application of various pretreatment methods to enhance biogas potential of waste chicken feathers
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This thesis comprises 30 ECTS credits and is a compulsory part in the Master of Science with a Major in Environmental Engineering, 120 ECTS credits
No. 8/2009
Application of Various Pretreatment Methods to Enhance Biogas Potential
of Waste Chicken Feathers
Azar Khorshidi Kashani
Application of various pretreatment methods to Enhance Biogas Potential of Waste Chicken feathers
AZAR KHORSHIDI KASHANI, az_khorshidi@yahoo.com
Master thesis
Subject Category: Environmental Engineering
University College of Borås School of Engineering SE-501 90 BORÅS Telephone +46 033 435 4640
Examiner: Ilona Sárvári Horváth
Supervisor,name: Ilona Sárvári Horváth
Supervisor,address: University of Borås, School of Engineering
SE-501 90 Borås
Date: 2009-09-21
Keywords: Chicken feathers, Keratin protein, Biogas potential, Lime treatment, Enzymatic treatment, Chemo-enzymatic treatment
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ACKNOWLEDGEMENTS
This thesis work has been performed at Department of Chemical Engineering at Faculty
of Engineering, University of Borås, Sweden. I would like to thank the supervisor of the
thesis Dr. Ilona Saravari Horvath for her guidance and assistance during this thesis work.
I'm also grateful to Dr. Dag Henriksson, Gergely Forgacs, Jonas Hanson and all other
enthusiastic people involved in helping me and supporting this work at the Department of Chemical Engineering.
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ABSTRACT Chicken feathers are the most abundant keratinous biomass in the world. Disposal of the
huge and increasing volume of waste feathers presents as a major concern for poultry
industry. On the other hand, energy and material recovery of this valuable protein source
is an important issue for organic solid waste treatment and bioenergy generation.
Anaerobic digestion is an environmentally and economically promising alternative
process for biogas production of waste feathers.
In this study in order to enhance the methane potential of batch anaerobic digestion of
chicken feathers this waste was treated by various kinds of pretreatments including
thermal, thermo-chemical, enzymatic, thermo-enzymatic and chemo-enzymatic methods.
Also the effect of different treatment conditions on the methane yield was investigated.
As a whole, thermo-chemical pretreatment with lime (Ca(OH)2) rendered the most
significant effect on enhancement of the chicken feathers methane potential. In particular
lime treated triplicate samples under treatment condition of 40g TS feather/l water, 0.1g
Ca (OH)2 /g TS feather, 100°C and 30 min produced the highest amount of methane (an
average maximum volume of 480 Nml/g VS, which is about 96.8% of the theoretical
methane potential of protein), during 50 days of anaerobic incubation. Increasing the
operational parameters such as feather concentration, lime loading, temperature and
reaction time improved the feathers solublisation resulting in a higher soluble chemical
oxygen demand (SCOD) concentration of the samples but inserted negative impacts on
the anaerobic digestion performance. Although other pretreatment methods improved the
SCOD concentrations of the feathers too, compared to the lime treatment those methods
didn’t show considerable effects on the enhancement of methane yield from the chicken
feathers. Thermo-enzymatic, enzymatic, and thermal pretreated triplicate samples
produced an average maximum of 185 Nml/g VS, 154 Nml/g VS, and 143 Nml/g VS
(37.3%, 31%, 28.8% of the theoretical methane potential) respectively, during 33 days of
50 days of anaerobic incubation. Especially, chemo-enzymatic pretreated sample showed
negative methane potential of only 41 Nml/g VS, i.e. 8% of the theoretical methane
potential. Consequently, lime pretreatment under the above recommended conditions can
be suggested for hydrolysis of chicken feathers to achieve significant enhancement of its
methane potential.
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TABLE OF CONTENTS
Acknowledgement ......……………………………………………………………………3
Abstract………….………………………………………………………………………...4
Table of content…………………………………………………………………………5-6
List of tables……………………………………………………………………………….7
List of figures……………………………………………………………………………8-9
Abbreviations…………………………………………………………………………….10 Chapter1. Introduction….………………………………………………………………..11
1.1 Background…………………………………………………………………………..11
1.1.1 Renewable Energy for a Sustainable Future……………………………………11-12
1.1.2 Biomass…………………………………….…………………………………..13-14
1.2. Biogas………………………...……………………………………………………..14
1.2.1 Biogas applications and benefits……………………………………………….14-16 1.2.2 Anaerobic Digestion Process……………………………………..…………….16-17
1.2.3 Environmental and operational parameters…………………………………….17-19
Chapter 2: Chicken Feather……………………………………………………………...20
2.1 Chicken Feather Waste Treatment……………………………………………….20-21
2.2 Anaerobic Digestion Process of Solid Poultry Slaughterhouse Waste…………..21-23
2.3 Specific Characteristic of Chicken Feathers and Keratin Protein …………………..23
2.4 Pretreatments methods for hydrolysis of poultry feathers…………………………...24
2.4.1 Hydrothermal pretreatments……………………………………………………26-26
2.4.2 Biological pretreatment………………………………………………………...26-27
2.4.3 Chemical-Biological pretreatment……………………………………………........27
2.5 Research Objectives…………………………………………………………….........28
Chapter 3: Materials and methods……………………………………………………….29
3.1 Equipments and apparatus………………………………….......................................28
3.2 Materials…………………………………………………...................................29-30
3.3 Methods………………………………………………………………………………30
3.3.1. Preparation of Waste Chicken Feathers…………………………………………...30
3.3.2. Inoculum ………………………………………………………….........................30
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3.3.3. Total Solids (TS%) and Volatile Solids (VS%) measurement………………...30-32
3.4 Pretreatment Methods………………………………………………………………..32
3.4.1 Thermo-Chemical Lime Pretreatment (Experiments 1, 2).. …………………...32-34
3.4.2 Biological Pretreatments (Experiment 3)…..…………………………………..34-35
3.5 Anaerobic Digestion Processes………………………………………………………36
3.5.1 Batch digestion process set-up for pretreated samples……................................36-38
Chapter 4: Calculation and Data Treatment………………………………………….39-40
Chapter 5: Results and discussion……………………………………………………….41
5.1 Effect of lime treatment on SCOD concentration (Exp.1, 2)…………………… 41-44
5.2 Effect of lime treatment on Anaerobic digestion performance (Exp. 1, 2) ….......44-51
5.3 Effect of biological treatments on SCOD concentration (Exp.3)...……………... 51-52
5.4 Effect of biological treatments on anaerobic digestion performance (Exp.3)……52-54
5.5 Conclusion……………………………………………………………………......55-56
5.6 Future work………………………………………………………………….........56-57
Reference……………………………………………………………………………..58-66
Appendices……………………………………………………………………………….67
Appendix A: Data Figures and Tables for the Results of TS% & VS% Measurement…67
Appendix B:
B.1 Data Figures and Tables for the Results of GC Measurements for Lime Treated
Samples………………………………………………………………………………..68-69
B.2 Data Figures and Tables for the Results of GC measurements for Biological and Combined Biological treated samples………………………………………………..69-70
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LIST OF TABLES Table page
1. Typical composition of biogas…………………………………………………...14
2. Some biogas equivalents………………………………………………………....14
3. Temperature ranges and optima for various anaerobic populations……………..17
4. Calculation of general theoretical methane potential for fat, protein and
carbohydrate using average chemical formulas ………........................................23
5. Thermo-chemical treated samples and treatment conditions…………………….33
6. Results of SCOD and average maximum methane yields of triplicate lime
treated samples of Exp.1, during 50 days of incubation…………………………44
7. Results of SCOD and average maximum methane yield of triplicate lime treated
samples 4 and 5 of Exp. 1, during 15 days of incubation, (liquid phase).……….47
8. Results of SCOD and average maximum methane yields of
triplicate lime treated samples of Exp.2 , during 15 days of incubation………..48
9. Results of SCOD and average maximum methane yields of
triplicate thermal, enzymatic and combined enzymatic samples
of Exp.3, during 50 days of incubation……………………………………….53
10. The recorded weighs during TS measurement and the results
for the TS% of the samples……………………………………………………...67
11. The recorded weighs during VS measurement and the results for the VS%
of the samples. ………………………………………………………………..67
12. Results of average methane yields for lime treated samples containing 40g TS
feather/l liquid, during 50 days incubation under thermophilic condition…...68-69
13. Results of average maximum methane yields for thermal, enzymatic
and combined enzymatic samples under thermophilic condition,
during 50 days incubation…………………………………………………….69-70
8
LIST OF FIGURES Figure Page
1. Global energy consumption from 1965 to 2030………………………………..11
2. Global energy consumption by fuel type from 1965 to 2030……………………..12
3. Potential pathway for biofuel production………………………………………13
4. Deployment of anaerobic digestion in the EU and the world…………………..15
5. Degradation of carbon in the anaerobic digestion process described by
4 steps: Hydrolysis, Acidogenesis, Acetogenesis and methanogenesis………...17
6. Chicken feathers image………………………………………………………......20
7. Degradation pathways during anaerobic digestion.……………………………...22
8. Keratin molecular structure………………………………………………………24
9. Protein hydrolysis during thermo-chemical treatment…………………………...25
10. COD Reactor with Direct Reading Spectrophotometer for SCOD
measurement of pre-treated samples……………………………………………..35
11. Samples maintained in the incubator at 55°C for anaerobic digestion process….37
12. Autosystem Gas Chromatograph with TCD for measurement of produced
methane and carbon-dioxide..................................................................................38
13. Results of SCOD measurement for lime treated samples containing 40gTS F/l
initial concentration (Exp.1) under various treatment conditions………………..41
14. Results of SCOD measurement for lime treated samples containing 40gTS F/l
initial concentration (Exp.1) with higher lime loadings at 120°C and for 2h....... 42
15. Results of SCOD measurement for lime treated samples containing 100gTS F/l
initial concentration (Exp. 2) under various treatment conditions.……………... 43
16. Results of SCOD measurement for lime treated samples of Exp. 2 with higher
lime loadings at 120°C and for 2h..……………...................................................43
17. Results of SCOD measurement for lime treated samples of Exp.1
selected for anaerobic digestion process………………………………………..44
18. Average maximum methane production curves for triplicate lime
treated samples of Exp.1, during 50 days of incubation………………………...45
19. Average maximum methane production curves for triplicate lime treated
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samples 4 and 5 of Exp. 1, during 50 days of incubation (liquid phase)… …...47
20. Average maximum methane production curves for triplicate lime
treated samples of Exp. 2, during 15 days of incubation………………………..49
21. Enzymatic, chemo-enzymatic and thermo-enzymatic pretreated samples
(Exp. 3)…………………………………………………………………………..51
22. Results of SCOD measurement for enzymatic and combined
enzymatic samples of Exp.3 containing …….…………………………………...52
23. Average maximum methane production curves for triplicate
thermal, enzymatic and combined enzymatic treated samples of Exp.3,
during 50 days of incubation……………………………………………………...53
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ABBREVIATIONS
F…………………………...Feathers
VS…………………………Volatile Solids
TS…………………………Total Solids
Std.………………………..Standard
R…………………………..Ideal Gas Constant
P(atm)…………………….Atmospheric Pressure
T…………………………..Temperature
K…………………………..Kelvin (Standard Temperature Unit)
COD………………………Chemical oxygen demand
SCOD……………………. Soluble Chemical Oxygen Demand AD…………………………Anaerobic Digestion
SSOFMSW………………..Source-Sorted Organic Fraction of Municipal Solid Waste OECD……………………..Countries that are members of the Organization for
Economic Co-operation and Development
TCD………………………..Thermal Conductivity Detector
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Chapter 1: Introduction
1.1 Background
The Rapid growth of the world population combined with concomitant economic
development exerts drastic increase in global energy demand. World energy consumption
is projected to expand by 50 percent from 2005 to 2030. Although in general developed
(OECD) countries consume the most energy, demand for energy is increasing faster in
developing and emerging (Non-OECD) countries, resulted from their robust economic
progress and expanding populations. Fig. 1 illustrates world total energy consumption
and contribution of OECD and Non-OECD in world energy consumption from 1965 to
2030 [1].
