Application of Various Pretreatment Methods to Enhance Biogas Potential of Waste Chicken Feathers

Azar Khorshidi Kashani

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, az_khorshidi@yahoo.com

Master thesis Subject Category: Environmental Engineering

University College of Bors School of Engineering SE-501 90 BORS Telephone +46 033 435 4640

Examiner: Supervisor,name: Supervisor,address:

Ilona Srvri Horvth Ilona Srvri Horvth University of Bors, School of Engineering SE-501 90 Bors

Date: Keywords:

2009-09-21 Chicken feathers, Keratin protein, Biogas potential, Lime treatment, Enzymatic treatment, Chemo-enzymatic treatment

ACKNOWLEDGEMENTS
This thesis work has been performed at Department of Chemical Engineering at Faculty of Engineering, University of Bors, 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.

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, 100C 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 didnt 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. 4

TABLE OF CONTENTS
Acknowledgement ......3 Abstract....4 Table of content5-6 List of tables.7 List of figures8-9 Abbreviations.10 Chapter1. Introduction...11 1.1 Background..11 1.1.1 Renewable Energy for a Sustainable Future11-12 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 pretreatments26-26 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 Methods30 3.3.1. Preparation of Waste Chicken Feathers...30 3.3.2. Inoculum .........................30

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 Processes36 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% Measurement67 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

LIST OF TABLES
Table 1. 2. 3. 4. 5. 6. 7. 8. 9. page Typical composition of biogas...14 Some biogas equivalents....14 Temperature ranges and optima for various anaerobic populations..17 Calculation of general theoretical methane potential for fat, protein and carbohydrate using average chemical formulas ........................................23 Thermo-chemical treated samples and treatment conditions.33 Results of SCOD and average maximum methane yields of triplicate lime treated samples of Exp.1, during 50 days of incubation44 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 Results of SCOD and average maximum methane yields of triplicate lime treated samples of Exp.2 , during 15 days of incubation..48 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. 11. 12. 13. The recorded weighs during TS measurement and the results for the TS% of the samples...67 The recorded weighs during VS measurement and the results for the VS% of the samples. ..67 Results of average methane yields for lime treated samples containing 40g TS feather/l liquid, during 50 days incubation under thermophilic condition...68-69 Results of average maximum methane yields for thermal, enzymatic and combined enzymatic samples under thermophilic condition, during 50 days incubation.69-70

LIST OF FIGURES
Figure 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Page Global energy consumption from 1965 to 2030..11 Global energy consumption by fuel type from 1965 to 2030..12 Potential pathway for biofuel production13 Deployment of anaerobic digestion in the EU and the world..15 Degradation of carbon in the anaerobic digestion process described by 4 steps: Hydrolysis, Acidogenesis, Acetogenesis and methanogenesis...17 Chicken feathers image......20 Degradation pathways during anaerobic digestion....22 Keratin molecular structure24 Protein hydrolysis during thermo-chemical treatment...25 COD Reactor with Direct Reading Spectrophotometer for SCOD measurement of pre-treated samples..35 Samples maintained in the incubator at 55C for anaerobic digestion process.37 Autosystem Gas Chromatograph with TCD for measurement of produced methane and carbon-dioxide..................................................................................38 Results of SCOD measurement for lime treated samples containing 40gTS F/l initial concentration (Exp.1) under various treatment conditions..41 Results of SCOD measurement for lime treated samples containing 40gTS F/l initial concentration (Exp.1) with higher lime loadings at 120C and for 2h....... 42 Results of SCOD measurement for lime treated samples containing 100gTS F/l initial concentration (Exp. 2) under various treatment conditions.... 43 Results of SCOD measurement for lime treated samples of Exp. 2 with higher lime loadings at 120C and for 2h.....................................................43 Results of SCOD measurement for lime treated samples of Exp.1 selected for anaerobic digestion process..44 Average maximum methane production curves for triplicate lime treated samples of Exp.1, during 50 days of incubation...45 Average maximum methane production curves for triplicate lime treated

samples 4 and 5 of Exp. 1, during 50 days of incubation (liquid phase) ...47 20. 21. 22. 23. Average maximum methane production curves for triplicate lime treated samples of Exp. 2, during 15 days of incubation..49 Enzymatic, chemo-enzymatic and thermo-enzymatic pretreated samples (Exp. 3)..51 Results of SCOD measurement for enzymatic and combined enzymatic samples of Exp.3 containing ....52 Average maximum methane production curves for triplicate thermal, enzymatic and combined enzymatic treated samples of Exp.3, during 50 days of incubation...53

