establish an anaerobic batch system by using guideline vdi 4630 and determine the biogas yield of different substrates in food processing villages

VNU UNIVERSITY OF SCIENCE - TECHNICAL UNIVERSITY OF DRESDEN




®

Bui Dieu Linh


ESTABLISH AN ANAEROBIC BATCH SYSTEM
BY USING GUIDELINE VDI 4630
AND DETERMINE THE BIOGAS YIELD OF
DIFFERENT SUBSTRATES
IN FOOD PROCESSING VILLAGES




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Hanoi - 2011

VNU UNIVERSITY OF SCIENCE - TECHNICAL UNIVERSITY OF DRESDEN



®




Bui Dieu Linh


ESTABLISH AN ANAEROBIC BATCH SYSTEM

BY USING GUIDELINE VDI 4630
AND DETERMINE THE BIOGAS YIELD OF
DIFFERENT SUBSTRATES
IN FOOD PROCESSING VILLAGES

Major: Waste Management and Contaminated Site Treatment
Code:



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SUPERVISORS:
ASSOC. PROF. DR. NGUYEN THI DIEM TRANG
PROF. DR. RER. NAT. DR. H.C. PETER WERNER




Hanoi - 2011


I
Topic
Establish an anaerobic batch system by using guideline VDI 4630 and
determine the biogas yield of different substrates in food processing villages
Task
The first task is to establish an anaerobic batch system in Environmental Chemistry
laboratory, Faculty of Chemistry, Hanoi University of Natural Sciences, Hanoi
National University by using German guideline - VDI 4630. With this system,
different inocula from various sources around Hanoi will be assessed their quality to
choose the good one for next experiments with substrates. Reference substrate is
sodium acetate. Substrates as wastes (cassava residues, rice residues, water

hyacinth, pig manure) in Dai Lam village - a craft village of wine production and
pig breeding will be sampled, prepared and fermented in free-oxygen environment.
The processing parameters are biogas production, biogas composition (CH
4
and
CO
2
), pH - value, TS (total solids), VS (volatile solids), COD (chemical oxygen
demand). The biogas yield (per amount of substrate, per VS of inoculum, per COD
of substrate) and the degradability of different substrates will be evaluated.
Objective
The aim of this thesis is from learning the methods of guideline VDI 4630 to
establish in practice an anaerobic batch system in the conditions of a Vietnamese
laboratory. Then it is to control this system to investigate the quality of inocula, the
fermentability/ the biogas potential/ the specific biogas activity of different organic
wastes from food processing and livestock of a Vietnamese craft village. It is also
close tied to the one objective of education and technology transfer of INHAND
project (project funded by the Federal Ministry for Education and Research of
Germany - BMBF; with the project coordinator Institute of Waste Management and
Contaminated Site Treatment, Dresden University of Technology) about Integrated
management of water, wastewater, waste and energy in craft villages in Vietnam.

Contents

II

Contents
Contents ………………………………………………………………. II
Abbreviations ………………………………………………………………. IV
List of figures …………………………………………………………… V

List of tables .…………………………………………………………… VII
Acknowledgements.……………………………………………………… IX
1. Introduction………………………………………………………………. 1
2. Theoretical Basics………………………………………………………… 4
2.1. Basics of Anaerobic Digestion……………………………………… 4
2.1.1. The four stages of decomposition………………………………… 4
2.1.2. Influence Factors of Digestion……………………………………. 7
2.1.3. Biogas potential of different substrates…………………………… 13
2.2. End products of fermentation…………………………………………. 15
3. Materials and methods…………………………………………………… 17
3.1. VDI survey about batchtests for biogas yield determination…………. 17
3.1.1. Scope and purpose of Fermentation batchtests…………………… 17
3.1.2. Methods of VDI 4630 in detecting the biogas yield……………… 17
3.1.3. Method of VDI 4630 in approaching the batchtests………………. 19
3.2. Analysis method ……………………………………………………… 19
3.2.1. Sampling………………………………………………………… 19
3.2.2. Sample preparation……………………………………………… 23
3.2.3. Determination of parameters: TS, VS, COD, and CH
4
and CO
2

Contents

III
content in biogas………………………………………………… 25
3.3. Experimental procedure………………………………………………. 30
3.3.1. Test condition and batch apparatus……………………………… 30
3.3.2. Method to calculate the biogas production, the biogas yield, the
biogas composition, the degree of degradation…………………… 37