Fig. 1. Global energy consumption from 1965 to 2030, [1].
1.1.1 Renewable Energy for a Sustainable Future
Currently the global mix of fuels comes from fossil (78%), renewable (18%) and nuclear
(4%) energy sources [2]. Fig. 2 demonstrates the global energy consumption by fuel
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type from 1965 to 2030. As indicated in Fig.2 conventional fossil fuels appropriate the
significant and highest portion of the global fuel consumption, likewise. However, these
fuels are non-renewable and finite resources releasing the highest amount of carbon
dioxide (CO2) and other greenhouse gases into the atmosphere and realized as the main
cause of global warming and climate change [3]. Fossil fuel combustion accounts for
62% of the global warming potential of all anthropogenic greenhouse gases [1].
Fig. 2.Global energy consumption by fuel type from 1965 to 2030, [1].
The above rising concerns beside the economical considerations such as increasing oil
price, reducing reliance on fossil fuels and worldwide potential economic development,
are potent incentives to incite global efforts and investments in promotion of sustainable
renewable and clean energy resources and technologies. Renewable energies including
geothermal, solar, wind, biomass, hydropower, ocean thermal, wave action, and tidal
action are utilizing in many energy fields such as electricity generation, transportation
fuels, industrial processes, heating , cooling and process steam. Although renewables
currently provide less than 10% of the world's energy, renewable energy sources have the
potential to exceed current global energy demands even with existing technologies [1].
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1.1.2 Biomass
Biomass as a major source of renewable energy accounts for about 14% of primary
energy consumption, and following oil, coal and natural gas is the fourth world-wide
energy resource. The world production of biomass is estimated at 146 billion metric tons
a year, mostly coming from wild plant growth [4,5].
The major resources of biomass are agricultural crops, plants and forestry residues,
organic components of municipal and industrial wastes and even the fumes from
landfills. Biomass can be converted to non-solid fuels form including liquid biofuel
(bioethanol and biodiesel) and gaseous biofuels (biogas, syngas,…). Fig. 3 Indicated
potential pathway for biofuel production.
Fig. 3. Potential pathway for biofuel production [6].
1.2 Biogas Biogas is the gaseous biofuel made through anaerobic digestion process or fermentation
of organic fraction of biomaterials. Biogas can be also captured from landfills. Almost all
kinds of organic and biodegradable materials such as municipal and industrial organic
wastes, sludge from sewage treatment plants and process water from the food industry,
energy crops and crop residues can be utilized as the resources for biogas production.
Biogas comprises from methane (CH4), carbon dioxide (CO2) and trace amounts of some
other components. Table 1 shows the typical composition of biogas.
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compound Percentage (%)
Methane, CH4 50-75
Carbon dioxide, CO2 25-50
Nitrogen, N2 0-10
Hydrogen, H2 0-1
Hydrogen sulfide, H2S 0-3
Oxygen, O2 0-2
Table 1.Typical composition of biogas [7]. 1.2.1 Biogas applications and benefits
Biogas is an environmentally friendly, clean, cheap and versatile fuel. Anaerobic
digestion substrate for biogas production can be obtained from almost all kinds of bio-
wastes and non-food based biomasses. Therefore biogas has no potential negative impact
on food chain products and prices, changes in land use and deforestation [8,9]. Combustion of biogas has less dangerous and neutral carbon dioxide emissions [10].
Moreover methane is a potent greenhouse gas, and hence capturing and burning it helps
environment from the global warming point of view. Biogas has a wide range of
applications e.g. in transportation, electricity production, cooking, space heating, water
heating and industrial process heating or even as a renewable feedstock to produce
hydrogen [8]. Table 2 shows some typical applications for one cubic meter of biogas.
Application 1m3 biogas equivalent
Lighting Cooking Fuel replacement Shaft power Electricity generation
equal to 60 -100 watt bulb for 6 hours can cook 3 meals for a family of 5 - 6 0.7 kg of petrol can run a one horse power motor for 2 hours can generate 1.25 kilowatt hours of electricity
Table 2. Some biogas equivalents [11,12].
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Europe seems to be the leader in the global production and use of biogas [10]. Fig. 4 shows
the deployment of anaerobic digestion in the EU and the world from 1995 to 2010.
Fig. 4. Deployment of anaerobic digestion in the EU and the world [9].
UK studies have shown that biogas is much cleaner and more efficient than biofuels for
use in transport. According to an EU well-to-wheel study of more than 70 different (fossil
and renewable) fuels and energy paths, biogas is the cleanest and most climate-neutral
transport fuel of all [10]. “A natural gas vehicle reduces CO2 over a gasoline car by 20-
30%. A car running on bio-methane reduces CO2 on a well-to-wheel basis by more than
100%over a petroleum-fuelled car [8].”
Biogas along with fossil natural gas is currently fuelling over 800,000 cars, truck and
buses in Europe and nearly 8 million vehicles worldwide [8]. Compressed biogas
is becoming widely used in vehicles in Sweden, Switzerland and Germany [7]. “Sweden
has led the world in the usage of biogas in transportation since 1996. Biogas producers
are operating a fleet of city buses in Sweden. Strong government support is important, it
includes 30 percent investment support, zero tax, reduced income tax for company car
users, and no congestion fees in the capital city of Stockholm [1].”
Among biomass sub-sectors, solid biomas (72.5% biomass electricity) has increased by
an avarage of 5.8% per year from 1997 to 2007. However, growth in biogas electricity
has been much more considerable (an average of + 12.9% per year) [14]. European
Biogas electricity production in 2006 was 17272GWh per year, of which 7338GWh was
produced by Germany alone [15]. Beside biogas, anaerobic digestion produces high
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nutrient content fertilizers to use in agriculture [11]. Furthermore, biogas production has
no geographical limitations and doesn’t need sophisticated technology [16]. Biogas can
be produced even by a very basic construction using mostly used materials providing a
few simple design rules are followed. Moreover, biogas production is possible in small
scale sites, to obtain for outlying areas [17]. Accordingly, biogas is a 100% sustainable
fuel playing also a very important role in environmental friendly waste management and
organic waste disposal [8].
1.2.2 Anaerobic Digestion Process
Anaerobic digestion process for generation biogas occurs in four steps: Hydrolysis,
Acidogenesis, Acetogenesis and Metanogenesis. In the first step, hydrolysis, insoluble
and complex organic compounds such as lipids, polysaccharides, proteins, fats, nucleic
acids, etc. transform into soluble and simpler organic materials such as amino acids,
sugars and fatty acids by strict anaerobic hydrolytic bacteria [18,19]. In the acidogenesis
step obligate and facultative anaerobic group of bacteria (acidogens) ferments and
breakdown soluble products from the first step into acetic acid, hydrogen, carbon dioxide,
some volatile fatty acids (VFA) and alcohols. In the third step, acetogenesis, long chain
fatty acids and volatile fatty acids will be converted to acetate, hydrogen and carbon
dioxide by obligate hydrogen-producing acetogens [18]. Finally in the methanogenesis
step strict anaerobic methanogens convert acetic acid, hydrogen, carbon dioxide,
methanol and other compounds into a mixture of methane and carbon dioxide and other
trace gases (Table 1), [18,19]. Fig. 5 shows anaerobic digestion process in four steps:
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Fig. 5. Degradation of carbon in the anaerobic digestion process described by four steps:
Hydrolysis, Acidogenesis, Acetogenesis and Methanogenesis [20,21,18].
1.2.3 Environmental and operational parameters
Governing parameters such as temperature, pH, C:N ratio, hydraulic retention time
(HTR), stirring, organic loading rate (OLR), pretreatment, particle size, the presence of
toxicants, etc. can affect and control the anaerobic digestion process. Some of these
parameters may differ between different processes and different plants with various
feedstocks [18].
Temperature
Temperature has a significant impact on the biogas production process. The range of the
temperature differs for diverse kinds of fermentative bacteria:
Table 3. Temperature ranges and optima for various anaerobic populations [22,23,18].
