
most well studied. The hydrolysis and acidogenesis steps are not affected much by the
temperature and can be operated in a wider range of temperatures than the
acetogenesis and methanogenesis. Different advantages and disadvantages is shown
in table 2(W. Parawira 2004).

Table 2:Comparison of mesophilic and thermophilic temperatures in the AD

process(W.Parawira, 2004)

2.5.2 pH value and optimal intervals

In the anaerobic digestion process the pH value has shown to be of great importance
for the performance of the system. The reason for this is, that each microbial group
operates in a specific range of pH to have an optimal growth(W. Parawira, 2004). The
pH value in a measurement of acidity and alkalinity in a solution measured in part per
million(ppm), and many factors in the AD process depend on a correct pH value to
perform on an optimal level(N.H. Basnet, 2014). For the methane producing
microorganisms the optimal pH range is 6.8 - 7.2, while for the acidic forming
microbes is lower an lies around a pH value of 6. Typically, in a one-step anaerobic
digestion, the pH is set to favor the methanogenic microorganisms to prevent an
dominance of the acid forming bacteria, which most likely would result in an

accumulation of VFA's. Even though maintaining the right pH is important it is only be

used as an indicator when treating waste with a low buffer capacity, such as
carbohydrate rich waste. Another important parameter in the AD process is the
alkalinity of the solution in the reactor. The measurement of alkalinity provides
information of the buffer capacity of the liquid solution. It is important that the buffer
capacity in the solution is high enough to neutralize VFA accumulations and keep the
pH in the range of 6.7 - 7.4 to keep a stable process. Compounds like hydrogen
sulphide, carbonic acid, dihydrogen phosphate and ammonia provide a good buffering
capacity in the useful region around pH 7. The capacity of the buffering compounds
depends on the composition substrate in the organic loading, which are injected into
the reactor(W. Parawira, 2004).
2.5.3 Volatile fatty acid(VFA) accumulation
Studies and research have recognized the accumulation of volatile fatty acids(VFA) to
be used as a good indicator of stress in the anaerobic digestion process systems. The
VFA's are produced during the acidogenesis and consist of intermediate compounds
like acetate, propionate, butyrate and lactate, which have carbon chains up to 6
carbon atoms. An instability of the AD process may lead to an accumulation of the
VFA's and as a result a drop in pH value of the aqueous solution. However, a drop the
pH may not always be the result of VFA accumulation due to the buffer capacity in
biomass. To detect an accumulation of VFA's the level of the concentration of VFA's
should exceed a certain level to cause a drop in pH value and at this point the process
may already be inhibited. The accumulation of VFA's is an important indicator, but it is
not recommended as an independent indicator for monitoring the process
stability(N.H. Basnet, 2014).
2.5.4 Ammonia
The degradation process of nitrogenous matter that produces ammonia generally
comes in the form of proteins and urea. The nitrogenous matter act with a significant
role in the anaerobic digestion process and is important for the formation of new
biomass. But even though nitrogen is essential for the growth of the microorganisms it
can also have a negative effect due to the products it forms like nitrite, nitrate,
ammonia, ammonium and nitrous oxide. As explained earlier, the methanogens are
the most intolerant microorganisms, that will show a decrease in growth because of
ammonia inhibition. Change in pH value, increase of maintenance of energy
requirement and specific enzyme reactions have all been proposed to be mechanisms
that changes as a result of ammonia inhibition. To control the ammonia inhibition and
enhance the biogas production different strategies have been proposed. These
strategies include adaptation of methanogens to higher ammoina concentration levels,
dilution of substrate, control of C:N ratio (carbon:nitrogen ratio) and pH control.
Chemical precipitation or air stripping can also be used as a method of removing the
ammonia from substrate and thereby control the concentration of ammonia (N.H.
Basnet, 2014).

2.5.5 Other factors (Sulphide, macro and micro nutrients (trace elements),
Light metals, Heavy metals)
Another important factor is the presence of inorganic sulphide in the waste, which is
treated. The sulphide is rapidly converted into a reduced form of sulphide(S -2) and
hydrogen sulphide(H2S) by the sulphide reducing bacteria(SRB). The SRB's and
methanogenic bacteria competes between different substrates in the AD process, such
as, acetate, butyrate, propionate and H 2. For the removal of sulphide/sulphate different
techniques are used like stripping, coagulation, precipitation and oxidation(N.H.
Basnet, 2014).
Nutrients is crucial for the growth and survival of all the microorganisms in the AD
process. The nutrients are divided up in two classifications, micro- and macro nutrients
and both are of equal importance. The micronutrients consist of the compounds iron,
nickel, cobalt, selenium, molybdenum and the macronutrients consist of carbon,
nitrogen, phosphorus and sulphur. A too high supply of nutrients will as well as an
insufficient supply result in inhibition of the whole AD process. A process, which has a
too low C/N ratio will result in a shortage of nitrogen supply for the microorganisms
and inhibit the synthesis. A too high C/N ratio could lead to an accumulation of
ammonia, which as explained earlier can lead to inhibition. Normally, the addition of
nutrients is not necessary while performing digestion of manure, since the manure
already contains these in the substrate(N.H. Basnet, 2014).
During the breakdown of the substrates different metals may be released. It may also
occur by the addition of compounds to the reactor for pH adjustment. The metals are
classified as either light metals or heavy metals. The common light metals ions
present in the digester are calcium, sodium, potassium, aluminum and magnesium.
The metal ions are required for the microbial growth and affect the specific growth
rate like any nutrient. The common heavy metals are cobalt, nickel, chromium, cupper,
zinc and cadmium. The heavy metals are as well required in a small amount to
activate enzymes and coenzymes, but too high concentration can be toxic for the
microbes(N.H. Basnet, 2014).