4. Evaluation and discussion of the batch experiments………………… 41
4.1. Activity potential of different inocula………………………………… 41
4.2. Biogas production and biogas composition of different substrates… 47
4.2.1. Biogas yield (ml
N
/gVS
inoculum
, ml
N
/gCOD
substrate
, ml
N
/ g substrate)…
47
4.2.1.1. Biogas yield of trial 6 with inoculum 5……………………… 47
4.2.1.2. Biogas yield of trial 7 with inoculum 5……………………… 50
4.2.1.3. Biogas yield review of different substrates with inoculum 5… 54
4.2.1.4. Biogas yield review of different substrates with inoculum 6… 56
4.2.2. Comparison of biogas yields with theory and literature………… 61
4.2.3. Biogas composition (CH
4
and CO
2
content)……………………… 64
4.3. Residue of biogas production…………………………………………. 66
4.3.1. Degree of degradation of volatile solids………………………… 66
4.3.2. Degree of degradation of total solids…………………………… 69
4.3.3. Degree of degradation of COD total……………………………… 71
4.4. Error analyis……………………………………… 77

5. Conclusion………………… 80
References………………… 83
Annex- Data records I
Statement under oath XI
Abbreviations

IV

Abbreviations
abs absolute
B Blank sample
C/ CR Cassava residues sample
COD Chemical oxygen demand
Inoc. Inoculum
l
N
standard liter, volume under normal condition
ml
N
standard milliliter, volume under normal condition
P/ PM Pig manure sample
R Reference sample
RR Rice residues sample
SA Sodium acetate
SLR Sludge loading rate or sludge loading ratio
TS Total solids
VS Volatile solids
W/ WH Water hyacinth sample

List of Figures

V

List of Figures
Figure 2-1: Four stages of anaerobic degradation (WEILAND, 2003) 4
Figure 3-1: Test apparatus according to DIN EN ISO 11734: Gas volume
measurement with a gas pressure measurement instrument
(VDI 4630) …………………………………………………
18
Figure 3-2: Gas volume measurement with a gas pressure measurement
instrument (S. Meier, 2009) 18
Figure 3-3: Map showing position of Dai Lam village………………… 21
Figure 3-4: Water hyacinth at main sewer… 21
Figure 3-5: Rice residues and cassava residues sampling at households… 22
Figure 3-6: Pig manure at a small swine farm of a household ……………. 22
Figure 3-7: Homogenizing cassava residues by blender…………………… 23
Figure 3-8: Homogenizing water hyacinth by blender…………………… 24
Figure 3-9: Filling the bottles by weighing method……………………… 30
Figure 3-10: Closing bottles with silicone stoppers 31
Figure 3-11: Creating the vacuum inside the bottles 31
Figure 3-12: Lovibond conditioning cabinet 32
Figure 3-13: K2000 Pressure table - EXTECH manometer 407910 32
Figure 3-14: MultiLabP4 33
Figure 3-15: MEMMERT drying cabinet 33
Figure 3-16: TDW muffle furnace 33
List of Figures

VI
Figure 3-17: Lovibond ET 108 and MERK spectroquant TR320 block

digesters 31
Figure 3-18: PI 722N instrument in measuring COD 34
Figure 3-19: Gas chromatograph Shimadzu GC-2010 34
Figure 4-1: Biogas yield without blank on different inocula [ml
N
Biogas/ g
COD sodium acetate] 44
Figure 4-2: Biogas yield with blank on different inocula [ml
N
Biogas/ g
COD sodium acetate] 45
Figure 4-3: Biogas yield with blank on inoculum 5 of trial 6 [ml
N
Biogas/ g
VS Inoculum] 48
Figure 4-4: Biogas yield without blank on inoculum 5 of trial 6 [ml
N

Biogas/ g COD substrate]
49
Figure 4-5: Biogas yield with blank on inoculum 5 of trial 7 [ml
N
Biogas/ g
VS Inoculum] 52
Figure 4-6: Biogas yield without blank on inoculum 5 of trial 7 [ml
N

Biogas/ g COD substrate]
52
Figure 4-7: Biogas yield with blank on inoculum 5 of trial 6-7 [ml

N

Biogas/ g VS Inoculum] 54
Figure 4-8: Biogas yield without blank on inoculum 5 of trial 6-7 [ml
N

Biogas/ g COD substrate] 55
Figure 4-9: Biogas yield with blank on inoculum 6 of trial 9 [ml
N
Biogas/ g
VS Inoculum] 58
Figure 4-10: Biogas yield with blank on inoculum 6 of trial 9 [ml
N
Biogas/ g
COD substrate] 59
Figure 4-11: The corrected biogas composition of different samples 65
List of Tables