ABBREVIATIONS F...Feathers VSVolatile Solids TSTotal Solids Std...Standard R..Ideal Gas Constant P(atm).Atmospheric Pressure T..Temperature K..Kelvin (Standard Temperature Unit) CODChemical oxygen demand SCOD. Soluble Chemical Oxygen Demand ADAnaerobic 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 Methane, CH4 Carbon dioxide, CO2 Nitrogen, N2 Hydrogen, H2 Hydrogen sulfide, H2S Oxygen, O2

Percentage 50-75 25-50 0-10 0-1 0-3 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 biowastes 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 Lighting Cooking Fuel replacement Shaft power Electricity generation

1m3 biogas equivalent 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 2030%. 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 15

nutrient content fertilizers to use in agriculture [11]. Furthermore, biogas production has no geographical limitations and doesnt 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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Particulate organic matter Protein Carbohydrates lipids

Hydrolysis
Soluble organic matter Amino acids Sugars Fatty acids

Acidogenesis
Intermediary products Alcohol and VFAs

Acetogenesis
Acetate H2, CO2 CH4, CO2

Aceticlastic methanogenesis

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:
Fermentation optimum Psychrophilic Mesophilic Thermophilic Temperature range 0-20 C 15-45 C 45-75 C Temperature 15C 35C 55C

Table 3. Temperature ranges and optima for various anaerobic populations [22,23,18]. 17

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 [68]. 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 -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 (-oxidation) is

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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:

Carbohydrates

Protein Amino acids

lipids Long-chain Fatty acids

Hydrolysis
Sugars Acidogenic fermentation Ammonia Volatile fatty acids other than acetic acid Beta oxidation

Acetogenic oxidation Acetic acid Homoacetogenesis Acetotrophic mathanogenesis Methane Hydrogenotrophic methanogenesis Hydrogen

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 Buswells formula [62] as shown in Table 4:

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Component Fat Protein Carbohydrate

Chemical formula C57H104O6 C5H7NO2 (C6H10O5)n

Theoretical biogas potential 3 (Nm CH4 per ton VS) 1014 496 415

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 -keratin, and birds feather contains -keratin. The polypeptides in -keratin are closely associated pairs of helices, whereas -keratin has high proportion of pleated sheets. This conformation confers an axial distance between adjacent residues of 0.35 nm in -sheets, compared to 0.15 nm in a-helices. The sheets have a far more extended conformation than the 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, waterinsoluble and protease-resistant [80]. Such a molecular structure makes feathers poorly degradable under anaerobic digestion condition [31,37]. Fig. 8 shows keratin molecular structure:

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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. Its 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 pretreatment 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].

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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].

PROTEIN

-keratin (hair), -keratin, animal tissue, plant matter.

HYDROLYSIS

DEAMIDATION GLN

Peptide bond is broken. Smaller peptides and free amino acids are generated.

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)

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].

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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 -keratin. These enzymes help the bacteria to obtain carbon, sulfur and energy for their growth and maintenance from the degradation of -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

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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].

27

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.

28

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 250l gas tight glass syringe with a pressure lock to take fixed volume and pressure samples from the reactors. Regulated incubator at 55C 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 1053C for TS measurement of feather Muffle furnace with temperature control of 550C for VS measurement of feather. Autoclave for thermal pre-treatments of samples Shaking water bath regulated at 37C 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-Bors. Lime for thermo-chemical treatment.

29

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 105C3. 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, SobackenBors and stored at 55C in an incubator for 3 days in order to readapt the inoculum to 55C, 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 Total Solid in Biomass (LAP-001) [98] as follows: -Crucibles were dried in drying oven 105C3 over the night and were weighed accurately to the nearest 0.1 mg and the weight was recorded. the

Laboratory Analytical Procedure (LAP-001), Standard Method for Determination of

30

-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 1053C 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 105C dried sample = weight 105C 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 Ash in Biomass [99] as follows: -Crucibles were heated at 550C10 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 (105C) 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 550C10 for 3 hours, reheated and reweighed to constant weight till varies by less than 0.3 mg. feather was measured according to the

Laboratory Analytical Procedure (LAP-005), Standard Method for Determination of

31

-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 105C 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 105C 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:

32

Exp. Feather Number Concentration (g TS F/l liquid)