2.6 Optional parameters

2.6.1 Particle size
The size of the particles in the substrate is important, since it affect the production of
the biogas during the AD process. The way is affect the process is by surface area,
since large pieces of substrate will cause problems for the microorganisms in the
sense of it would take longer for them to degrade and may also cause blockage of the
digestion system. In contrast, smaller pieces of substrate will be more accessible for
the microbes and result in a better absorption of the substrate, which consequently
will result in more microbial activity and thus more biogas production(N.H. Basnet,
2014).
2.6.2 Water content

The water content also plays an important role for the growth and activity of the
microbes in the reactor. The water content determines the bacterial movement and
the extracellular enzyme activity within the reactor. A too low water content would
result in disabling the movement of the bacteria so they would not be able to reach
the substrate. The water content should be kept in a range of 60-95% depending on
the substrate composition, to achieve the optimal process conditions(N.H. Basnet,
2014).
2.6.3 Reactor type
There exist a range of different types of reactors are available for the treatment of
organic waste and depending on the desired result, and can roughly be divided into
batch or continuous processes. In a batch process all materials are added to the
system at once in the beginning of the experiment and first after process completion
the products are removed. In the continuous processes the substrate flows in and out
of the system in a continuous flow. The system is operated in a steady state with a
constant liquid flow rate and a constant gas production(N.H. Basnet, 2014).
2.6.4 Organic loading rate (OLR)
The organic loading rate describe the rate of which the reactor is supplied with
substrate. The OLR is an important operational parameter for having a successful
digestion process. It is important to have the correct organic loading rate to obtain the
maximum biogas yield. If the reactor is feed more organic matter than the
microorganisms can degrade it would result in an overload, which initially could
increase the biogas production but quickly would result in a drop of methane
production in the biogas.
2.6.5 Hydraulic retention time (HRT)
The hydraulic retention time (HRT) describes the average time that the biomass spent
in the continuous reactor before it comes out again. The HRT is important for the
biogas production, since the biomass has to stay in the reactor enough time to be fully
degraded into products. The HRT depends on the feed composition and the treatment
technology. A lower digestion rate would mean the microorganisms have a slower
doubling time and a higher HRT. In general the AD process works in a range of 20-40
days retention time and an organic loading rate of 1kg VS/m 3 per day. The HRT
depends on the OLR, but also on the composition of the substrate, type of digester
and environmental conditions. There should be enough time for the microbes to digest
and degrade the organic material. In a mesophilic environment the average HRT for
cattle manure is 12-25 days and for pig manure it is 10-20 days.

2.7 Biogas
2.7.1 Definition of biogas
Biogas is defined as a renewable clean form of energy, that could be a substitute for
conventional forms of energy (fossil fuels, oil, etc.), all of which are depleting at a
faster rate due to an increasing demand for energy (Yadvika, Santosh, et al., 2004).

The main components of biogas are methane(CH 4), carbon dioxide(CO2) and sulfuric
components (H2S) (Coelho et al., 2006). In general the biogas is composed of methane
(5565%), carbon dioxide (3545%), nitrogen (03%), hydrogen (01%), and hydrogen
sulfide (01%) (Anunputtikul and Rodtong, 2004). In table 3 the general composition of
biogas is presented. The composition can vary and in for example manure the
composition will depend on the diet the animal has digested.
Composition of biogas
_
Typical analysis
(% by volume)
Methane (CH4)
55-65
Carbon dioxide (CO2)
35-45
Hydrogen sulphide (H2S)
0-1
Nitrogen (N2)
0-3
Hydrogen (H2)
0-1
Oxygen (O2)
0-2
Ammonia (NH3)
0-1
Table 3: Composition of biogas (M. Balat & H. Balat, 2009)

2.7.2 Sources of biogas:

The production of biogas is usually applied in waste treatments from mainly sewage
sludge, agricultural waste(mostly animal manure) and in industrial organic waste
streams. The primary source for microorganisms used for the digestion of the organic
wastes comes from animal manure, mainly from cattle and pigs farms. A large amount
of manure is produces each year and alone in EU-27 a production of 1500 million
tons(Mt) is produces every year. Table 4 gives an overview of the production different
countries and as shown most countries could benefit from utilizing the manure in a
productive way(M. Balat and H. Balat, 2009).