Fermentation Temperature range Temperature optimum Psychrophilic 0-20° C 15°C Mesophilic 15-45° C 35°C Thermophilic 45-75 °C 55°C
Particulate organic matter Protein Carbohydrates lipids
Soluble organic matter Amino acids Sugars Fatty acids
Intermediary products Alcohol and VFAs
Acetate H2, CO2
CH4, CO2
Hydrolysis
Acidogenesis
Acetogenesis
Aceticlastic methanogenesis
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Although anaerobic digestion can be carried out both in the mesophilic and thermophilic
temperature range, thermophilic digestion systems results in more and faster biogas
production, and better pathogen and virus kill [9].
pH and Buffering Capacity
pH is an essential factor affecting the growth of microbes during anaerobic digestion. “To
maintain a dynamic equilibrium in the anaerobic system a pH between 6.5 and 7.5 is
desirable. [18]” ( or between 7 and 8, according to another literature [23].) At PH<6.5 the
growth of the methanogens is very low [24]. Buffering capacity or alkalinity that is the
resistance of an anaerobic digestion process against change in pH is primarily based on
the carbonate-bicarbonate-carbonic acid system, but other compounds such as ammonia
and volatile fatty acids may have significant buffering capacity and change the pH of the
AD system [18]. “In a normal proceeding anaerobic digestion system concentration of
volatile fatty acids, acetic acid in particular should be below 2000 mg/l, [19].”
C:N Ratio
Anaerobic microorganisms in fermentation process utilize both carbon and nitrogen
elements to live. However, their carbon consumption is usually 20-30 times higher than
nitrogen. Hence, C:N ratio for digestion process should be about 20-30:1 [19].
Hydraulic Retention Time (HRT)
Hydraulic Retention Time (HRT) is the average time to degrade all organic matters inside
the digester. “In tropical countries like India, HRT varies from 30-50 days while in
countries with colder climate it may go up to 100 days.” Shorter retention time may lead
to washout of active bacteria and longer retention time needs a larger volume of the
digester and increases the capital cost [19].
Agitation
Adequate stirring of the digester contents provides desired contact between bacteria and
substrates and improves the digestion process. Agitation can be done through different
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methods such as gas recirculation, mechanically stirring by mixing devices such as mixer,
scraper, piston, etc [19].
Particle size
Reducing the particle size of the feedstock by a physical pretreatment such as grinding
and milling increases the surface area for the contact between the substrate and active
bacteria [25], reduces the volume of digester [26,27] and enhances biogas yield.
Moreover, too large particles may result in clogging of the digester and making digestion
process difficult for bacteria [19].
Pretreatment
Due to the complexity of organic material, hydrolysis can be the rate limiting step for
anaerobic digestion process in cases that the substrate is in particulate form [18].
Therefore in this step physical, chemical and biological pretreatment of feedstock are
required to break down high molecular mass organic compounds into the simple and
more susceptible monomers for biodegradation. Pretreatment of substrate in rate limiting
step optimizes digestion process and increases the methane yield [19]. Pretreatment
methods are usually classified in following ways [18]:
(a) Chemical or thermo-chemical pretreatment of the feedstock with alkali or acid
(b) Biological pretreatment of fresh substrate through bacterial hydrolysis or enzyme
addition.
(c) Physical methods such as thermal treatment, high pressure, ultrasonic treatment,
milling, etc.
Toxicants
During digestion process some toxicant materials can have inhibitory effects on
methanogenic bacteria and consequently reduce the biogas yield. Toxicant may be
originated from the substrate or be produced during microbial breakdown [107]. The
most common and important toxic materials are free ammonia, high level of volatile fatty
acids, hydrogen, hydrogen sulphide (H2S). Besides, salts and xenobiotics can also be
inhibitory [18].
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Chapter 2: Chicken Feathers 2.1 Chicken Feathers Waste Treatment Poultry industry is continuously producing increasing amount of poultry meat and
noticeable quantities of organic residues such as feather, bone meal, blood, offal and so
on. Chicken feathers, making up about 5% of the body weight of poultry, is a
considerable waste product of the poultry industry being produced about 4 million tons
per year world-wide [30,31]. Disposal of waste feathers is a major concern for poultry
industry and accumulation of this huge volume of the waste feathers results in
environmental pollution and protein wastage.
Fig.6. Chicken feathers image [29].
Currently a minor quantity of waste feathers is used in other industrial applications such
as clothing, insulation and bedding [32], producing biodegradeable polymers [33] and
enzymes [34] and also as a medium for culturing microbes.
A higher quantity of pretreated feather is utilized to produce a digestible dietary protein
feedstuff for poultry and livestock [35-39]. However, to decrease the risk of disease
transmission via feed and food chain legislation on the recovery of organic materials for
animal feed is becoming tighter (Commission of the European Communities, 2000),
[40,110].
Hence development of other alternative methods to utilize enormous amount of feathers
and practical processes to fulfill these usages is inevitable [37]. Anaerobic digestion is an
environmentally and economically promising process to recover feather waste and other
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solid organic wastes to valuable materials such as biogas and fertilizers [40]. However,
slaughterhouse wastes are in general considered as difficult substrates for anaerobic
digestion because of their high protein and lipid content leading in production of some
by-products such as unionised ammonia, floating scum and accumulated log chain fatty
acids (LCFA) during anaerobic degradation, which are toxic and inhibitory to anaerobic
microorganisms in high concentrations [6–8]. Such practical difficulties have limited and
hindered the successful efforts on anaerobic digestion of feathers and other solid poultry
slaughterhouse wastes [31].
2.2 Anaerobic Digestion Process of Solid Poultry Slaughterhouse Waste
Solid poultry slaughterhouse waste is a complex substrate containing high quantity of
different proteins and lipids. Various bacteria take part in different steps of anaerobic
digestion of this waste. “In the hydrolysis step fermentative bacteria, especially the
proteolytic clostridium species, solublise proteins to polypeptides and amino acids. Lipids
are hydrolyzed to long chain fatty acids (LCFA) by β-oxidation and glycerol [41-43] and
polycarbohydrates to sugars and alcohols (Fig. 7), [41,44,43]. In the second step
“fermentative bacteria convert the intermediates to volatile fatty acids (VFAs), hydrogen
(H2), and carbon dioxide (CO2). Ammonia and sulphide are the by-products of amino
acid fermentation [41-43]. Hydrogen- producing acetogenic bacteria metabolize LCFAs,
VFAs with three or more carbons and neutral compounds larger than methanol to acetate,
H2, and CO2 (Fig. 7). As these reactions require an H2 partial pressure of ca. 10-3 atm,
they are obligately linked with micro-organisms consuming H2, methanogens, and some
acetogenic bacteria [45,43]. Methanogens ultimately convert acetate, H2 and, CO2 to
methane and CO2 (Fig. 7) [46,43]. In the presence of high concentrations of sulphate, H2
consuming acetogenic bacteria and sulphate reducing bacteria compete with methanogens
for H2 [47,43,40].
During this process produced ammonium from protein degradation dissociates to
unionised ammonia which is toxic and inhibitory to anaerobic microorganisms in high
concentrations [48-50]. Meanwhile Lipid degradation produces floating scum and
accumulated long-chain fatty acids (LCFA) [50-53]. “LCFA degradation (β-oxidation) is
22
considered a limiting step in the anaerobic degradation of complex organic substrates
[50-52,54] because LCFA oxidizing bacteria are slow growers [55] and because as
syntrophic substrates, like volatile fatty acids (VFA), their anaerobic microbial
degradation is limited by high hydrogen (H2) partial pressure [55, 43]. H2 is produced in
several steps in the anaerobic degradation of complex organic substrates and removed
from the process mainly by hydrogen-consuming methanogens and some acetogenic
bacteria [43]. Furthermore, in high concentrations LCFA [52,6-60] and unionized VFA
[61,62] are inhibitory to anaerobic microorganisms.” Consequently, to successfully
prevent LCFA and VFA accumulation in the anaerobic digestion of slaughterhouse
wastes determination the effect of the substrate loading and hydraulic retention time
(HRT) is in particular important [31]. Fig.7 illustrates degradation pathways in anaerobic
digestion process:
Hydrolysis Acidogenic Ammonia fermentation
Homoacetogenesis Acetotrophic mathanogenesis
Fig.7. Degradation pathways during anaerobic digestion. [41,44,43,40]
The theoretical methane potential for proteins, fat and carbohydrates can be calculated
using their component composition in Buswell’s formula [62] as shown in Table 4:
Carbohydrates Protein lipids
Sugars Amino acids Long-chain Fatty acids
Volatile fatty acids
other than acetic acid Beta oxidation
Hydrogen Acetic acid
Acetogenic oxidation
Hydrogenotrophic methanogenesis
Methane
23
Table 4. Calculation of general theoretical methane potential for fat, protein and
carbohydrate using average chemical formulas [63,64,18].
2.3 Specific Characteristic of Chicken Feathers and Keratin Protein
Chicken feathers are composed of over 90% of keratin protein, small amounts of lipids
and water. Feathers keratin consists of high quantities of small and essential amino acid
residues such as glycyl, alanyl and seryl as well as cysteinyl and valyl [65,66,30].
Keratin is also the main protein components of hair, wool, nails, horn, and hoofs. Animal
hair, hoofs, horns and wool contain α-keratin, and bird’s feather contains β-keratin. The
polypeptides in α-keratin are closely associated pairs of α helices, whereas β-keratin has
high proportion of β pleated sheets. “This conformation confers an axial distance between
adjacent residues of 0.35 nm in β -sheets, compared to 0.15 nm in a-helices. The β sheets
have a far more extended conformation than the α –helices” [67,108, 80].
Keratins are insoluble macromolecule comprises a tight packing of supercoiled long
polypeptide chains with a molecular weight of approximately 10 kDa. High degree of
cross linked cystin disulphide bonds between contiguous chains in keratinous material
imparts high stability and resistance to degradation [35-37,33]. Hence, a keratinous
material is a tough, fibrous matrix being mechanically firm, chemically unreactive, water-
insoluble and protease-resistant [80]. Such a molecular structure makes feathers poorly
degradable under anaerobic digestion condition [31,37]. Fig. 8 shows keratin molecular
structure:
Component Chemical formula Theoretical biogas potential (Nm3 CH4 per ton VS) Fat C57H104O6 1014 Protein C5H7NO2 496 Carbohydrate (C6H10O5)n 415
24
Fig. 8. Keratin molecular structure [68]. 2.4 Pretreatments methods for hydrolysis of poultry feathers
Because of the complex, rigid and fibrous structure of keratin, poultry feather is a
challenge to anaerobic digestion. It’s poorly degradable under anaerobic conditions.
[69,33] However, application of appropriate pretreatments methods hydrolyzes feather
and breaks down its tough structure to corresponding amino acids and small peptides
[70,35].