VII

List of Tables
Table 2-1: Average composition of biogas (FNR, 2005) 6
Table 2-2: Factors influencing the anaerobic degradation (WEILAND,
2001) modified 8
Table 2-3: Micro-nutrients for the anaerobic degradation 10
Table 2-4: Inhibitory concentrations of various elements (WESSELAK, 2009) 11
Table 2-5: Inhibitory concentrations of various heavy metals
(WESSELAK, 2009) 12
Table 2-6: Biogas composition and yield of different groups of
substances (Biogas Guide 2006) 13

Table 2-7: Properties of renewable resources (FNR, 2005) 14
Table 3-1: Description about sources of six inocula 20
Table 3-2: Description of the batchtests for investigating the quality of six
inoculums 36
Table 3-3: Description of the batchtests for investigating the biogas
potential of different substrates
37
Table 4-1: Description of the experimental approach and parameters in
trials investigating quality of six inocula 42
Table 4-2: Summary of biogas yield without blank and SLR on different
inocula
46
Table 4-3: Description of the experimental approach and parameters in trial 6 47
Table 4-4: Summary of biogas yield and SLR of trial 6 49
Table 4-5: Description of the experimental approach and parameters in trial 7 51
List of Tables

VIII
Table 4-6: Summary of biogas yield and SLR of trial 7 53
Table 4-7: Description of the experimental approach and parameters in trial 9 56
Table 4-8. Summary of biogas yield and SLR of trial 9 60
Table 4-9: Comparison of biogas yields with theory 62
Table 4-10: Comparison biogas yields of water hyacinth samples with literature. 63
Table 4-11: Biogas composition of different samples in trials 6, 7, 9 64
Table 4-12: Volatile solids of different samples 66
Table 4-13: Degree of degradation of volatile solids 68
Table 4-14: Total solids content of different samples 69
Table 4-15: Degree of degradation of total solids 70
Table 4-16: Chemical oxygen demand of different samples 72
Table 4-17: Degree of degradation of COD 73

Table 4-18: Degree of degradation of COD 75
Table 4-19: Degree of degradation of COD 76






Acknowledgements

IX

Acknowledgements
Special thanks I would like to say to my supervisor Prof. Dr. Nguyen Thi
Diem Trang for giving me the opportunity to make this thesis at Faculty of
Chemistry, Hanoi University of Science in the Double-Degree-Program between the
Hanoi National University and the Dresden University of Technology. Many thanks
also to my German supervisor Prof. Dr. Peter Werner.
Special thanks also to Msc. Tran Thi Nguyet (from Institute of Waste
Management and Contaminated Site Treatment, Dresden University of Technology)
and Dipl Ing. Sebastian Meier (from Institute for Water Quality and Waste
Management, Leibniz University Hannover) – team members of INHAND project
who enthusiastically supervised me during the course of this thesis.
Thanks to the help in inocula collecting of Prof. Dr. Nguyen Viet Anh, from
the Centre for Environmental Engineering of Towns and Industrial areas (CEETIA),
Hanoi University of Civil Engineering and the help in gas composition analysis of
Mr. Thai Ha Vinh, from Monitoring and Environmental Analysis Department,
Monitoring and Analysis of Working Environment Station, National Institute of
Labor Protection. Thanks also to the help in sample preparation of the staff (Mrs.
Nguyen Thi Diem Huong, etc) in the laboratory of Monitoring Centre for Natural

Resources and Environment of Bac Ninh province.
Thanks to the students of the Environmental Chemistry laboratory (Thang,
Cham, Thao, Lan, etc) for their friendship with me during the experimental period
time.
Special thanks also to my family members for their great support in all the
time of this master course!
Introduction
Bui Dieu Linh
1
1. Introduction
In view of the challenges in the global and regional energy markets and facing the
needs for global climate protection as well as the increasing efforts in developing
rural areas, the utilization of renewable energies has to be advanced. In this context,
the usage of biogas plays an exceptional role as it is a highly flexible fuel with
respect to a wide range of input substrates. Biogas also offers various options in
providing and using energy on a local, regional and global scale.
Vietnam is no exception to the trend biogas applications to replace fossil energy.
Biogas production has been studied and applied long in Vietnam, but until 2003, it
became the real attention when the Ministry of Agriculture and Rural Development
collaborated with the Netherlands Development Organization to build renewable
energy project, the “Support Project to the Biogas Program for the Animal
Husbandry Sector in Some Provinces of Vietnam.”. By the end of 2008, the project
has supported construction of over 56,000 household biogas plants, provided
training for 500 provincial and district technicians, 700 biogas mason teams, and
organized thousands of promotion workshops and trainings for biogas users. Up to
now, the project has become a national program “Biogas program for the Animal
Husbandry Sector of Vietnam” that supports the implementation of household
biogas digesters throughout Vietnam. By the end of 2012, the team aims to
complete 164,000 biogas plants (including large plants) in 58 provinces throughout
Vietnam and gain 1.5-3 tradable emission rights per year per digester