Table 4: Overview of production of manure in EU-27 (M. Balat & H. Balat, 2009)

2.7.3 Uses of biogas:

Traditional biogas has been utilized for burning in internal combustion engines for the
production of electricity and heat energy. But the biogases potential in fuel cells could
increase the electricity efficiency especially in low scale with the benefit of diminishing
the nitrogen emissions to the atmosphere.
To use biogas in fuel transport it first needs to purified, usually into 97% methane,
which is known as CH4-enriched biogas. Once the biogas is purified into CH 4-enriched
3

biogas it has an energy value of 36.6 MJ/ m n

3

and will replace 1 liter of petrol. Normally 1 m n

of biogas will generate 0.57 m n

of

CH4-enriched biogas, which will replace 0.57 liter of petrol (M. Balat and H.
Balat(2009)).

3. The study
3.1 Objective of this study
The main objective of this study was to design a system that would monitor the biogas
yield from organic cattle manure. Furthermore, to get an understanding of the different
processes taking place in the experiment. To achieve the goal different parameters

were controlled and a set of analysis was performed to understand the efficiency of
the system. The analysis of the experiment were the following:

To perform TS/VS % analysis on both influent and effluent to observe how much
of the organic matter that was converted during the anaerobic digestion into
biogas.
Analysis of the methane content in the biogas produced by AD process by
performing a gas chromatography (GC) analysis.
Measurement of the pH, which will be significant for the stability of the AD
process.
Volatile fatty acid (VFA) analysis to study process stability and possible
inhibition.
Ammonium nitrogen analysis for further evaluation of process inhibition.
And Chemical oxygen demand (COD) analysis to evaluate the conversion of the
organic matter in the AD process.

3.2 Structure of this report

The report is systematically divided into four different main parts to make it more
comprehensible.

The first part(1-3) consist of an introduction, a definition of the biogas and

theory related to the anaerobic digestion process, objective of the study and
structure of the report.
The second part describes the materials and methods. This section provides
information about
the substrate, experimental set-up, analytic methods and the experiment, that
were used to accomplish the objective of the study.
The third part includes the results from all the experiments along with a
discussion in which the outcome of the different experiment are explained and
compared to previous findings within the same fields. After discussion of the
each experiment a conclusion of each experiment are given.

In the fourth part an overall conclusion of the whole experiment are presented based
on the finding in the study. Furthermore, recommendations for future work are also
presented in this section.

4. Materials and Method

Preparation/Setup of experiment:

4.1 Substrate
The cattle manure was obtained from an organic farm in Denmark. When the cattle
manure was first retrieved it contained a lot of fibers. Because the manure would be
too dense to be able to pass through the tubes of the reactor the initial step was to
filtrate it with a 3mm strainer, which resulted in a liquid form. And so was the
preparation of the substrate that was used in this experiment. The biomass was stored
in a freezer at -20C and the required amount was taken out and dried after necessity.

The reactor was filled up with 1,5L manure (cow) and 1,5L inoculum. The inoculum was
a digestate of a biogas plant obtained from Hashj biogas plant.

4.2 Reactor set-up (mixing and time settings):

After preparing the manure for the reactor the system was set up. The reactor was
cleaned from previous usage and connected to the reactor stirrer. Furthermore it was
connected to the pump in one end and to the gas-meter in the other end. Between the
gas-meter and the reactor was a flask that collected the manure effluent. In the
influent beaker (3L) a stirrer was set up to mix the injected manure, since new manure
was added frequently. The stirrer of the influent mixture was set to be stirred once a
day at 11:58 until 12:01.The reactor was also connected to a stirrer, which was
connected to a timer that was set to be stirred every 8 hour at 04:00, 12:00 and 20:00
o'clock in intervals of 5 minutes. Furthermore The pumping of the influent into the
reactor was controlled by a program, which was set to inject the substrate once a day
at 12:00 and was pumped for 8,47 second to inject 120mL manure. The 8,47 seconds
was determined when making the calibration curve for the pumps. Furthermore, a
water bath was connected to the reactor with a flow in and a flow out of the reactor.
This was to keep the reactor at the desired temperature (37C), and to keep further
control of the temperature in the reactor a thermometer was placed in the reactor.
When the set-up of the system was finished the calibration of the pump and gas-meter
could be performed.

4.3 Calibration of pump and gas-meter

4.3.1 Calibration of peristaltic pump
The calibration of the pump was performed with two different substrates, water and
cow manure respectively. A beaker (2000mL) with an outlet was connected to the
suction inlet of the pump from, which the substrate was filled and pumped into a
graduated cylinder, which was placed on the outlet of the pump. The time of the
pumping was programmed at the automatic meter in three different intervals (5sec,
10sec and 15sec). Each interval was done in triplicates to get an average of the pump
efficiency. Each pumping interval was initiated with a initial pumping to fill the tubes
before measuring the efficiency of the system. The volume of the liquid was measured
each time to account for the flow of the pump. The measurements of the calibrations
are shown is table 5 and 6 along with the calibration curves in figure 2 and 3.
Experiment

Time
(second)

1
2
3

5
10
15

Average
volume of
pumped water
(mL)
63 0
102.67 11.72
142 15.39

Required
volume
(mL)