For more than half a century many studies have been performed and various pre-
treatment methods have been applied to improve the digestibility of feather meal as well
as development of its nutritional value for production of a dietary protein feedstuff for
animals [30,72,75]. These pretreatments methods may also enhance feather biogas
potential. However, only a few studies have been reported on this subject [30]. Feather
meal treatment methods are usually categorized into two groups: hydrothermal treatments
and microbial keratinolysis [74,35].
25
2.4.1 Hydrothermal pretreatments
Hydrothermal pre-treatment includes thermo-chemical treatment methods (such as acidic
hydrolysis and alkali hydrolysis), and also steam pressure cooking [35,73]. These
methods usually need high temperatures [75] or high pressure [76,77] with addition of
diluted acids such as hydrochloric acid [76] or alkali such as sodium hydroxide [78,35].
“Acidic solutions promote the loss of some amino acids such as tryptophan. [79]”
Although alkaline reactions are sometimes slower and may not go to completion,
degradation of some amino acids with hydroxide is less. Hence the use of bases is
recommended. A stepwise diagram for the hydrolysis of protein rich material under
alkaline condition is indicated in Fig. 9 [80].
Fig. 9. Protein hydrolysis during thermo-chemical treatment [80].
As a whole, hydrothermal hydrolysis usually consumes high amount of energy and
employs expensive equipment during lengthy processes (8 to 12 hrs), [65,37].
PROTEIN α-keratin (hair), β-keratin, animal tissue, plant matter.
HYDROLYSIS Peptide bond is broken.
Smaller peptides and free amino acids are generated.
DEAMIDATION GLN and ASN residue in protein
react and form GLU and ASP residues, with ammonia as a
product.
SMALLER PEPTIDES & FREE AMINO ACIDS Smaller peptides with a higher digestibility (structure) and
free amino acids are dissolved in the liquid phase.
DEGRADATION Several amino acids are not stable under alkaline conditions and
undergo reactions that generate different products (e.g. other amino acids, ammonia)
26
Thus, optimization of the treatment conditions is an important issue from technological
and economical points of view when applying this method.
2.4.2 Biological pretreatment
Biodegradation of feathers is another alternative method. Some bacterial strains can
produce keratinase proteases which have keratinolytic activity and are capable to
keratinolyse feather β-keratin. These enzymes help the bacteria to obtain carbon, sulfur
and energy for their growth and maintenance from the degradation of β-keratin [81].
Various keratinases from different microorganisms such as Bacillus sp. [84] Bacillus
licheniformis [85-88] Burkholderia, Chryseobacterium, Pseudomonas, Microbacterium
sp. [89] Chryseobacterium sp. [90,91] Streptomyces sp. [92,93] has been isolated and
studied to date [72, 81-83].
Microbial proteases are classified into acidic, neutral, or alkaline groups, depends on the
required conditions for their activity and on the characteristics of the active site group of
the enzyme, i.e. metallo-, aspartic- , cysteine- or sulphydryl- or serine-type. Alkaline
proteases which are active in a neutral to alkaline pH, especially serine-types, are the
most important group of enzymes used in protein hydrolysis, waste treatment and many
other industrial applications. Alkaline protease from Bacillus subtilis was used for the
keratinolysis of waste feathers [109].
Subtilisins are extracellular alkaline serine proteases, which catalyse the hydrolysis of
proteins and peptide amides. Savinase is one of these enzymes; Alcalase, Esperase and
Maxatase are others. These enzymes are all produced using species of Bacillus. Maxatase
and Alcalase come from B. licheniformis, Esperase from an alkalophilic strain of a B.
licheniformis, and Savinase from an alkalophilic strain of B. amyloliquefaciens [109]. An
important advantage of enzyme treatment method is fully biodegradability of enzymes by
themselves as proteins. Hence, unlike other remediation methods, there is no buildup of
unrecovered enzymes or chemicals that must be removed from the system at the end of
degradation process. Although enzymatic treatment is a promising technology; it has
some limitations and disadvantages, as well. Currently, the main disadvantage of using
alkaline proteases is the high cost of the enzymes production. Much of the cost of
27
producing enzymes is related to high purification of enzymes solutions to avoid the side
effects and side activities of the crude enzyme solution which is cheaper. Furthermore, in
contrast with microbes which can reproduce themselves and increase their population to
be able to consume a large quantity of substrate and survive in harsh environments,
extracellular enzymes like alkaline protease do not have reproducibility. Namely,
increasing the enzyme population must be done through adding new enzymes from
outside into the system. On the other hand, these alkaline proteases lose some reactivity
after they interact with pollutants and could eventually become completely inactive.
Hence they do not have the adaptability to the harsh environment even though they can
survive in a wide range of environmental conditions. This means that the enzyme
concentrations must be monitored and controlled during the process in order to optimize
enzyme kinetics for site-specific conditions [109].
2.4.3 Chemical-Biological pretreatment
Keratins are insoluble macromolecule comprises super coiled long polypeptide chains
with high degree of cross linked disulphide bonds between contiguous chains. According
to the literatures disulfide bonds in keratin significantly decrease protein digestibility
[94]. And “for complete easy degradation of feather all enzymatic keratinolysis from any
organism essentially needs to be assisted by a suitable redox [95].” therefore, it has been
suggested that some reductants, such as thioglycollate, copper sulphate , ammonia and
sodium sulphite [96] and others, might cleavage the disulfide bonds in keratin and allows
the proteases to have access to their peptide bond substrates [97], and consequently
improve the degradability of feathers [94,35,12,65]. For instance Ramnani et al., 2007
found that savinase is capable of near complete feather degradation (up to 96%) in the
presence of sodium sulfite [95].
28
2.5 Research Objectives
Considering the abundance and continual increase in the production of chicken feather
waste as a high value resource of protein and also the hard degrading structure of feather
keratin, the objective of this study was to investigate the feasibility and the effects of
various pretreatment methods on the hydrolysis of chicken feather for enhancement of its
methane potential.
For this purpose chicken feathers were pretreated by thermal, thermo-chemical,
enzymatic, thermo-enzymatic and chemo-enzymatic methods followed by anaerobic
digestion of pretreated feathers. Besides, the effects of the variation in treatment
conditions during thermo-chemical treatment on the methane yield of chicken feathers
and optimization of these conditions were studied.
29
Chapter 3: Materials and methods
3.1 Equipments and apparatus
The following equipments and supplies were applied for the experiments:
• 118 ml glass bottles (flasks) with rubber septum, as bioreactors
• 250µl gas tight glass syringe with a pressure lock to take fixed volume and
pressure samples from the reactors.
• Regulated incubator at 55°C for incubation the samples in a thermophilic
condition.
• Autosystem Gas Chromatograph equipped with thermal conductivity detector
(TCD), for the measurement of CH4 and CO2.
• COD Reactor with Direct Reading Spectrophotometer for SCOD measurement of
the pre-treated samples.
• Convection drying oven with temperature control of 105±3°C for TS
measurement of feather
• Muffle furnace with temperature control of 550°C for VS measurement of feather.
• Autoclave for thermal pre-treatments of samples
• Shaking water bath regulated at 37°C and 150 rpm for chemical pretreatment of
samples
• Centrifuge for separation the suspended solid and liquid phase of samples for
SCOD (soluble chemical oxygen demand) measurement of pre-treated samples.
• Digital pH meter to measure and adjustment of the pH of the pre-treated samples
for digestion and final pH of digested samples at the end of experiments.
3.2 Materials
• Waste Chicken Feather as a bioresource for biogas production.
• Inoculum from thermophilic Biogas Plant, Sobacken-Borås.
• Lime for thermo-chemical treatment.
30
• Sodium sulfate for chemo-enzymatic treatment.
• Savinase ®ClEA for enzymatic treatment.
• Gas mixture of 80% N2 and 20% CO2 for air removal of the samples head space.
• 100% CO2 gas as CO2 standard for gas chromatography and also carbonation of
lime treated samples.
• 100% CH4 gas as methane standard for gas chromatography
• Phosphate buffer to adjust the pH of the samples in experiment 3.
3.3 Methods
3.3.1 Preparation of Waste Chicken Feathers
Waste chicken feathers were cleaned and washed with lukewarm water a few times, and
then air-dried at room temperature followed by drying in the oven at 105°C±3.
After drying the feathers were grinded and stored in capped dishes in cooling room.
3.3.2 Inoculum
Active thermophilic inoculum was obtained from thermophilic Biogas Plant, Sobacken-
Borås and stored at 55°C in an incubator for 3 days in order to readapt the inoculum to
55°C, ensure degradation of easy degradable organic matters still present in the inoculum
and remove dissolved methane.
3.3.3 Total Solids (TS%) and Volatile Solids (VS%) measurement
Total Solids percentage (TS%) of the feathers was measured according to the
“Laboratory Analytical Procedure (LAP-001), Standard Method for Determination of
Total Solid in Biomass (LAP-001)” [98] as follows:
-Crucibles were dried in drying oven 105°C±3 over the night and were weighed
accurately to the nearest 0.1 mg and the weight was recorded.
31
-Air dried; milled feathers were weighed into the dried crucibles to the nearest 0.1 mg.
The total weight of the -each sample and crucible were recorded.
-Samples were placed into the convection oven at 105±3°C and were dried for overnight
to constant weight.
-Samples were removed from the oven and placed in a desiccator to cool to room
temperature.
-The total weight of the crucibles and oven dried samples were measured to the nearest
0.1 mg and recorded.
Total Solid (TS%) of the samples were calculated according to the following equation:
Total Solids percentage (TS %) = (W2 /W1) x 100
Where:
W1 = weight air dried sample
W2 = weight 105°C dried sample = weight 105°C dried sample plus dish – weight dish
Data figures for TS% measurement are shown in table 11 appendix A. And hence,
Average Measured TS% of Chicken Feather was:
Feather (TS%) = 91.29%
Volatile Solids percentage (VS%) of feather was measured according to the
“Laboratory Analytical Procedure (LAP-005), Standard Method for Determination of
Ash in Biomass” [99] as follows:
-Crucibles were heated at 550°C±10 for 4 hours and placed in a desiccator to cool to the
room temperature. Then crucibles were weighed accurately to the nearest 0.1 mg and the
weight was recorded.
-Oven dried (105°C) feathers were weighed into the dried crucibles to the nearest 0.1 mg.