40

Lime Autoclave loading Temperature (g/g TS F) (C) 0.1 0.2 0.4 100, 110, 120 1 2 4 0.1 0.2 0.4 1 2 100, 110, 120

Time (min)

30,60,120

100

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:

33

1- 0.1g lime /g TS feather, 30 min, 100C: 2g TS feather + 48 ml water + 0.2g lime 2- 0.1g lime /g TS feather, 30 min, 120C: 2g TS feather + 48 ml water + 0.2g 3- 0.2g lime /g TS feather, 1 h, 120C: 2g TS feather + 48 ml water + 0.4g lime 4- 0.2g lime /g TS feather, 2 h, 120C: 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, 120C: 5g TS feather + 45 ml water + 0.5g lime 2- 0.2g lime /g TS feather, 2h, 120C: 5g TS feather + 45 ml water + 1g lime 3- 1g lime /g TS feather, 2h, 120C: 5g TS feather + 45 ml water + 5g lime 4- 2g lime /g TS feather, 2h, 120C: 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: 34

1- Thermal treatment: autoclaving for 5min at 120C 0.9g TS feather + 9.1 g potassium phosphate buffer solution 2- Enzymatic treatment: incubation for 2h at 55C 0.9g TS feather + 9g potassium phosphate buffer solution + 100mg enzyme (1% w enzyme/vial) 3- Thermal-Enzymatic treatment: autoclaving feather for 5min, at 120C, followed by buffer and enzyme addition and incubation for 24h at 55C 0.9g TS feather + 9g potassium phosphate buffer solution + 100mg enzyme (1% w enzyme/vial) 4- Chemical-Enzymatic treatment: water bath for 60h at 37C 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.

35

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 36

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 waterphase [101]. The samples were then incubated at 55C for 50 days (Fig. 11).

Fig. 11. Samples maintained in the incubator at 55C 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).

37

Gas samples of 250l 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.

38

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 ( p (s)) in the sample and ( p(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 = 22C = 295 K, Atmospheric Standard Pressure, Patm= 101325 Pa, R (Ideal Standard Gas Constant) =8.314, Sample (syringe) Volume (Vs) = 250 l,

39

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.

40

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:

SCOD Concentration
60000 50000 40000 100C 110C 120C

SCOD (mg/l) 30000

20000 10000 0

0g/g 0,1g/g 0,2g/g (30m) (30m) (30m)

0g/g 0,1g/g 0,2g/g (1h) (1h) (1h)

0g/g 0,1g/g 0,2g/g (2h) (2h) (2h)

Lime conc., Time

Fig. 13. Results of SCOD measurement for lime treated samples containing 40gTS F/l initial concentration (Exp. 1), under various treatment conditions.

41

SCOD concentration (120C, 2h)

53000 52000 51000 50000 0,4 g/g 1 g/g 2 g/g 4 g/g

SCOD (mg/l) 49000

48000 47000 46000 45000 0,4 g/g 1 g/g 2 g/g 4 g/g

Lime Conc. (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 120C 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, 100C, 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, 120C, 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 120C 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 didnt 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) 42

SCOD Concentration
160000 140000 120000 100000

SCOD (mg/l)

80000 60000 40000 20000 0 0g/g (1h) 0,1g/g (1h) 0,2g/g (1h) 0g/g (2h) 0,1g/g (2h) 0,2g/g (2h) 110C 120C

Lime conc., Time

Fig. 15. Results of SCOD measurement for lime treated samples containing 100gTS F/l initial concentration (Exp. 2) under various treatment conditions.

SCOD Concentration (120C, 2h)

170000 165000 160000 155000 SCOD 150000 (mg/l) 145000 140000 135000 130000 125000 0,4 g/g 1 g/g 2 g/g

0,4 g/g 1 g/g 2 g/g

Lime conc. (g/g TS)

Fig. 16. Results of SCOD measurement for lime treated samples of Exp. 2 with higher lime loadings at 120C 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, 120C, 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.