Required
time
(second)

120

12.21

Table 5: Calibration for water

Water
150
f(x) = 7.9x + 23.56
R = 1

100
Volume (mL) of Water

50
0
4

10

12

14

16

Time (s)

Figure 2: Calibration graph for pump(water)

Experime
nt

Time
(second)

1
2
3

5
10
15

Average volume
of pumped
water (mL)
74.67 7.51
143 11.36
200 2

Required
volume
(mL)
120

Required
time
(second)
8.47

Table 6: Calibration for manure

Manure
250
200

f(x) = 12.53x + 13.89

R = 1

150
Volume (mL) of manure 100
50
0
4

10

12

Time (s)

Figure 3: Calibration graph for pump(manure)

14

16

4.3.2 Calibration of Gas-meter

The calibration of the volumetric gas-meter was adjusted so the count was locked in a
10mL intervals. The adjustments of the calibration was carried out by adding/removing
paraffin oil in the gas-meters U tube and by pushing air though the system using a
60mL syringe. When the volume of paraffin oil displaced by air was equal to one count
on the gas-meter for every 10mL of air, it meant the gas-meter was calibrated. This
was repeated in triplicates to ensure that the displacement of paraffin with air
continuously was the same.
4.2.3 Continuous Experiment:
The Continuous Stirred Tank Reactor (CSTR) was made of stainless steel and had a 5L
capacity
with the working volume of 3L, which was operated with a Hydraulic Retention Time
(HRT) of 25 days. The temperature of the reactor was maintained at 37C by
circulating hot water in the heating channel using a water bath. Furthermore the
temperature in the reactor was controlled by using a thermometer that was placed in
the reactor. The CSTR experiment was performed by feeding organic cattle manure.
The feeding was stirred 3 minutes before each feeding to achieve the most
homogenous substrate. The reactor was fed once a day (120mL) using a peristaltic
pump. The biogas produced in the reactor was registered by using a volumetric gasmeter, which was logging the gas production automatically in 10mL intervals. The
reactor was stirred 5 minutes 3 times a day.

Figure 4: Picture of the Continous stirred tank reactor system

4.2.3.1 HRT and OLR

The hydraulic retention time (HRT) was calculated by using the formula for HRT.

The formula for HRT is

HRT (days)=

Volume of (working) reactor (mL)

Influent flow rate(mL /day )

and was determined to be 25 days. As mentioned earlier this is a normal HRT for cattle
manure in mesophilic conditions. From this formula the flow rate of the influent, which
also is known as the organic loading rate (OLR) was also determined to be 120 mL per
day.

The formula for the OLR is

OLR(

Volume of (working )reactor (mL)

ml
)=
day
Hydraulic retention time(days) .

The HRT and OLR are both important parameters in the AD process. The HRT holds
important value for the economics of the AD process, where a low HRT leads to a
smaller reactor size, which is more industrially favorable. The HRT and OLR are directly
connected and these parameters depends on each other. A too high OLR can result in
an overload, which would cause a wash out of the biomass an ruin the production.

4.3 Analytic methods

4.3.1 Determination of TS/VS and measurement of pH
The total solids (TS) and volatile solids (VS) are a method that reviles the organic
matter content in the biomass. The total solids (TS) content, volatile solids (VS)
content and ash content were determined by the standard method given by AAU-CPH
University (SNJ, 2009), based on Standard Methods.
An amount of organic cattle manure was taken out and measured by weight in
crucibles, which also was measured by weight before the manure was placed into
them. The crucibles containing the manure were kept for 24 hours in a 105C oven
and then weight again to obtain the TS content. The crucibles were then moved to a
oven at 550C for 3 hours and weight again to obtain the amount of ashes of the
biomass. The VS was then determined by the difference in weight of the TS and the
ashes. The TS/VS content of the effluent was measured twice a week at Wednesdays
and Fridays, but since the influent probably did not change much in two days it was
only measured once a week at Wednesdays.
Besides measuring the TS/VS of the influent and effluent the pH value was also
measured. The pH measurement was performed with a pH-meter and served the
purpose of checking the acidity of the influent/effluent, which as explained earlier
holds important value for the production of biogas.
4.3.2 Ammonium
For the measurement of the ammonia content in the samples the first step was to
dilute with water. The capacity of the ammonia determination solution was in a range
of 1mg N-NH4/L - 12mg N-NH4/L. The estimated amount of ammonia in the samples
was 2 g N-NH4/L, which meant the solution had to be diluted in a ratio of 1:100. When