The total weight of the each sample and crucible were recorded.
-Samples were placed into the muffle furnace at 550°C±10 for 3 hours, reheated and
reweighed to constant weight till varies by less than 0.3 mg.
32
-Samples were removed from the oven and placed in a desiccator to cool to the room
temperature.
-The total weight of the crucibles and burned residue were measured to the nearest 0.1
mg and recorded.
Volatile Solids (VS %) of the samples were calculated according to the following
equation:
%Volatile Solids (VS % of TS) = (W1-W2/W1) x 100
Where:
W1 = weight 105°C dried sample, and
W2 = weight of ash (burned residue) = weight burned residue plus dish – weight dish
Data figures for TS% measurement are shown in table 10 appendix A.
And hence, average Measured Volatile Solid% of TS Feather was:
Feather VS% (of TS) = 99.34% of TS
And Average Measured Volatile Solid% of Air Dried Feather was:
Feather (VS%) = 90.69%
3.4 Pretreatment Methods
3.4.1 Thermo-Chemical Lime Pretreatment (Experiments 1, 2)
Various concentrations of Lime (Ca (OH)2 g/g TS F) were added to the mixtures of 2
different concentrations (40 &100g TS/l water) of milled and 105°C dried chicken
feathers. 50 ml of each sample was prepared in duplicate. Afterward, samples were
closed with aluminum foil loosely and were heated in the autoclave at different
temperatures for different treatment times according to the Table 5:
33
Exp. Number
Feather Concentration (g TS F/l liquid)
Lime loading (g/g TS F)
Autoclave Temperature
(°C)
Time (min)
0.1 0.2
0.4 1 2
1
40
4
100, 110, 120
30,60,120
0.1 0.2 0.4 1
2
100
2
100, 110, 120
60,120
Table 5. Thermo-chemical treated samples and treatment conditions (Exps.1 and 2) After cooling the samples to the room temperature in a desiccator, pH measurement for
the samples was carried out. In general, due to the presence of the lime pH values of the
treated samples has been maintained around 11.5-12.5.
To adjust the pH of the samples to the suitable value for anaerobic digestion and also to
convert the existing lime in the samples to the water-soluble Ca(HCO3)2 (as much as
possible), samples were carbonated with pure CO2 gas while the pH were controlled
continuously. In this way the pH of the samples decreased to about 8-8.5 and major
amount of the lime was converted to water-soluble calcium bicarbonate (Ca(HCO3)2) and
also low soluble calcium carbonate (CaCO3) [35].
One of each duplicated samples were centrifuged and the liquid phase of them were used
for soluble chemical oxygen demand (SCOD) concentration measurement.
Considering the SCOD measurement results, the following uncentrifuged samples which
their centrifuged couples had revealed high SCOD concentration and also contented
much lower amount of the precipitated lime and calcium carbonate (CaCO3) were
selected to use for the anaerobic digestion process (samples had been made in 50ml
volume):
- For experiment 1, using 40 g TS feather/l concentration, the selected samples had been
treated under the following conditions:
34
1- 0.1g lime /g TS feather, 30 min, 100°C:
2g TS feather + 48 ml water + 0.2g lime
2- 0.1g lime /g TS feather, 30 min, 120°C:
2g TS feather + 48 ml water + 0.2g
3- 0.2g lime /g TS feather, 1 h, 120°C:
2g TS feather + 48 ml water + 0.4g lime
4- 0.2g lime /g TS feather, 2 h, 120°C:
2g TS feather + 48 ml water + 0.4g lime
- For experiment 2, using 100 g TS feather/l concentration, the selected samples had been
treated under the following conditions:
1- 0.1g lime /g TS feather, 2h, 120°C:
5g TS feather + 45 ml water + 0.5g lime
2- 0.2g lime /g TS feather, 2h, 120°C:
5g TS feather + 45 ml water + 1g lime
3- 1g lime /g TS feather, 2h, 120°C:
5g TS feather + 45 ml water + 5g lime
4- 2g lime /g TS feather, 2h, 120°C:
5g TS feather + 45 ml water + 10g lime
3.4.2 Biological Pretreatments (Experiment 3)
In this series of experiment the effect of thermal, enzymatic, combined thermo-enzymatic
and combined chemo-enzymatic pretreatments on hydrolysis of feather were examined.
Milled and oven dried feathers, 0.9g TS F/vial, (1g F/vial) were pre-treated in the small
flasks (118 ml), in triplicate and one excess sample for SCOD measurement. For the
enzymatic treatment an alkaline endopeptidase enzyme, Savinase, was used.
Furthermore, for chemo-enzymatic treatment sodium sulfite was also added as chemical
reductant agent to cleavage disulphide bonds. The pH of the samples was adjusted to
pH=8.0 using phosphate buffer. The total volume of each sample was 10 ml.
Pretreatments were conducted using the following conditions and materials:
35
1- Thermal treatment: autoclaving for 5min at 120°C
0.9g TS feather + 9.1 g potassium phosphate buffer solution
2- Enzymatic treatment: incubation for 2h at 55°C
0.9g TS feather + 9g potassium phosphate buffer solution + 100mg enzyme (1% w
enzyme/vial)
3- Thermal-Enzymatic treatment: autoclaving feather for 5min, at 120°C, followed
by buffer and enzyme addition and incubation for 24h at 55°C
0.9g TS feather + 9g potassium phosphate buffer solution + 100mg enzyme (1% w
enzyme/vial)
4- Chemical-Enzymatic treatment: water bath for 60h at 37°C 150 rpm
0.9g TS feather + 9g potassium phosphate buffer solution + 100mg enzyme (1% w
enzyme/vial or 100mg/10ml) + 0.0252g Na2SO3 (20 mM/l)
The extra pretreated samples were centrifuged and the liquid phase of them was used for
SCOD measurement (Fig. 10):
Fig. 10. COD Reactor with Direct Reading Spectrophotometer for SCOD
measurements of the pre-treated samples.
36
3.5 Anaerobic Digestion Processes
3.5.1 Batch digestion process set-up for pretreated samples
In this step for lime treated samples (Exps.1 and 2) 5g of each sample consisting of both
proportional liquid and solid phases were transferred to 3 small flasks (118 ml) to make
triplicate samples for anaerobic digestion process. Then, during stirring of the inoculum
20 ml of the inoculum was transferred to each of the flasks. Total volume of each sample
was 25 ml. Hence, the VS content of pretreated feathers in each flask for samples of
experiment 1 was 0.191g VS F/Vial (0.765%VS) and respectively, for pretreated samples
of experiment 2 it was 0.453g VS F/Vial (1.8% VS). 3 untreated samples (control
samples) and 3 blanks were also prepared with the following materials:
- For experiment 1:
Untreated samples:
0.191g oven dried (TS) feather + 4.8 ml water +20 ml inoculum
Blank samples:
5ml water + 20 ml inoculum
- For experiment 2:
Untreated samples:
0.453g oven dried (TS) feather + 4.55 ml water +20 ml inoculum
Blank samples:
5ml water + 20 ml inoculum
To evaluate the effect of the solid phase on the methane productivity of pretreated
samples with 40g TS F/l and 0.2g lime /g TS F which contained negligible amount of
insolublised substrate and more amount of precipitated lime and carbonate calcium in
their solid phase, anaerobic digestion was also performed using just liquid phase of those
samples (samples 4 and 5 in Table 6).
For biological pretreated samples (experiment 3) also during stirring of the inoculum 50
ml of the inoculum was transferred to each flask which contained 10ml pretreated
37
feathers. The total volume of each sample was 60 ml. 3 untreated samples and 3 blanks
were also prepared, as following:
Untreated samples:
0.9g TS feather /vial (1g F/vial) + 9.1 g phosphate buffer solution + 50 ml inoculum
Blank samples:
10 ml phosphate buffer solution + 50 ml inoculum
In the final step the sample flasks, prepared for the anaerobic digestion on the above
described ways, were closed with a rubber septum and an aluminum cap and were
flushed with a mixture of gas containing 8o% N2 and 20% CO2 for 2 minutes to provide
anaerobic condition in the headspace of the reactors and prevent pH-change in the water-
phase [101]. The samples were then incubated at 55°C for 50 days (Fig. 11).
Fig. 11. Samples maintained in the incubator at 55°C for anaerobic digestion process.
Volume of the produced CH4 and CO2 were measured at least twice a week using a Gas
Chromatograph equipped with TCD (Fig. 12).
38
Gas samples of 250µl were taken from the headspace of the flasks through the septum
using a gas tight syringe equipped with a pressure lock, and then were injected directly
into the gas chromatograph (GC). Pure CH4 and CO2 gases were used as standard gases
in GC measurements. To avoid build-up of the gas over pressure in the flasks leading to
gas leakage, gas pressure inside of the flasks was usually kept below 2 bars and the over
pressure was released under a hood by inserting a hospital needle in the rubber septum.
After the release an additional gas sample was taken and measured in a similar way as
described previously. During the incubation period the samples were regularly shaken
and moved around in the incubator to compensate any minor temperature variations at the
different parts of the incubator. Samples were shaken also before each GC measurement.
Fig. 12. Autosystem Gas Chromatograph with TCD for measurement of produced
methane and carbon-dioxide.
39
Chapter 4: Calculation and Data Treatment
The produced amount of methane was determined according to the “GC External
Standard Method” [100]. In this standard, assuming, the response index of the detector is
unity, if the (p)th gas component in the mixture is at a concentration of (cp (s)) in the
sample and (cp(st)) in the standard gas, then:
cp(s) = (ap(s)/ap(st)) * cp(st)
Where:
cp(s) is the concentration of the component (p) in the sample,
(ap(s)) is the area of the peak for the component (p) in the sample chromatogram,
(ap(st)) is the area of the peak for the component (p) in the reference chromatogram,
And (cp(st)) is the concentration of the standard in the reference.