43

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 didnt 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:
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

2 3 4 5

40

Control, untreated 0.1g lime/gTS, 100C, 30 min 0.1g lime/gTS, 120C, 30 min 0.2g lime/gTS, 120C, 60 min 0.2g lime/gTS, 120C,120 min

--41600 55400 63100 67200 0.191 (0.765%)

47.4 480 338 230 123

9.6% 96.8% 68.1% 46.4% 24.8%

Table 6. Results of SCOD and average maximum methane yields of triplicate lime treated samples of Exp.1 during 50 days of incubation. 44

SCOD Concentration

70000 60000 50000 0.1g/g, 100C, 30min 0.1g/g, 120C, 30min 0.2g/g, 120C, 1h 0.2g/g, 120C, 2h

SCOD (mg/l)

40000 30000 20000 10000 0 0.1g/g, 100C, 30min 0.1g/g, 0.2g/g, 120C, 120C, 30min 1h 0.2g/g, 120C, 2h

Lime conc., te mp., time

Fig. 17. Results of SCOD measurement for lime treated samples of Exp. 1, selected for anaerobic digestion process.

Methane Normal Volume

0.6 Untreated 0.5

N VOL (m3/kg VS)

0.4 0.3 0.2 0.1 0 0 -0.1 10 20 30 40 50 60

0,1g/g 30min 100C 0,1g/g 30min 120C 0,2g/g 1h 120C 0,2g/g 2h 120C

Time (days)

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, 100C and 30 minutes) conditions demonstrated the highest 45

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 100C) 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

46

the methane yields of these samples were observed, up to 15.7% increase for sample 4 and 51.2% for sample 5.

Sample

Pretreatment Feathers Concentration (g TS/l liquid)

Pretreatment Conditions

SCOD (Mg/L)

Concentration of substrate in vials

Maximum Methane yield Nml/gVS

Percentage of theoretical methane potential

g VS/Vial Control, untreated --63100 67200 0.191 (0.765%)

1 40

47.4

9.6%

0.2g lime/g TS, 120C, 60 min 0.2g lime /g TS,

266 186

53.6% 37.5%

120C, 120min

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

0.3 0.25

N VOL (m3/kg VS)

0.2 0.15 0.1 0.05 0 0 -0.05 10 20 30 40 50 60

Untreated

0,2g/g 1h 120C 0,2g/g 2h 120C

Time (days)

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).

47

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:

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

Control, untreated 0.1g lime /g TS,

--114200 153800 162500 168500

0.453

118 139 53 23 20

23.8% 28% 10.7% 4.6% 4.0%

2 100 3

120C, 60 min 0.2g lime/g TS, 120C, 60 min 1g lime /g TS, 120C, 60 min 2g lime /g TS, 120C, 60min (1.8%)

Table 8. Results of SCOD and average maximum methane yields of triplicate lime treated samples of Exp. 2, during 15 days of incubation.

48

Methane Normal Volume

0.16 0.14 Untreated 0,0g/g 1h 120C 0,2g/g 1h 120C 0,1g/g 1h 120C 1g/g 1h 120C 2g/g 1h 120C 0 5 10 15 20

N VOL (m3/kg VS)

0.12 0.1 0.08 0.06 0.04 0.02 0

Time (days)

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

49

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]. Lopez 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 Lopez 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, 100C, 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, 100C and

50

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 thermalenzymatic 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.

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COD Concentration

40000 35000 30000 25000 Enzymatic Thermo-Enzymatic Chemo-Enzymatic

SCOD (mg/l) 20000

15000 10000 5000 0 Enzymatic ThermoEnzymatic ChemoEnzymatic

Treated Samples

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 chemoenzymatic 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, 100C 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:

52

samples

Feathers concentration

Treatment Conditions

SCOD Mg/L

Maximum Methane yield Nml/gVS

Percent of theoretical methane potential

1 2 3 1g F/vial 4
(1.5% VS)

Control, Untreated Thermal, 120C, 5 min Enzymatic, 1%w enzyme/vial, 55C, 2 h Thermal-Enzymatic, 120C, 5min1%w enzyme/vial, 55C, 24 h Chemical-Enzymatic, 1%w enzyme /vial, 20mM/L Na2SO3 37C, 150 rpm, 60 h

------18,640

135 143 154

27.2% 28.8% 31%

32,760 36,760

185 41

37.3% 8%

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

0.2

0.15

N VOL (m3/kg VS)

Untreated 0.1 Thermal Enzymatic Thermo-Enzymatic 0 0 -0.05 10 20 30 40 50 60 Chemo-Enzymatic

0.05

-0.1

Time (days)

Fig. 23. Average maximum methane production curves fort triplicate thermal, enzymatic and combined enzymatic treated samples of Exp.3, during 50 days incubation.