the samples were diluted they were put into the centrifuge at 10.000 rpm for 10
minutes. After centrifugation the supernatant was filtrated by using a high flow filter
with a pore size of 0,45 m. After filtration the samples was added to the analysis
solution in the Hach Lange kit LCK-305(1-12mg/L range). The mixing of the samples
with the Hach Lange kit was performed according to the guide in the kit. First a
component from the kit was mixed in the vials with the solution by shaking them
together. The samples were then added (2mL) and mixed again by shaking. The
samples were then allowed to react with the solution for 15min before they were
analyzed in the spectrophotometer.
4.3.3 Chemical oxygen demand (COD)
For the measurement of the chemical oxygen demand (COD) the samples were first
diluted. The COD solution had a capacity of 5-60 g/L of organic matter. According to
the VS calculation it was know that the samples needed to be diluted 1/5 for the
influents and 1/2 for the effluents. The COD kit (Hach Lange kit LCK-914) also included
a guide on how to perform the mixing of samples and solution. The mixing solution
was first shaken and then the diluted samples was added in a 2mL quantity and then
shaken again to mix the samples with the reactant. The solution allowed to react for
15 minutes and then put in the heating device (148C) for 2 hours. After the heating
the samples was cooled down for 10 minutes before they were analyzed in the
spectrophotometer.
4.3.4 Volatile fatty acids(VFA)
The samples for the volatile fatty acids (VFA) analysis were first diluted in a 1:10 ratio.
After dilution the samples was put in the centrifuge at 10.000 rpm for 10 minutes and
the supernatant from the samples was put into 2 mL eppendorf tubes. Into the
eppendorf tubes was added 200 l of phosphoric acid H 3PO4 at 17% concentration. The
solution in the eppendorf tubes was mixing using a 1mL syringe and the pH was then
checked to be below a pH value of 3. The samples were then filtrated with a 0,45 m
high flow filter and transferred into vials, which were sent to the gas chromatograph
analyzer. The VFA were analyzed using a gas chromatograph (PerkinElmer, Clarus
400) equipped with an Agilent HP-FFAP capillary column of 30-m length and 0.53 mm
i.d. followed by a flame ionization detector (FID). The carrier gas was nitrogen (13
ml/min). The temperatures of the detector and the injector were 230 and 240 C,
respectively.
4.3.5 Biogas measurement
The measurement of the amount of methane in biogas was performed with a gas
chromatograph (SRI GC model 310) with a thermal conductivity detector and a packed
column (Porapak-Q, length 6ft and
inner diameter 2.1 mm). The temperature of the gas chromatograph was 80C. A
standard of 30% CH4 and 70% of N2 was used for comparison with the actual
measurement of methane production. The result was analyzed by the use of the
software Peak simple version 3.9.3.

Figure 5: Gas chromatograph (GC) for measurement of biogas production and methane
composition

5. Results and discussion

5.1 Continuous reactor (CSTR)
During the weeks the reactor (CSTR) was operated there did not occur any problems
with blockage, fall in temperature or any other setbacks. All the systems setting
worked properly such as the settings of the timers stirring of influent and reactor, the
pumping programming and the reactor temperature, which was controlled by the
water bath. The reactor was checked every second day and the measurement of the
temperature, gas-meter counting's, the amount of effluent and pH values of
influent/effluents was observed and measured twice a week. In table 7 below is an
overview of the measurement presented.
Date
Temperature (C)
10-nov
14-nov
19-nov
21-nov
26-nov
28-nov
03-dec

37
37
37
37
37
37
37

Countings(10m
Effluent
L)
Time
(mL)
207
13:00
275
488
13:30
340
842
11:51
285
357
12:29
226
567
13:24
410
182
12:44
188
502
12:34
460

Table 7: Observations and measurements of the reactor

5.2 Biogas and Methane yields

In figure 6 below a graphically view of the biogas production compared to the methane
production is shown. As it can be seen that the methane production almost
consistently consist of half of the biogas produced. This was expected as a normal
composition of biogas is composed of 55-65% methane, 35-45% carbon dioxide, 0-3%
nitrogen, 0-1% hydrogen and 0-1% hydrogen sulfide (Anunputtikul and Rodtong,
2004). The maximum biogas yield was achieved after 19 days in the AD process and

544,73mL/gVS and the maximum methane yield was also achieved after 19 days in an
amount of 363,54mL/gVS, which was equal to 66,55% of the biogas. Other studies has
shown a methane yield in a range of 229-450 mL/CH 4/gVS, which places this methane
production somewhere in the good end of the middle(B.M. Salces et al, 2014).
As seen from the graph in figure 6 the production of the gasses are increasing right
until the last period.
As it is shown in the table 7 a change occur after the 26 of November. The reason for
the sudden drop in biogas production was caused by the input of a new filtrated
manure. After 2 weeks the initially used manure was used up and a portion of manure
was filtrated( 3mm) and mixed with remaining influent manure in the 3L beaker, that
was connected to the inlet of the reactor. This change in substrate also shows a
sudden change in the other measurement (COD, ammonia and VFA).
600
500
400
ml CH4/g VS added

300
200
100
0
0

10

15

20

25

30

Time (days)
Biogas yield

Methane yield

Figure 6: Development of biogas and methane gas during the period of the AD process

5.3 Methane content

In figure 7 a graphically evaluation of the methane production is describes during the
AD process in the reactor. Since the goal of this experiment was to evaluate the
accumulation of methane from the biomass(manure) it was comforting to see an
increasing production of the gas.
As expected in the experiment the methane yield increased during the first couple of
days as a result of the reactor fitting to the biomass. As the microbes adjusted the
methane yield increased and reached a maximum of 66,5%, that indicated a stable
process as the standard composition of a biogas contains 55-65% of
methane(Anunputtikul and Rodtong, 2004). This result indicates a successful
conversion and therefore also a successfully AD process.