Assuming ideal gas mixtures and using the ideal gas law, from the mole numbers of each
gas components measured in the sample of known volume, the mole numbers of each gas
components in the head space can be calculated without measuring the actual pressure in
the flasks. Furthermore, t he amount of CH4 (or CO2) produced between two subsequent
sampling in the head space of each flask was calculated from the difference of mole
numbers of methane (or carbon-dioxide) determined after releasing the overpressure and
the mole numbers of methane (or carbon-dioxide) determined at next sampling time
before the release. To calculate the produced methane volumes the following
experimental conditions were considered:
T = 22°C = 295 K, Atmospheric Standard Pressure, Patm= 101325 Pa,
R (Ideal Standard Gas Constant) =8.314, Sample (syringe) Volume (Vs) = 250 µl,
40
Finally, Normal Volume of the produced methane per gram VS (Nm3 CH4/kg VS) was
calculated for each sample at standard conditions of 273 K and 101325 Pa and the data
are presented as produced methane (Nm3 CH4/kg VS) versus time (days). Calculations
for all triplicates were computed and analyzed using MS Excel-Sheet and the blank
samples performance (gas production of the inoculum) was subtracted from the
performance (gas production) of the other samples.
41
Chapter 5: Results and discussion
5.1 Effect of lime treatment on SCOD concentration (Experiments. 1, 2)
In this study thermo-chemical treatment with lime exerted the most significant effect on
solublisation of the complex and rigid structure of feather keratin and generated a rich
mixture of small peptides and free amino acids resulting in high concentrations of soluble
chemical oxygen demand (SCOD). The average values for SCOD of the samples under
various pretreatment conditions such as different feather concentration, lime loading,
temperature and reaction time are shown in the Figs. 13-16:
0
10000
20000
30000
40000
50000
60000
SCOD (mg/l)
0g/g(30m)
0,1g/g(30m)
0,2g/g(30m)
0g/g(1h)
0,1g/g(1h)
0,2g/g(1h)
0g/g(2h)
0,1g/g(2h)
0,2g/g(2h)
Lime conc., Time
SCOD Concentration
100°C
110°C
120°C
Fig. 13. Results of SCOD measurement for lime treated samples containing 40gTS F/l
initial concentration (Exp. 1), under various treatment conditions.
42
45000460004700048000
49000500005100052000
53000
SCOD (mg/l)
0,4 g/g 1 g/g 2 g/g 4 g/g
Lime Conc. (g/g)
SCOD concentration (120°C, 2h)
0,4 g/g
1 g/g
2 g/g
4 g/g
Fig. 14. Results of SCOD measurement for lime treated samples containing 40gTS F/l
initial concentration (Exp. 1) with higher lime loadings at 120°C for 2h.
As seen in Figs. 13 and 14 for the samples of experiment 1, containing 40gTS F/l liquid
concentration, SCOD concentration increased drastically from a minimum of 850 mg/l
under 0g Ca(OH)2/g TS F, 100°C, 30min treatment conditions i.e. with no lime addition
to a maximum of 59450 mg/l under 0.2g Ca(OH)2/g TS F, 120°C, 2h treatment
conditions. However, further increase in the lime loading to 0.4, 1.0, 2.0, and 4.0g
Ca(OH)2/g TS F at 120°C with a reaction time of 2h reduced the SCOD concentration of
the samples, comparatively. The lowest value of 47675 mg/l SCOD was obtained with
addition of the highest amount of lime (4g Ca(OH)2/g TS F). Increasing some other
pretreatment conditions such as reaction time and temperature didn’t change SCOD
concentration significantly. Previously, Coward-Kelly et al. 2005 [35], studied
pretreatment of feather with lime to generate an amino acid rich foodstuff for animals.
They found that feather solublisation significantly increases from 0 to 0.1 g Ca(OH)2/g
TS F, but does not change considerably for higher lime loadings. Hence, lime loading
shows a critical value below which the digestibility greatly declines and above which the
digestibility does not change substantially [35]. However, as expected, increasing feather
concentration from 40 to 100g TS F/l liquid in experiment 2 increased the SCOD
concentration. (Figs. 15 and 16)
43
0
20000
40000
60000
80000
100000
120000
140000
160000
SCOD (mg/l)
0g/g(1h)
0,1g/g(1h)
0,2g/g(1h)
0g/g(2h)
0,1g/g(2h)
0,2g/g(2h)
Lime conc., Time
SCOD Concentration
110°C
120°C
Fig. 15. Results of SCOD measurement for lime treated samples containing 100gTS F/l
initial concentration (Exp. 2) under various treatment conditions.
125000130000135000140000145000150000155000160000165000170000
SCOD (mg/l)
0,4 g/g 1 g/g 2 g/g Lime conc. (g/g TS)
SCOD Concentration (120°C, 2h)
0,4 g/g
1 g/g
2 g/g
Fig. 16. Results of SCOD measurement for lime treated samples of Exp. 2 with
higher lime loadings at 120°C and for 2h.
For instance, as indicated in Figs.16and 14, sample with 100 g TS F/l concentration under
2g Ca(OH)2/g TS F, 120°C, and 2h treatment conditions, revealed the highest SCOD
concentration, of 168500 mg/l, while for the sample with 40 g TS F/l concentration
treated at the same conditions the SCOD concentration was 52000 mg/l respectively i.e.
the relative SCOD releases for these samples were 1685 and 1300mg SCOD/g TS F.
44
Therefore we can conclude that the relative SCOD release could be increased by about
30% when higher concentration of feathers was used for the treatment.
Meanwhile, the effects of the variation of other pretreatment conditions on 100 g TS F/l
concentrated samples (experiment 2) were similar to those of 40 g TS F/l samples
(experiment 1). i.e. increasing the lime loading from 0 g/g TS F to 0.2 g/g TS F improved
SCOD concentration drastically but further increase in the lime loading (from 0.2g Ca
(OH)2/g TS F to 2 g Ca (OH)2/g TS F) could improve SCOD only slightly . And the same
as in the experiment 1, increasing the other pretreatment conditions such as temperature
and reaction time didn’t exert noticeable positive effect on increasing of SCOD
concentration.
5.2 Effect of lime treatment on Anaerobic digestion performance
(Experiments 1, 2)
Regarding the objectives of this study and the results obtained by SCOD measurements,
the best pretreated samples containing high SCOD concentrations, optimal pretreatment
conditions and the least content of precipitated lime and calcium carbonate had been
selected for anaerobic digestion process. Table 6 and Figs. 17, 18 illustrate the SCOD
concentration (after treatment) and maximum methane yield of the selected samples
containing 40 g TS F/l concentration, during 50 days of anaerobic incubation:
Table 6. Results of SCOD and average maximum methane yields of triplicate lime treated
samples of Exp.1 during 50 days of incubation.
Sample pretreatment Feathers Concentration (g TS/l liquid)
Pretreatment Conditions
SCOD (mg/L)
Concentration of substrate in vials (g VS/Vial)
Maximum Methane yield (Nml/g VS)
Percentage of theoretical methane potential
1 Control, untreated
--- 47.4 9.6%
2
0.1g lime/gTS, 100°C, 30 min
41600 480 96.8%
3
0.1g lime/gTS, 120°C, 30 min
55400 338 68.1%
4
0.2g lime/gTS, 120°C, 60 min
63100 230 46.4%
5
40
0.2g lime/gTS, 120°C,120 min
67200
0.191 (0.765%)
123 24.8%
45
0
10000
20000
30000
40000
50000
60000
70000
SCOD (mg/l)
0.1g/g,100°C,30min
0.1g/g,120°C,30min
0.2g/g, 120°C,
1h
0.2g/g, 120°C,
2h
Lime conc., temp., time
SCOD Concentration
0.1g/g,100°C, 30min0.1g/g,120°C, 30min0.2g/g, 120°C, 1h0.2g/g, 120°C, 2h
Fig. 17. Results of SCOD measurement for lime treated samples of Exp. 1, selected
for anaerobic digestion process.
Methane Normal Volume
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0 10 20 30 40 50 60
Time (days)
N VO
L (m
3/kg
VS
)
Untreated
0,1g/g30min100°C0,1g/g30min120°C0,2g/g 1h120°C
0,2g/g 2h120°C
Fig. 18. Average maximum methane production curves for triplicate lime treated
samples of Exp. 1, during 50 days of incubation.
According to these results, beside the considerable improvement of SCOD concentration,
lime treatment showed the most significant effect on increasing the methane productivity
of chicken feathers. In particular for sample 2 of this experiment, pretreatment under
(0.1g Ca(OH)2 /g TS F, 100°C and 30 minutes) conditions demonstrated the highest
46
increase in the methane yield of 480 N ml CH4/g VS which is about 96.8 % of the
theoretical methane potential. General theoretical methane potentials for fat, protein and
carbohydrates were illustrated in Table 4.
Although increasing the pretreatment conditions such as feather concentration, lime
loading, reaction time and temperature showed an overall positive effect on SCOD
enhancement, exert negative effect on the methane yield. For instance increasing the lime
loading from 0.1 g to 0.2 g/g TS feather for samples 4 and 5 also increased the SCOD to
some extent, but resulted in the highly increased amount of precipitated lime and
carbonate calcium, unstable anaerobic digestion performance and much less efficiency in
the methane productivity of those samples (Figs. 17,18 and Table 6). According to the
Coward-Kelly et al. (2006), protein and amino acid degradation are associated with
ammonia production which is the most important toxicant for anaerobic digestion of
proteins (e.g., deamidation of asparagine and glutamine, generating asparatate and
glutamate and ammonia) (Figure 9) [80,35].
Therefore shorter reaction time and lower temperatures (approximately 100°C) in
treatment of chicken feathers are preferred because the degradation of susceptible amino
acids and ammonia production may be reduced to a minimum (35,80,102). It means that
increasing the treatment temperature and time in this experiment has led in more feathers
solublisation. The increased solublised feathers, which compared to the sample 1 were
observable in the lime treated samples of 2-5, have increased the SCOD concentration
and also overloaded these samples with amino acids. Meanwhile, increasing the treatment
temperature and time has resulted in more amino acid degradation associated with
accumulated ammonia. This accumulated ammonia has inhibited the methane
productivity of the samples 2-5.
To evaluate the effect of the precipitated lime and carbonate calcium in the solid phase of
these samples on the methane productivity, extra anaerobic digestion assay was done for
samples 4 and 5 using just liquid phase of these samples which contained negligible
amount of insolublised substrate and high amount of precipitated lime and carbonate
calcium in their solid phase. As seen in table 7 and Fig. 19 bellow, some improvements in
47
the methane yields of these samples were observed, up to 15.7% increase for sample 4
and 51.2% for sample 5.