(1)
. This
program raises the effective movement of production technology and application of
biological energy, reduces environmental pollution in rural areas, creates jobs and
improves living standard for Vietnamese farmers and minimizes the greenhouse
effects. Along with that, the World Bank is also currently funding an array of
manure management demonstration projects in Vietnam, ranging from small
household -scale systems to village-scale systems
(2)
.

(1)


(2)

Introduction
Bui Dieu Linh
2
The studies
(3)
of biological energy have been simultaneous developing, as well as
Prof. Dr. Bui Van Ga – director of Da Nang University and his colleagues at the
Research Center for Environmental Protection (Da Nang University) with research
focused on biogas refining and motor applications, or the teams from Hanoi
Polytechnic University and Ho Chi Minh Polytechnic University with research on
completed conversion of gasoline and diesel to biogas fuel running engines
Therefore, Vietnamese farmers now are utilizing the produced biogas not only in
cooking, lighting, heating but also generating electricity for their own farms by their
own capital. The only pity is that the farmers can not sell surplus generated power

into general grid. Besides, the Bio-Gas Project in the framework of the "Go Green -
Green Journey" by Toyota Vietnam (TMV) in collaboration with the General
Department of Environment and Ministry of Education and Training has been
installing 500 generator powered by bio-gas in households, farms, small and
medium enterprises from 2008 up to 2012.
Based on those, biological energy has developed at a larger scale, focusing on the
factories, farms, producing biogas from waste water of tapioca starch plants,
seafood processing plants, rubber production plants, etc. Up to now, Vietnam has
achieved 11 CDM
(4)
Projects
(5)
in validation, with the credit period from 2009/2010
to the end of 2020/2030, in the field of waste/ waste water treatment and biogas
capture (mainly in tapioca starch sector) with the parties involved as limited
companies, corporations from Japan(4), France(1), Netherlands (5), Germany(1).


(3)


(4)
CDM (Clean development mechanism) is the one of three market-based mechanisms in Kyoto Protocol
which requires developed countries to reduce green house gas (GHG) emission. CDM enables emission
reduction projects, like biogas capture projects, in developing countries to get CER (certified emission
reduction) credits which can be traded with developed countries to meet part of their emission reduction
commitments.

(5)

Introduction
Bui Dieu Linh
3
The development of biogas production and application with all the domestic
potential and overseas support has been bringing great benefits for Vietnam’s
economic development and environmental improvements. For that, contributing to
the studies of biogas in Vietnam, this thesis focused on the method of German
guideline (VDI 4630) to assess the potential of recovery of organic waste in a
Vietnamese food processing village by biogas production. Firstly, it is to establish
an anaerobic batch system for biogas production in the laboratory in the North of
Vietnam. Then, experiments with this system were set up to investigate the quality
of inocula from different sources around Hanoi. And with chosen inocula, substrates
as organic waste of Dai Lam village - a wine production and pig breeding village in
Bac Ninh province were assessed. Parameters of process were pH, temperature, TS,
VS, and COD. Biogas potential and the fermentability of these substrates were
evaluated and interpreted by using VDI- 4630 guideline.







Theoretical Basics
Bui Dieu Linh
4
2. Theoretical Basics
2.1. Basics of Anaerobic Digestion
2.1.1. The four stages of decomposition
The methane fermentation process comprises four stages that organic materials

being degraded by anaerobic microorganisms in the absence of oxygen. The
degradation of high molecular weight starting substrates such as carbohydrates, fats
and protein via low molecular weight compounds (fatty acids and alcohols) to
methane, which is the main component of biogas. Figure 1 shows a degradation
process which is described below:

Figure 2-1: Four stages of anaerobic degradation (WEILAND, 2003)
In the first stage (hydrolysis), facultative anaerobic microorganisms hydrolyze the
biomass with the aid of extracellular enzymes (exoenzymes
(1)
) to low molecular
weight components. Here, the organic substance is transferred by addition or
temporary storage of water molecules in a dissolved form. The facultative anaerobic

(1)
Exoenzymes are synthesized by the bacteria-consuming enzymes, which serve to break up nutrients.