Methane yield
80.00%
60.00%
Percentage of methane production

40.00%
20.00%
0.00%
0.00 20.00 40.00
Days (Time)

Figure 7: Yield of the methane production during the AD process

5.4 pH
For each sample the measurement of the gas and effluent produced by the AD system
were taken. Beside this, the pH value of each sample (influent/effluent) was also
measured. As shown in figure 8 the pH value is kept in a range of 7,5 - 7,9 which
indicates a stable process and as explained earlier this is also the favorable pH value
(6,0-8,0 pH range(N.H. Basnet, 2014)) with respect to the all the microbes involved in
the AD process. From figure 8 it is also observed that a little drop in the pH value occur
between the influents and effluent. This is also what was expected since a production
of VFA and other acid substances are produced during the AD process.
9
8.5
8
7.5
pH value

7
IN R1

6.5

OUT R1

6
5.5
5
0.00

5.00 10.00 15.00 20.00 25.00

Days (Time)

Figure 8: Measurement of pH during the period of the AD process

In figure 8 a graphical development of the pH value in the influent and effluent are
shown. Normally, in a optimal AD process the pH of the effluents should be higher than
in the influent. But since a small accumulation of acids was present it was expected to
be lower. But as it is also seen in figure 8 there is only a small variation between the
influent and effluent, which indicates the AD process was not inhibit significantly.

5.5 TS/VS measurements

From the digested manure TS/VS content was measured frequently to obtain an
understanding of the composition of the manure in both influent and effluent. The
TS/VS describes the amount of inorganic and organic material in the biomass, which
hold significant value for determination of other parameters in the AD process, such as
HRT and OLR. It was also important to know the TS/VS content for further
measurements of products of the AD process because of the parameters for the
different analyzing kits, such as the kits for COD and ammonia measurements. In
figure 9 and 10 the development of the TS/VS during the period of the AD process.

TS

mg/L

100

100

50

50

TS Removal (%)

0
0

10

15

20

25

Time (days)
TS IN R1

TS OUT R1

TS Removal R1 (%)

Figure 9: Development in Total solids(TS) in the influent and effluent during the AD process

VS

mg/L

VS Removal (%)
0

10

15

20

25

Time (days)
VS IN R1

VS OUT R1

VS REMOVAL R1 (%)

Figure 10: Development in Volatile solids(VS) in the influent and effluent during the AD
process

As seen in figure 9 the TS removal percentages are in the range of 17,45 - 74,05%,
but shows a decreasing value during the period of the AD process. Normally the TS
value in the influent should be the same during the whole period. As shown in the
graph the value of TS decreases, which indicates an non-homogenous influent. This
could be caused by improper stirring of the influent, a problem with the filtration of the
manure or an error when sampling. Around the 4 day of the process a break in the
graph is observed, which could be explained by an extra high conversion rate properly
caused by the composition of the particular manure injected that day or more properly
it may be a mistake in the measurement or in the equipment. The TS value was
measured with a range of 59,13 g/L to 39,42 g/L for the influent and in a range of
33,76 g/L to 13,04 g/L for the effluent. It was highly expected to see a lower value in
the effluent, since organic matter was converted in the AD process.
Figure 10 shows the VS content in and out of the reactor from the influent and effluent
respectively. Similar to the TS graph the VS follows the same graphically pattern and
ranges in amount 22,40 - 83,75% of VS removal. Since the optimal condition should be
to have the same amount of VS content going into the reactor, it is observed that this
is not the case and it can be concluded that the influent was non-homogenous. The
peak around the 4th day is as explained for TS properly a mistake in measurement or
the equipment, but maybe also be caused by variation in the injected manure
composition. Similar to the TS graph the graph for VS removal also shows a steady
decrease except around the 4th day. This is obvious, since the TS and VS are directly
connected as the amount of VS in a fraction of TS. The VS removal is a important
parameter as it gives an indication of the efficiency of the AD process. The more VS
that is removed from the biomass the more biomass is converted into biogas. The VS
value was measured with a range of 40,05 g/L to 25,04 g/L for the influents and in a
range of 21,21 g/L to 5,37 g/L in the effluents. This was also expected as organic
matter was converted into products and it can be seen that approximately half of the
organic matter was successfully converted into products.

5.6 Chemical oxygen demand (COD)

The total chemical oxygen demand (tCOD) was analyzed for the all the samples and as
shown below a graphically development was created. Like the VS removal the tCOD
describes the conversion of the organic matter in the biomass. As the biomass is
converted into products during the AD process the COD value decreases.

COD
100

100

80

50

60
mg/L

40

20
0

COD Removal (%)

-50
0

10

15

20

25

Time (days)
C OD IN R1

C OD OUT R1

C OD REMOVAL R1 (%)

Figure 11: Development of the Chemical oxygen demand(COD) during the AD process

In figure 11 a graphically development of the COD is described during the AD process.