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As seen in Fig. 23 and Table 9 among these samples, sample 4 hydrolyzed under combined thermal (120C, 5min) and enzymatic (1%w enzyme/vial, 55C, 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 chemicalenzymatic 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.

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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 100C 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 didnt 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 (120C, 5min) and enzymatic (1% w enzyme/vial, 55C, 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 chemicalenzymatic treatment by combination of savinase and sodium sulfite (1% w enzyme/vial, 20mM/L Na2SO3 , 37C, 150 rpm, 60 h) rendered a noticeable and also higher increase in SCOD concentration than those of thermal and other combined enzymatic treated samples, chemicalenzymatic pretreated sample showed negative

55

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

56

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.

57

REFERENCES
[1] Overview of World Energy, Sigma Xi, the Scientific Research Society (2009) http://energy.sigmaxi.org/?p=551 [2] Energy facts, SEI (Solar Energy International), (2009) http://www.solarenergy.org/resources/energyfacts.html [3] Jingura R. M. and Matengaifa R., Optimization of biogas production by anaerobic digestion for sustainable energy development in Zimbabwe., Renewable and Sustainable Energy Reviews, 13, 11161120 (2009). [4] Egger C., hlinger C. and Dell G., Fourteen percent of the total primary energy produced from biomass a success story from the highly industrialized region of Upper Austria., O.. Energiesparverband, http://www.esv.or.at [5] Ibrahim A., SCIENCE: In pursuit of sustainable energy. New Strait Times, Online (2009) [6] Biomass, Inter Academy Council (2009), http://www.interacademycouncil.net/CMS/Reports/11840/11928/11943.aspx [7] What is biogas?, Biogas Congress And Expo, (2009) http://eng.biogasinfo.ru/biogas/ [8]Dr.M. Seisle J., Rethinking sustainability and the biofuels policy.IANGV(International Association For Natural Gas Vehicles) (2008), [9] Demirbas M.F. and Balat M., Recent advances on the production and utilization trends of bio-fuels: A global perspective., Energy Conversion and Management 47, 23712381 (2006) [10] B ANANA M ETHANE P OWER ED C ARS , P IG P OO P OWER A ND O THER U SES F OR B IOGAS , PEAK ENERGY, http://peakenergy.blogspot.com/ [11] Biogas and liquid, Practical Action Technology challenging poverty (2009) http://www.wcasfmra.org/biogas_docs/biogas_liquid_fuels.pdf [12] - Kristoferson L. A., and Bokalders V., Renewable Energy Technologies - their application in developing countries. ITDG Publishing (1991) [13] Biogas Germany for Europe from energy maize, Institute of Science in Society (2008) http://www.i-sis.org.uk/theBiogasEconomyArrives.php

58

[14] Worldwide electricity production from renewable source, general forecasts, Stats and figures series, Tenth Inventory Edition 2008, chap 1. ObservER [15] Biogas electricity production hits 17 272GWh a year in Europe, Power Engineer Online Magasin Waste To Energy; Engineer live (2009) http://www.engineerlive.com/Power [16] Taleghani, G. and Kia, A.S., Technicaleconomical analysis of the Saveh biogas power plant, Renewable Energy 30, pp. 441446 (2005) [17]The Modern Importance of Biogas, http://www.habmigern2003.info/biogas/biogas.html [18] DAVIDSSON ., Increase of biogas production at wastewater treatment plants. Ph.D thesis, Lund University, Lund, Sweden (2007) [19] Yadvika, Santosh, Sreekrishnan T.R., Kohli S. and Rana V., Enhancement of biogas production from solid substrates using different technique-a review., Bioresource Technology 95, 1-10 (2004) [20] Garcia-Heras J.L., Reactor sizing, process kinetics and modeling of anaerobic digestion of complex wastes. In Mata-Alvarez, j. (ed.). Biomethanization of the organic fraction of municipal solid wastes. IWA publishing. ISBN 1-90022-14-0 (2002) [21] Gujer W. and Zender A.J.B., Conversion processes in anaerobic digestion. Water Science and Technology, Vol. 15, No. 8-9, pp. 127-167 (1983). [22] Tchobanoglous G., Theisen H. and Vigil S., Integrated Solid Waste Management: Engineering Principles and Management Issues, McGraw Hill, New York. ISBN 0-07112865-4 (1993) [23] Prescott L.M., Harley J.p. and Klein D.A., Microbiology, 4th ed. WCB7 McGraw Hill, ISBN 0-697-35439-3 (1999) [23] Chynoweth D. P. and Isaacson R., Anaerobic digestion of biomass. Agricultural engineering department, University of Florida, Gainesville, Florida, USA. (1987) [24] Mosey F.E. and Fernandez X.A. Patterns of hydrogen in bio gas from the anaerobic digestion of milk-sugars. Water science and Technology, Vol. 21. No. 4-5, pp. 187-196 (1989). [25] S.K., Mishra, I.M., Sharma, M.P. and Saini, J.S.,. Effect of particle size on biogas generation from biomass residues. Biomass 17, pp. 251263 (1988) [26] Gollakota K.G. and Meher, K.K., Effect of particle size, temperature, loading rate and stirring on biogas production from castor cake. Biol. Wastes 24, pp. 243249 (1988)