Stage 1
Hydrolytic phase
Stage 2
Acidogenic Phase
Stage 3
Acetogenenic Phase

Stage 4
Methanogenic Phase

Hydrolytic Bacteria
Fermentative
Bacteria

Fermentative
Bacteria
Acetogenic
Bacteria
Methanogenic
Bacteria
Biomass
Polysacharides
Proteins
Fats
Sugars
Amino acids
Fatty acids
Carboxylic
acids
Alcohols

Acetate


H
2
/ CO
2



Biogas
CH
4

/ CO
2

Theoretical Basics
Bui Dieu Linh
5
bacteria which were created to consume the remaining dissolved oxygen present
with low required redox potential (less than - 330 mV) are the obligatory anaerobic
bacteria (methanogens). The rate of hydrolysis is determined by the substrate
composition, temperature, pH value (optimum pH = 6, (BISCHOFSBERGER, et
al., 2005)), and the concentration of microorganisms. For example, sugar and
hemicelluloses are hydrolyzed very well, whereas pectin and lignin are difficult to
hydrolyze. Therefore, the higher the proportion of shares in the easily cleavable
substrate is, the faster the digestion process of the bacterial cells run. The hydrolysis
is therefore generally regarded as rate-limiting step of anaerobic (and aerobic)
degradation (ROEDIGER et al., 1990).
Subsequently, the hydrolysis products in the second stage of fermentation
(acidification) fermented intracellularly by the bacteria, the bacteria excrete mainly
carboxylic acids, ethanol, ammonia, hydrogen sulphide and carbon dioxide. The
acidogenic bacteria here have a large tolerance, so that the pH value for the
acidification of carbohydrates may fall below 4.0 (BISCHOFSBERGER et al.,
2005). The degradation of the hydrolysis and acidification can be inhibited by its
own metabolic products. One hand, the bacteria produce only insufficiently
dissolved substrate exoenzymes, on the other hand, these exoenzymes are sensitive
to pH values less than 6.5 (ROEDIGER et al., 1990).
Because methanogenic bacteria produce methane only from acetic acid, hydrogen
and carbon dioxide in the third stage (acetogenesis), the carboxylic acid and alcohol
of the second stage can be converted to acetic acid, carbon dioxide, water and
hydrogen. Ammonia is mineralized to ammonium.
In the fourth stage (methanogenesis) methanogenic bacteria convert acetic acid,

hydrogen and carbon dioxide to methane. The methane formation is carried out for
70% of the acetic acid degradation (also called acetate-degrading, see equation 2-1)
and about 30% of the conversion of carbon dioxide and hydrogen (ROEDIGER et
al., 1990). See equation 2-2.
CH
3
-COO
-
+ H
2
O Æ CH
4
+ HCO
3
-
(Equation 2-1)
Theoretical Basics
Bui Dieu Linh
6
4 H
2
+ CO
2
Æ CH
4
+ 2 H
2
O (Equation 2-2)
The methane formation can also occur with a few methanogenic bacteria from
formic acid, methanol and methyl amine (CH

3
NH
3
) (BISCHOFSBERGER et al.,
2005). For methanogenesis, the optimum pH is 7 and the optimum temperature is at
35 ° C and 55 ° C (ROEDIGER et al., 1990).
There are 13 species of methane-forming bacteria, of which 11 species have their
optimum working environment in the mesophilic range and one type in the
thermophilic environment. Another type is thermo-tolerant and can operate in
mesophilic and thermophilic range.
Throughout the fermentation process, hydrogen inhibits the acetogenic bacteria. The
close symbiosis between the acetogenic bacteria and the methanogenic bacteria is
an important prerequisite, since methanogenic bacteria prevent the inhibitory effects
of excess hydrogen, by converting the hydrogen with carbon dioxide to methane
(see equation 2-2). The symbiosis should be adjusted by the mixing process in the
reactor with registered shear forces, in order not to destroy it.
After a trouble-free decomposition of organic substrates, there is an energy-rich gas
mixture which consists mainly of methane and carbon dioxide. An average biogas
composition is given in Table 2-1.
Table 2-1: Average composition of biogas (FNR, 2005)
Ingredients Concentration
Methane(CH
4
) 50 – 75 Vol %
Carbon dioxide (CO
2
) 25 – 45 Vol %
Water (H
2
O) 2 – 7 Vol % (20 – 40 °C)