The graph shows changing COD removal percentages, which properly is caused by a
mistake, especially around the 4th day where the measurements shows a higher value
in the effluent than in the influent. This should not be possible since the COD value is
not accumulated but in contrary reduced as organic matter gets oxidized. Beside the
mistake on the 4th day the graph shows a lower value in the effluent than in the
influent which is what is expected. The maximum rate of COD removal was measured
after 16 days of the experiment with a 66,62% of removal. From this it can be
concluded that organic matter has been successfully oxidized. During the last days of
the experiment the COD also has a sudden change, which can be explained by the
change in influencing manure.

5.7 Ammonia production

The figure 12 shows a development of the accumulation of the amount of ammonia in
the influent and effluent during the period of the AD process. As seen in figure 12 the
ammonia nitrogen is consistently higher in the effluent than in the influent. This
should not be the case in an optimal AD process.
The amount of ammonia nitrogen was measured for both the influent and effluent to
evaluate whether an accumulation and inhibition of the AD process were present. At
the graph the effluent measurements shows a low steady amount of ammonia
accumulation, which indicates a stable process without significant inhibition. But as
the graph also shows there was an accumulation of ammonia present during the AD
process, which is explained by the proteins in substrate(influent) broken down but not
fully further converted into biogas(W. Parawira, 2004).

Ammonia
1500
1000
mg/L

500
0
0

10

15

20

25

Time (days)
N-NH4 IN R1

N-NH4 OUT R1

Figure 12: Measurements of the Ammonia accumulation during the AD process

5.8 Volatile fatty acids (VFA's)

For measurement of volatile fatty acids (VFA) 6 different measurement of VFA's was
analyzed. The VFA's content of the AD process is known to be the most common
parameter for process stability.
In the figures below a graphically development of the analyzed results achieved from
the measurement of VFA's is presented for acetate, propionate, iso-butyrate, butyrate,
iso-valerate and valerate. The concentration of VFA's in the effluent is lower than the
influent, with exception made for propionate, iso-butyrate and butyrate, probably as a
result of an experimental errors when processing the samples.
For a stable reactor performance a range of concentration of acetate (1.0 g/L),
propionate (0.25 g/L), butyrate and valerate (0.05g /L) is considered as the favorable
(N.H. Basnet, 2014). For this experiment the removal percentages for the VFA's were
22,81 - 95,31% for acetate, 0 - 94,5% for propionate, 49,52 - 100% for iso-butyrate, 0 100% for butyrate, 27,83 - 100% for iso-valerate and 54,55 - 77,23% for valerate. The
removal percentages for all the compounds started out very low, as the microbes were
fitting to the substrate in the reactor, but as they adapted the removal increased
significantly.
For this experiment the measurement of acetate, propionate, iso-butyrate, butyrate,
iso-valerate and valerate during digestion (effluent samples) were in a range of
1,97g/L - 0,16g/L for acetate, 0,76g/L - 0,05g/L for propionate, 1,13g/L - 0g/L for isobutyrate, 0,14g/L - 0g/L for butyrate, 0,15g/L - 0g/L for iso-valerate and 0,04g/L 0,02g/L for valerate. Even though, some of the measurement turned out to be of little
higher concentrations than the most favorable concentration. But for most cases was
in the favorable range and as an overall the VFA removal was successfully removed.
The high concentrations of all the VFA's were only present in the beginning of the
experiment(first 10 days) and rapidly dropped below the limit as the microbes
adjusted to the substrate and the VFA's was almost fully removed. In the last period of
the AD process, when the initial manure was changed, a small accumulations of VFA's

appears, which can also be seen in the figure 12. This can also be seen in figure 8 that
describes the pH value, in which the pH values drop a little partly because of the
presence of VFA's. This can be further related to the biogas/methane production, which
also have a drop in production as seen in figure 7.
120.00

100.00

80.00
Acetate removal (%)

60.00

40.00

g/L

20.00
0.00

0
0

10

15

20

25

Time (Days)
Acetate Removal (%) R1

Acetate IN R1

Acetate OUT R1

Figure 13: Development of Acetate during the period of the AD process

120.00

100.00

0.8

80.00
Propionate removal (%)

0.6

60.00

0.4

40.00

g/L

0.2

20.00
0.00

0
0

10

15

20

25

Time (Days)
Propionate Removal (%) R1

Propionate IN R1

Propionate OUT R1

Figure 14: Development of Propionate during the period of the AD process

Iso-butyrate removal (%)

120.00

0.3

100.00

0.25

80.00

0.2

60.00

0.15

40.00

0.1

20.00

0.05

0.00

g/L

0
0

10

15

20

25

Time (Days)
Iso-butyrate Removal (%) R1

Iso-butyrate IN R1

Iso-butyrate OUT R1

Figure 15: Development of Iso-butyrate during the period of the AD process

120.00

0.4

100.00
0.3

80.00
Butyrate removal (%)

60.00

0.2

40.00
20.00

g/L

0.1

0.00
-20.00 0

10

15

20

25 0

Time (Days)
Butyrate Removal (%) R1

Butyrate IN R1

Butyrate OUT R1

Figure 16: Development of Butyrate during the period of the AD process

Iso-valerate removal (%)