Table 7. Results of SCOD and average maximum methane yield of triplicate lime treated
samples 4 and 5 of Exp. 1, during 15 days of incubation, (liquid phase).
Methane Normal Volume
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0 10 20 30 40 50 60
Time (days)
N V
OL
(m3/
kg V
S)
Untreated
0,2g/g 1h120°C
0,2g/g 2h120°C
Fig. 19. Average maximum methane production curves for triplicate lime treated samples
4 and 5 of Exp. 1, during 50 days of incubation (liquid phase).
Sample Pretreatment
Feathers
Concentration
(g TS/l liquid)
Pretreatment
Conditions
SCOD
(Mg/L)
Concentration
of substrate in
vials
g VS/Vial
Maximum
Methane
yield
Nml/gVS
Percentage
of theoretical
methane
potential
1 Control,
untreated
--- 47.4 9.6%
2 0.2g lime/g TS,
120°C, 60 min
63100
266
53.6%
3
40
0.2g lime /g TS,
120°C, 120min
67200
0.191 (0.765%)
186 37.5%
48
However, for samples 2 and 3 of experiment 1, because of the presence of more
insolublised substrate in the solid phase, using both solid and liquid phase of the sample
is inevitable. Meanwhile, for these samples almost no visible precipitated lime and
calcium carbonate were observed to be separated.
Increasing the feather concentration to 100g TS F/l in experiment 2, which also resulted
in increasing of SCOD (Figs. 15, 16), led in much lower and even depressed methane
productivity of the most samples during 15 days of anaerobic incubation. Table 8 and
Fig. 20 illustrate the SCOD concentration (after treatment) and maximum methane yield
of the selected samples containing 100 g TS F/l concentration, during 15 days of
anaerobic incubation:
Table 8. Results of SCOD and average maximum methane yields of triplicate lime treated
samples of Exp. 2, during 15 days of incubation.
Sample Pretreatment
Feathers
Concentration
(g TS/l liquid)
Pretreatment
Conditions
SCOD
(mg/L)
Concentration of substrate in vials(g VS/Vial)
Maximum
Methane
yield
( Nml/g VS)
Percentage
of theoretical
methane
potential
1 Control,
untreated
--- 118 23.8%
2
0.1g lime /g TS,
120°C, 60 min
114200 139 28%
3
0.2g lime/g TS,
120°C, 60 min
153800 53 10.7%
4
1g lime /g TS,
120°C, 60 min
162500 23 4.6%
5
100
2g lime /g TS,
120°C, 60min
168500
0.453
(1.8%)
20 4.0%
49
Methane Normal Volume
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0 5 10 15 20Time (days)
N V
OL
(m3/
kg V
S)
Untreated
0,0g/g 1h120°C 0,2g/g 1h120°C 0,1g/g 1h120°C1g/g 1h120°C2g/g 1h120°C
Fig. 20. Average maximum methane production curves for triplicate lime treated
sample of Exp. 2, during 15 days of incubation.
The increased SCOD concentration and meanwhile decreased methane yield of these
samples probably reflect the effect of the overloading of the system with organic
substrate leading in accumulation of ammonia which inhibited CH4 productivity of the
protein material during anaerobic digestion process. Accordingly, less feathers loading
may result in more efficient anaerobic digestion process.
It is mentionable that the same considerations i.e. high SCOD content, least precipitation
of lime and calcium carbonate (CaCO3) and optimal conditions had been applied in
selection of the samples of experiment 2 for anaerobic digestion process.
After lime treatment the measured pH for the samples was around 11.5-12.5. Carbonating
samples with CO2 gas before digestion process decreased the pH to about 8-8.5. Although
no buffer was used to adjust the pH during the digestion process, final measurement of
the pH indicated that the pH of the samples had been maintained almost at the same level
of the starting of the AD process (pH of 8-8.5). According to the literatures “Calcium
hydroxide is an alkaline material poorly soluble in water that maintains a relatively
constant pH (~12), provided enough lime is in suspension. This low solubility ensures a
constant pH during the thermo-chemical treatment and relatively weaker conditions
(compared to sodium hydroxide and other strong bases) that helps in reducing the
50
degradation of susceptible amino acids. The new carboxylic acid ends react in the
alkaline medium to generate carboxylate ions, consuming lime in the process [35].”
Lo´pez Torres et al.,2007 [104] found also the similar point and reported that in contrast
with the other AD systems the digesters fed with lime pretreated waste maintained its
alkalinity and neutral pH during digestion process without necessity of continuously
addition of alkali.
Samples 2 and 3 of experiment 1 had a fast onset in methane production but samples 4
and 5 had a one week lag phase. However, in repetition of the AD process using liquid
phase of samples 4 and 5 no delay was observed in the start of the methane production.
The ammonia production of in vitro rumen digested lime soluble chicken feather keratin
was also previously studied by Coward-Kelly et al. (2005) [35]. They found that
ammonia production from soluble keratin in rumen fluid was similar to that of soybean
and cottonseed meals and was greatly less than that of urea. Soybean and cottonseed
meals are the most popular protein sources for cattle which do not result in ammonia
toxicity. Therefore, soluble feather keratin is likely more readily digested than the other
proteins and no ammonia toxicity will result from cattle being fed soluble keratin [35].
Similar performance might be expected from lime treated samples during anaerobic
digestion of feather for biogas production, namely no ammonia toxicity is produced and
inhibits the anaerobic microorganisms for the recommended condition.
According to Lo´pez Torres et al. 2007, Alkaline pretreatment of organic materials with
Ca(OH)2 not only increases the level of soluble COD but also surface area of complex
organic matter, due to fiber swelling. These facts make these materials more susceptible
to enzymatic attack by microorganisms and enhance anaerobic digestion processes [104].
Another significant advantage of alkaline treatment is disruption of the disulphide bonds
in feather which was previously noticed by Salminen et al. [30]. All of the above results support the positive effect of lime pretreatment on hydrolysis of
the chicken feather and other organic materials, and according to the achieved results in
the present study pretreatment of chicken feather under (40g TS feather/l, 0.1g Ca(OH)2/g
dry feather, 100°C, 30 min) condition is the optimum condition to exert the most
significant effect on increasing the methane yield of chicken feather. Coward-Kelly et
al., 2005 [35] found that pretreatment of feather under 0.1g Ca(OH)2/g dry F, 100°C and
51
300 min treatment conditions can solublise 80% of feather keratin to produce an amino
acid rich foodstuff for animals and in this study the pretreatment times was modified to
30 min for anaerobic digestion of feathers resulted in 96.8 % of the theoretical potential
methane productivity. This shorter treatment time is safer for AD process and more
profitable from economical point of view.
5.3 Effect of biological treatments on SCOD concentration (Exp.3)
In this series of the experiments the effect of the thermal, enzymatic, combined thermal-
enzymatic and combined chemical-enzymatic pretreatment on solublisation and methane
yield of chicken feather were investigated.
Fig. 21 shows the samples after enzymatic, chemo-enzymatic and thermo enzymatic
pretreatment.
Fig. 21. Enzymatic, chemo-enzymatic and thermo-enzymatic pretreated samples (Exp.3).
The average values for SCOD concentration of the pretreated samples are demonstrated
in the Fig. 22.
52
0
5000
10000
15000
20000
25000
30000
35000
40000
SCOD (mg/l)
Enzymatic Thermo-Enzymatic
Chemo-Enzymatic
Treated Samples
COD Concentration
Enzymatic
Thermo-Enzymatic
Chemo-Enzymatic
Fig. 22. Results of SCOD measurement for enzymatic and combined enzymatic
pretreated samples of Exp.3.
As seen in the Fig. 22 these methods of pretreatment solublised the feather and showed
positive effect on increasing the SCOD concentration of the samples. As seen in the
Fig.22 these methods of pretreatment solublised the feather and showed positive effect on
increasing the SCOD concentration of the samples. But in contrast to lime treatment,
where the highest relative SCOD release was around 1680 mg SCOD/g TS F here the
highest relative SCOD release value was 407 mg SCOD/g TS F produced by the chemo-
enzymatic treatments. It is still much lower than the relative SCOD release of 1040 mg
SCOD/g TS F for the recommended lime treatment conditions of 40g TS F/l liquid, 0.1g
Ca(OH)2/g TS F, 100°C and 30 min.
5.4 Effect of biological treatments on anaerobic digestion performance
Although combined enzymatic pretreatments could solublise feather and increase the
SCOD concentration, methane yield enhancement by these methods were also much
lower than those of lime pretreatment. Table 9 and Fig. 23 illustrate maximum methane
productivity of these samples during 50 days of anaerobic incubation:
53
Table 9. Results of SCOD and average maximum methane yield of triplicate thermal, enzymatic and combined enzymatic pretreated samples of Exp.3.
Average Normal vol CH4 EXP6- Feather
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0 10 20 30 40 50 60
Time (days)
N V
OL
(m3/
kg V
S) Untreated
Thermal
Enzymatic
Thermo-Enzymatic
Chemo-Enzymatic
Fig. 23. Average maximum methane production curves fort triplicate thermal, enzymatic
and combined enzymatic treated samples of Exp.3, during 50 days incubation.
samples
Feathers concentration
Treatment Conditions
SCOD Mg/L
Maximum Methane yield Nml/gVS
Percent of theoretical methane potential
1 Control, Untreated ---- 135 27.2%
2 Thermal, 120°C, 5 min
---- 143 28.8%
3
Enzymatic, 1%w enzyme/vial, 55°C, 2 h
18,640
154
31%
4
Thermal-Enzymatic, 120°C, 5min- 1%w enzyme/vial, 55°C, 24 h
32,760
185
37.3%
5
1g F/vial (1.5% VS)
Chemical-Enzymatic, 1%w enzyme /vial, 20mM/L Na2SO3 37°C, 150 rpm, 60 h
36,760
41
8%
54
As seen in Fig. 23 and Table 9 among these samples, sample 4 hydrolyzed under
combined thermal (120°C, 5min) and enzymatic (1%w enzyme/vial, 55°C, 24 h)
conditions , produced the highest volume of methane of 185 Nml/g VS, which is about
37.3% of the theoretical methane potential. Salminen et al. (2003) [30] have already done
similar thermal and combined biological assays, using another alkaline endopeptidase
[30]. They also studied the effect of the different pretreatment conditions such as time,
temperature, chemical and enzyme loading on the methane yield..