Hydrogen sulfide (H
2
S) 20 – 20000 ppm
Nitrogen (N
2
) < 2 Vol %
Oxygen (O
2
) < 2 Vol %
Hydrogen (H
2
) < 1 Vol %
Theoretical Basics
Bui Dieu Linh
7
2.1.2. Influence Factors of Digestion
Anaerobic degradation processes involve a number of factors that determine the growth
and activity of microorganisms significantly. We distinguish effects due to operational
factors (temperature, mixing, residence time, organic loading rate) and the influence of
substrate components (C: N: P ratio, pH - value, concentration of inhibitory substances
and nutrients, etc). Some of these factors influence each other and are mutually
dependent. Selected factors are briefly explained in the following sections.
Mixing
Optimum mixing of the reactor is set to achieve sufficient contact between bacteria
and fermentation substrate. Temperature and concentration differences are
compensated in the fermenter by mixing. Without or with insufficient mixing, the
mass of bacteria would decrease and easily demolished at the bottom to rise up the
fermentation and form a floating layer through which the gas outlet would escape
more difficult. On the contrary, with vigorous mixing, there is the danger to those
living in small communities, symbiosis acetogenic and methanogenic bacteria

communities are affected by too high shear forces (GRONAU et al., 2007).
Temperature
The maximum metabolic rate and the maximum growth rate of organism is a
function of the temperature. For example, different optimal temperature possesses
involved anaerobic microorganisms. Therefore, a classification of organisms is
based on temperature ranges, micro-organisms can be distinguished with
psychrophilic (< 25°C), mesophilic (25 - 40°C) and thermophilic (45 - 60°C)
(BISCHOFSBERGER et al., 2005). In industrial processes there are mainly
mesophilic or thermophilic microorganisms. The choice of the temperature range
depends on factors such as the substrate properties, the hygiene degree, or the
residence. Since mesophilic fermentation processes have a greater diversity of
methanogens (12 species), they are shown against thermophilic fermentation
processes (only two species of methane bacteria) as a stable. It should be noted that
Theoretical Basics
Bui Dieu Linh
8
the species Methanosarcina barkeri(2) are both the mesophilic and thermophilic
methane bacteria (DORNACK, 2001). Since methane bacteria are very temperature-
sensitive, and are adversely affected even at low temperature, a constant
temperature control of anaerobic reactors is necessary (ROEDIGER et al., 1990).
With extreme temperature fluctuations, the bacteria respond with lower metabolic
rates and reproductive performance (DORNACK, 2001).
pH value
The pH - value arises from the metabolic products of naturally present
microorganisms and the different self-regulating buffer system. The predominant
organisms in the degradation phase species have different optimal pH. During
hydrolysis and acid-forming, bacteria find their pH optimum at 4.5 to 6.3, methane
bacteria thrive best in a very narrow pH - 6.8 to 7.5 (FNR, 2005). A shift in the pH -
value out of the narrow range from 6.6 to 8.0 can be rough interference to substrate
degradation, result in the bacterial growth rate and metabolism (FAULSTICH,

1995). For example, the stable of hydrolyzing substrates can be inhibited by an
excess of lime, because the required optimum pH is not created by the buffering
action of the lime. The consequence is lower gas yields.
Factors influencing the anaerobic degradation are summarized in table 2-2 below.
Table 2-2: Factors influencing the anaerobic degradation (WEILAND, 2001) modified
Hydrolysis / acidification Methane fermentation
pH 5.2 – 6.3 6.7 – 7.5
C: N – ratio 10 – 45 20 – 30
Solids < 40% TS < 30% TS
Nutrient Requirements
- C: N: P: S
500 : 15 : 5 : 3 600 : 15 : 5 : 3
Red-ox potential +400 – (-)300 mV < -250 mV
Trace elements No special Claims essential: Ni, Co, Mo, Se

(2)
 />
Theoretical Basics
Bui Dieu Linh
9
Ratio of nutrient elements
The C/N ratio and the C/N/P/S ratio can be used as an indicator of the optimal
supply of carbon and nutrients. If the C/N ratio is too high then the used carbon is
not completely eliminated and therefore the methane potential not fully exploited.
This ratio is shifted to nitrogen, there is the danger of the formation of ammonia
which is toxic in too high on the bacteria. The C/N/P/S ratio is used to draw
conclusions about the nutrient supply of the bacteria.
Trace elements
For the growth and survival of the bacteria, trace elements that do not exceed
certain levels should also be required (see Table 2-3). The trace elements must

present in dissolved form. The natural sources of entry for the trace elements in
agricultural biogas plants are agricultural soils, etc, which can be distinguished
between natural and anthropogenic influences such as pedogenesis fertilizer,
emissions from industry and traffic.
The incorporation of trace elements in plant and animal feces is based on feeding
and additives and other input sources. Lacking of trace elements, so as at too high
concentrations, a delay occurs that can lead to the stoppage of the anaerobic
degradation.