120.00

0.3

100.00

0.25

80.00

0.2

60.00

0.15

40.00

0.1

20.00

0.05

0.00

g/L

0
0

10

15

20

25

Time (Days)
Iso-valerate Removal (%) R1

Iso-valerate IN R1

Iso-valerate OUT R1

Figure 17: Development of Iso-valerate during the period of the AD process

120.00

0.1

100.00

0.08

80.00
Valerate removal (%)

0.06

60.00

0.04

40.00

0.02

20.00
0.00

0
0

10

15

20

25

Time (Days)
Valerate Removal (%) R1

Valerate IN R1

Valerate OUT R1

Figure 18: Development of Valerate during the period of the AD process

g/L

To sum up the development of all the VFA's a graph was made for all the different
VFA's. In figure 19 below the overview of all VFA are presented. It can be seen that the
percentages of removal is low in the beginning of the experiment but after a period of
10 days rapidly increases to almost being fully converted into methane. This also fits
nicely with all the graphs for the individual VFA compounds, which is acetate,
propionate, iso-butyrate, butyrate, iso-valerate and valerate. The total VFA removal
ranged from 21,76 - 95,32% of removal in amount ranging from 2,83 - 0,25g/L. Also
here was the concentration of the total VFA only high in the beginning of the
experiment, and as the process proceeded the values rapidly dropped.
120.00

10

100.00

80.00
VFA's removal (%)

60.00

40.00
20.00

0.00

0
0

10

15

20

g/L

25

Time (Days)
VFA Removal (%) R1

VFA IN R1

VFA OUT R1

Figure 19: Development of VFA during the period of the AD process

6. Conclusion
The production of biogas is a reflection of the whole AD process. And as explained not
all parameters of the process was the most favorable, which mean there is room for
improvements. The yield of the methane reached 66,55% of the biogas with a
maximum after 19 days at an amount of 363,54mL/gVS. This is considered as a
successful production as production in general produces 229-450mL/gVS of methane.
As it is seen from the results of the TS/VS removal the percentages of removal
decreases as the experiment proceeds. This could have lead to a lower production of
biogas/methane as less organic matter gets converted. But in contrast the methane
production increased, which can be explained by the microbes adjusting to the
environment and becoming more efficient converting the biomass into gas. The
highest measured TS/VS removal was measured after 4 days in percentages of 74,01%
and 83,75%, respectively.
The COD measurement showed variations in the results, but like the TS/VS it showed
a lower value in the effluent than in the influent, which indicates that organic matter
was successfully converted. The TS/VS measurement were constantly decreasing,

whereas the COD measurements was increasing and deceasing. At some point(after
16 days) the COD showed increase with a 66,62% removal, whereas the TS/VS
removal were decreasing at a percentage 30,92% and 36,10%, respectively.
The ammonia results turned out be higher in the effluent than in the influent by
approximately 2mg/L. This might have caused the small drop in the pH value, which
only was a drop of about 0,2 in pH.
From analyzing the VFA content in the effluent of the system, that with a almost fully
conversion of VFA's into methane, it can be concluded that the no significant inhibition
was caused by VFA accumulation. The VFA removal ranged from 21,76 - 95,32%, but
showed only low removal values in the beginning(first 10 days) of the experiment.
Beside small inhibition problems in the AD system, the overall process can be
concluded to be successful as a production of biogas, and furthermore a production of
methane were obtained from the experiment. The inhibitions was caused by the input
of new manure, which clearly seen in the biogas/methane production graph.
Furthermore a small accumulation of ammonia also appeared, that may have caused
the small drop in pH, which may also has inhibited the process a little. The goal of this
study was to design a system, which would anaerobically digest the injected biomass.
Moreover, was the goal to get an insight in the microbial processes and reactions
taken place within the process. These goals was both obtained and lays ground for a
further understanding and investigation of how this process could be enhanced.

7. Recommendations for further work

Anaerobically digestion of cattle manure has shown to be productive and a possibility
of making a greener world in the sense of better utilization of wastes. This has already
implanted in many countries in a successful way. However, there is still many
improvement that can be made. Studies has shown that pretreatment and codigestate can help improving and increasing the productivity of the AD process. An
example is enzymatic pretreatment, which has showed to improve the methane yields
from digested manure(N.H. Basnet, 2014). Although cattle manure has proven to hold
good qualities as substrate for anaerobic digestion with a high buffer capacity and also
providing all the suitable microorganisms , the high content of water and its content of
recalcitrant organic matter(the fibers) results in a low methane yield per biomass(B.M.
Salces, 2014). This makes it a poor substrate on its own, but if added with other
substrates like sugar roots, catch crops or other high carbon content organic material.
If these material contains cellulose, lignin or other compound, which are difficult to
degrade, they would need a pretreatment to be successfully in an AD process.

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