The methane production of all of these samples declined gradually after 33 days of 50
days of anaerobic incubation, probably due to the inhibition by ammonia resulted from
overloading of the system by organic substrate, and other toxicants [101].
According to many literatures disulfide bonds in keratin significantly reduces protein
digestibility [94] and Ramnani et al. (2006) found that “for complete easy degradation of
feather all enzymatic keratinolysis from any organism essentially needs to be assisted by
a suitable redox.” For instance savinase is capable of near complete feather degradation
(up to 96%) in the presence of sodium sulfite [95]. However, in the this study, although
chemical–enzymatic treatment by combination of savinase and sodium sulfite rendered a
considerable and higher increase in SCOD concentration of the sample than that of other
combined enzymatic treatments, its pretreated sample (sample 5) showed negative
methane potential and produced an average maximum of 41 ml methane/g VS (3 times
less than untreated sample and 8% of the theoretical methane potential) likely due to the
high degradation of some amino acids under the effect of the pretreatment method and
also quick formation of some inhibitory compounds during anaerobic digestion process
[101]. As a whole, methane productivity of this sample demonstrated a fast onset, a short
increasing period, and a few days steady state followed by a fast and continuous drop
after 12 days of 50 days anaerobic incubation.
55
5.5 Conclusion
Chemical treatment of chicken feather with lime rendered the most significant positive
effect on the enhancement of its methane yield during 50 days of anaerobic digestion.
Methane production was continuing even after 50 days incubation. In particular the
highest methane volume, 480 Nml/g VS, up to 96.8% of the theoretical methane
potential, was produced by pretreated sample under treatment conditions of 40g TS
feather/l initial feather concentration, and 0.1g Ca(OH)2/g TS F addition, at 100°C for 30
min, i.e. in the lowest concentration of feather, lime loading, treatment temperature and
shortest treatment time.
Moreover, according to the literatures the least amino acid degradation and also no
ammonia toxicity formation are expected under the recommended condition of
pretreatment [102,35].
Increasing the operational factors of the pretreatment, such as feather concentration, lime
loading, reaction time and temperature exerted positive effect on increasing the feather
degradation resulting in higher SCOD concentrations in the samples but rendered
negative impact on their methane yield. Probably the overloading of the system with
degraded feathers and amino acids resulted in ammonia accumulation and toxicity under
those conditions.
Compared to the lime treatment, other pretreatment methods such as thermal, enzymatic
and combined thermal-enzymatic didn’t show considerable positive effect on increasing
the methane productivity of pretreated chicken feathers, in contrast with their positive
effect on increasing SCOD concentration. Among the pretreated samples with these
methods, combined thermal (120°C, 5min) and enzymatic (1% w enzyme/vial, 55°C, 24
h) pretreated sample showed a comparatively higher methane yield than that of the others
and produced an average maximum of 185 Nml CH4/g VS (about 37.3% of the
theoretical methane potential) during 33 days of 50 days anaerobic digestion.
Although chemical–enzymatic treatment by combination of savinase and sodium sulfite
(1% w enzyme/vial, 20mM/L Na2SO3 , 37°C, 150 rpm, 60 h) rendered a noticeable and
also higher increase in SCOD concentration than those of thermal and other combined
enzymatic treated samples, chemical–enzymatic pretreated sample showed negative
56
methane potential and produced an average maximum of 41 Nml CH4/g VS i.e. 3 times
less than untreated sample and only 8% of the theoretical methane potential during 50
days of anaerobic incubation, likely due to the high degradation of some amino acids
under the effect of the pretreatment conditions which leaded to more and quick formation
of some inhibitory compounds (e.g. ammonia and H2S) during anaerobic digestion
process [41-43]. Further experiments must be performed to determine the inhibitory
agents and reasons for the low methane production of these samples, as well. Also the
effect of the treatment conditions such as temperature, reaction time, enzyme and sodium
sulfite loading, etc. on the anaerobic digestion performance of these samples should be
investigated in the future works.
As a whole, the results of the experiments performed in this study revealed that the less
feather loading results in more efficient anaerobic digestion process.
Therefore, considering the results of this study, simplicity of the treatment method and
also the low price of lime, lime treatment under the above mentioned optimal condition
can be suggested as the most feasible and the highest efficient pretreatment method to
enhance chicken feather methane potential through anaerobic digestion process.
5.6 Future work
In the present study anaerobic digestion for lime treated samples were carried out in a
batch mode. The effects of the lime treatment on the methane efficiency of the chicken
feathers can also be evaluated in a fill-and-draw or semicontinuous anaerobic digestion
process suggested in previous studies and literatures as a more efficient process than
batch system [105,103,106]. Application of this method in anaerobic digestion of lime
treated feather would also demonstrate the long time anaerobic digestion performance of
the treated samples.
Inhibitory agents for anaerobic digestion of thermal and enzymatic and combined
thermo-enzymatic pretreated samples and other probable reasons of the declining of their
methane productivity after 33 days, and also fast deviation in the methane yield of the
chemical enzymatic pretreated sample after 12 days should be determined through
57
performing further experiments. Moreover, optimization of the feather loading can be
performed and then the effect of the variation of the other treatment condition such as
temperature, reaction time, enzyme and sodium sulfite loading, etc. on the anaerobic
digestion performance can be further investigated.
58
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APPENDICES APPENDIX A: Tables and Data Figures for the Results of TS% & VS%
Measurement:
Table 10. The recorded weighs during TS measurement and the results for the TS% of the samples.
Table 11. The recorded weighs during VS measurement and the results for the VS%
of the samples.
Sample
Weight of oven dried (105°C) crucibles (g)
Total weight of air dried sample and dried(105°C) crucible (g)
Total weight of oven dried sample and crucible (105°C) (g)
Weight of dried sample (105°C) (g)
TS%
1
48.78
51.25
51.04
2.2593
91.40
2
47.67
50.21
49.98
2.3120
91.12
3
48.90
51.37
51.15
2.2535
91.35
Sample
Weight of oven dried (550°C) crucibles (g)
Total weight of oven dried (105°C) sample and dried (550°C) crucible (g)
Total weight of burned sample and crucible (550°C) (g)
Weight of burned sample (g)
VS% of TS%
VS%
1
44.84
45.62
44.67
0.0066
99.15
90.62
2
44.67
45.41
44.85
0.0041
99.44
91.06
3
45.00
45.74
45.00
0.0041
99.44
91.29
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APPENDIX B:
B.1 Data Figures and Tables for the Results of GC Measurements for
Lime Treated Samples: Tables below shows the average volume of methane produced by lime treated samples
containing 40g TS feather/l liquid during 50 days incubation, under thermophilic
condition:
SAMPLES
DAYS 4 7 12 15
Blank
0.061545110 0.107782159 0.166422791 0.254385275
Untreated -0.004482785 0.005514148 0.007283593 0.011975649
0,1g/g 30min
100°C 0.040896956 0.097728058 0.237959717 0.270855041
0,1g/g 30min
120°C 0.024326879 0.062333417 0.189068178 0.206595321
0,2g/g 1h 120°C 0.014278450 0.027579944 0.149519133 0.148542139
0,2g/g 2h 120°C 0.010159925 0.018492598 0.122586108 0.127801511
SAMPLES
DAYS 18 21 25 28
Blank 0.295161441 0.355546697 0.368813312 0.378083595
Untreated 0.008422816 0.004018749 0.011778914 0.025134312
0,1g/g 30min
100°C 0.315416032 0.323729309 0.350435115 0.371622572
0,1g/g 30min
120°C 0.237553138 0.227847347 0.278952618 0.278944583
0,2g/g 1h 120°C 0.140260389 0.107803545 0.157972583 0.145736381
0,2g/g 2h 120°C 0.132080761 0.093856274 0.149245047 0.120585316
69
Table 12. Results of average methane yields for lime treated samples containing 40g TS
feather/l liquid, during 50 days incubation under thermophilic condition.
B.2 Data Figures and Tables for the Results of GC measurements for
Biological and Combined Biological treated samples:
Tables below shows the average volume of methane produced by thermal, enzymatic and
combined enzymatic samples sample under thermophilic condition, during 50 days
anaerobic digestion:
SAMPLES
DAYS 18 22 29 33
Blank 0.009014246 0.014617669 0.021896883 0.035878597
Untreated 0.006225877 0.009859087 0.022840392 0.040670314
Thermal 0.007200646 0.011673398 0.023989006 0.040484616
Enzymatic 0.020873279 0.037026724 0.062039793 0.089158365
Thermo-Enzymatic 0.022282808 0.044268167 0.079669038 0.117232248
Chemo-Enzymatic 0.030937420 0.040294133 0.041024846 0.032207047
SAMPLES
DAYS 32 39 47 50
Blank 0.411118003 0.433809509 0.463857722 0.510750945
Untreated 0.047009350 0.007942775 0.027654587 0.047366399
0,1g/g 30min 100°C 0.367779852 0.403629101 0.456906043 0.480531477
0,1g/g 30min 120°C 0.299425868 0.296685305 0.316122637 0.338409710
0,2g/g 1h 120°C 0.121931338 0.173767625 0.209098577 0.230633498
0,2g/g 2h 120°C 0.094253524 0.123459981 0.158209156 0.122153496
70
SAMPLES
DAYS 18 22 29 33
Blank 0.044965396 0.058197332 0.076712876 0.085083086
Untreated 0.057649437 0.087810093 0.120080353 0.135370181
Thermal 0.059607799 0.086200427 0.1239123 0.14380032
Enzymatic 0.112290231 0.135679498 0.1482558 0.153980185
Thermo-Enzymatic 0.145019907 0.16877803 0.179538023 0.185265871
Chemo-Enzymatic 0.022700818 0.015066339 0.005187397 0.015728094
Table 13. Results of average maximum methane yields for thermal, enzymatic and
combined enzymatic samples under thermophilic condition, during 50 days incubation.
SAMPLES
DAYS 38 43 50
Blank
0.102888315 0.117800289 0.135924701
Untreated 0.134891874 0.12598066 0.116355492
Thermal 0.141166233 0.13913784 0.132497512
Enzymatic 0.150328111 0.147217018 0.134521632
Thermo-Enzyma 0.180084479 0.178922111 0.164682812
Chemo-Enzymat -0.028445423 0.044213761 -0.059780228
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