Theoretical Basics
Bui Dieu Linh
10
Table 2-3: Micro-nutrients for the anaerobic degradation
Concentration (mg/l organic matter)
Element
according to SAHM3 according to KLOSS
(4)
According to SEYFRIED
(5)
Fe
not analyzed (n.a.) 10 – 200 1 – 10
Ni
0.006 0.5 – 30 0.005 – 0.5
Co
0.06 0.5 – 20 0.003 – 0.06

Mo
0.05 0.1 – 0.35 0.005 – 0.05
Se
0.008 0.1 – 0.35 0.08
W
n.a. 0.1 – 0.35 0.1 – 0.4
Cr
0.005 – 50 n.a. n.a.
Mn
0.005 – 50 n.a. n.a.
Pb
0.02 – 200 n.a. n.a.

Inhibiting and toxic substances
During anaerobic degradation, a variety of substances in high enough
concentrations have inhibitory effects. The inhibitors can be distinguished
according to their origin such as inhibitors in the degradation by addition of
substrate or inhibitors as intermediates. Inhibitors which are introduced by adding
substrate, include substances which were used for cleaning and disinfection
purposes or for the treatment of animal diseases. Also, herbicides, salts or heavy
metals, including essential heavy metals can be toxic in high enough concentrations
(WESSELAK et al., 2009).

(3)
SAHM, H. 1981: Required concentrations for optimal anaerobic degradation
(4)
KLOSS, R. 1986: Physico-chemical minimum requirements for the biogas process
(5)
SEYFRIED, C.F. 1990: Low concentrations of dissolved trace elements in the anaerobic reactor


Theoretical Basics
Bui Dieu Linh
11
Table 2-4: Inhibitory concentrations of various elements (WESSELAK, 2009)
Inhibitor Limiting concentration (mg/l)
Sodium
from 3000
Calcium
from 2800
Magnesium
from 2400
Ammonium
from 2700
Ammonia
from 150
Sulfur
from 50 (H
2
S)
from 100 (S
2-
)
from 160 (Na
2
S)
up to 600 mg/l Na
2
S (in adapted cultures)
up to 1000 mg/l H
2

S (in adapted cultures)
Iso butyric
from 50

Because bacteria can adapt well to different populations of individual inhibitors,
very different maximum allowable concentrations of toxic substances are published
in the literature. The different threshold concentrations also result from the
interaction between the ingredients, the type of fermenter and the operation of the
plant (KALTSCHMITT et al., 2009).
The addition of substrates with high proportions of carbohydrates and fats may lead
to the inhibition process, when the hydrolytic and acid-forming bacteria break down
organic matter faster than the acetogenic and methanogenic bacteria convert the
resulting acids into biogas. This process can be determined by the ratio of the
organic acids to alkalinity (VOA
(6)
/ TAC
(7)
). Values below 0.4 indicate a stable

(6)
Volatile organic acids

(7)
Total anorganic carbon
Theoretical Basics
Bui Dieu Linh
12
process and interpret values greater than 0.8 indicate a process of inhibition
(MAEHNERT, 2007). The degradation of proteins forms end products of acetic
acid and ammonia, and there may be an increase of hydrogen sulfide. If there are

acetic acid, ammonia and hydrogen sulphide in dissolved, undissociated form and in
high concentrations, these substances act as cytotoxins and the methane formation is
inhibited. Methane-forming bacteria take on only the acid in undissociated form
(see Eq. 2-3). The acidity constant of acetic acid is given as pKa = 4.75, ie in the pH
range of methanogenic bacteria between 6.8 to 7.5 acetic acid is largely dissociated
acetate as before (BRANDENBURG, 2008). Concentrations of 50 mg / l H
2
S are
considered directly inhibiting process, while the failure of essential trace elements
as insoluble sulfides is possible the indirect inhibition.
CH
3
COOH ' CH
3
COO
-
+ H
+
(Equation 2-3)
The inhibitory effect of ammonia increases with higher pH values and higher
temperatures, because the equilibrium between ammonium and ammonia is strongly
pH and temperature dependent. For the anaerobic fermentation, a total
concentration of NH
3
/NH
3
+ H
+
greater than 3 g / l is critical (MAEHNERT, 2007).
Table 2-5: Inhibitory concentrations of various heavy metals

(WESSELAK, 2009)
Heavy metals
(ions)
Limiting
concentration (mg/l)
Heavy metals
(carbonates)
Limiting concentration
(mg/l)
Ni
from 10
Zn
from 160
Cu
from 40
Cu
from 170
Cr
from 130
Cd
from 180
Pb
from 340
Cr
3+

from 530
Zn
from 400
Fe

from 1750

Theoretical Basics
Bui Dieu Linh
13
2.1.3. Biogas potential of different substrates
The anaerobic degradation of glucose to carbon dioxide and methane can be
described approximately by (Eq. 2-4).
C
6
H
12
O
6
Æ 3CH
4
+ 3CO
2
(Equation 2-4)
Biomass is not only from carbohydrates and biogas consist not only carbon dioxide
and methane. If the exact composition of the fermentation substrate was determined
in terms of the number of carbon, oxygen and hydrogen atoms, then BUSWELLS
and MUELLER (BUSWELL, et al., 1952) simplified the equation
C
n
H
a
O
b
+ (n– a/4 – b/2)H

2
O Æ (n/2 + a/8 – b/4) CO
2
+ (n/2 – a/8 + b/4) CH
4

(Equation 2-5)
C
n
H
a
O
b
N
c
S
d
+ (n– a/4 –b/2 + 3c/4 + d/2)H
2
O Æ
(n/2 – a/8 + b/4 + 3c/8 + d/4) CO
2
+ (n/2 + a/8 – b/4 – 3c/8 – d/4) CH
4
+ c NH
3
+ d H
2
S
(Equation 2-6)

(Equation after BOYLE (BOYLE, 1976))
Table 2-6: Biogas composition and yield of different groups of substances
(Biogas Guide 2006)
Groups of
substances
Molecular
Weight (g/mol)
Mole fractions in
Biogas (mol)
Molar volume in
Biogas (g/mol)
Possible biogas
yield (l/g)
Carbohydrates
C
6
H
12
O
6
or
(CH
2
O)
n

180 3 CO
2
+ 3 CH
4

134,4 0,746
Fats
C
16
H
32
O
2

256 4,5 CO
2
+ 11,5 CH
4
358,4 1,39
Proteins
C
13
H
25
O
7

(N
3
S)
367 5,125 CO
2
+ 7,857 CH
4
291 0,79

Vegetable
Biomass
C
38
H
60
O
26

932 18 CO
2
+ 20 CH
4
851 0,91
Theoretical Basics
Bui Dieu Linh
14
The equation for BUSWELLS and BOYLE (Eq. 2-6) is used to determine the
respective proportions of carbon dioxide and methane in the biogas. The calculation of
the biogas potential of the three main groups of carbohydrates, fats and proteins after
BUSWELLS equation provides the results in Table 2-6. Under the full anaerobic
reduction a higher methane content in biogas in fats and proteins is expected than the
carbohydrates. The average methane content in biogas is 50 - 75 vol.%.
Table 2-7: Properties of renewable resources (FNR, 2005)
Substrate
TS
(8)

[%]
VS

(9)

[%]
N
[% TS]
Biogas yield
[m³/t VS]
CH
4
-content
[Vol %]
Cattle
manure
8 - 11 75 - 82 2,6 – 6,7 200 – 500 60
Pig manure approx. 7 75 - 86 6 - 18 300 – 700 60 – 70
Corn silage 20 - 35 85 - 95 1,1 - 2 450 – 700 50 – 55
Rye - whole
crop silage
30 - 35 92 - 98 4,0 550 – 680 approx. 55
Grass silage 25 - 50 70 - 95 3,5 - 6,9 550 – 620 54 – 55
Sugar beet 23 90 - 95 2,6 800 – 860 53 – 54
Beet leaf 16 75 - 80 0,2 - 0,4 550 – 600 54 – 55

Renewable resources offer in terms of the fermentation substrate, a number of
favorable properties. Such are, for example, a high organic content, the amount of
nutrients and trace elements, and a low impurity content. Some selected substrates
and their properties are listed in Table 2-7. The very high specific gas yield of
renewable resources as compared to cattle or pig manure makes sense with
fermentation biogas plants. Furthermore, the substrates brought nutrient and trace
element content into the fermenter for optimal performance of important

microorganisms.

(8)
TS: Total Solids

(9)
VS: Volatile Solids