59

[27] Moorhead K.K. and Nordstedt, R.A.,. Batch anaerobic digestion of water hyacinth: effects of particle size, plant nitrogen content and inoculum volume. Bioresour. Technol. 44 1, pp. 7176 (1993) [28] Hobson, P.N., Problems and solution in full-scale biogas plant concerned with feedstocks and effluents. Anaerobic digestion 1988, 5th International Symposium on anaerobic digestion, eds Hall, E.R. and Hobson, P.N. (1988) [29] http://grendelgravenstein.com/Grimoires.html [30] Salminen E., Einola J. and Rintala J., The methane production of poultry slaughtering residues and effect of pretreatments on the methane productivity of poultry feather., Environmental Technology 24, 1079-1086 (2003) [31] Salminen E. and Rintala J., Semi-continuous anaerobic digestion of solid poultry slaughterhouse waste: effect of hydraulic retention time and loading., Water Research 36 31753182 (2002) [32] Poopathi S, Abidha S., "Use of feather-based culture media for the production of mosquitocidal bacteria". Biological Control 43 (1): 4955 (2007) doi:10.1016/j.biocontrol.2007.04.019. [33] Schmidt W.F., Barone J.R., New uses for chicken feathers keratin fiber. Poultry Waste Management Symposium Proceedings. pp. 99101 (2004) [34] Casarin, Franciani; Florencia Cladera-Olivera & Adriano Brandelli, "Use of Poultry Byproduct for Production of Keratinolytic Enzymes". Food and Bioprocess Technology 1 (3): 301305. doi:10.1007/s11947-008-0091-9 26 (2008) [35] Coward-Kelly G., Vincent S. Chang, Frank K. Agbogbo, Mark T. Holtzapple, Lime treatment of keratinous materials for the generation of highly digestible animal feed: 1. Chicken feathers., Bioresource Technology 97,13371343 (2006) [36] Tamilmani1 P., Umamaheswari A., Vinayagam1 A. and B. Prakash, Production of an Extra Cellular Feather Degrading Enzyme by Bacillus licheniformis Isolated from Poultry Farm Soil in Namakkal District (Tamilnadu), International Journal of Poultry Science 7 (2): 184-188, (2008) [37] Weidele. T., Methode for using biomass in biogas process, US patent online, Pub. No. US 2009/0035834 A1 (2009) [38] Onifade A.A., Al-Sane N.A., Al-Musallam, A.A., Al-Zarban, S., A review, potential for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins of livestock feed resources. Bioresour. Technol. 66, 111.l (1998)

60

[39] Papadopoulos M.C., El Boushy A.R., Ketelaars, E.H., Effect of different processing conditions on amino acid digestibility of feather meal determined by chicken assay. Poult. Sci. 64, 17291741 (1985) [40] Salminen E. and Rintala J., Anaerobic digestion of organic solid poultry slaughterhouse waste a review, Bioresource Technology 83, 1326 (2002) [41] Koster I.W., Toxicity in anaerobic digestion with emphasis on the effect of ammonia, sulfide and long-chain fatty acids on methanogenesis. Ph.D. Thesis, Wageningen Agricultural University (1989) [42] McInerney M.J., Anaerobic hydrolysis and fermentation of fats and proteins. In: Zehnder, J.B. (Ed.), Biology of Anaerobic Microorganisms. Wiley, New York, pp. 373 415 (1988) [43] Zinder S.H., Microbiology of anaerobic conversion of organic wastes to methane: recent developments. ASM News 50, 294298 (1984) [44] Pavlostathis S.G., Giraldo-Gomez, E., Kinetics of anaerobic treatment: a critical review. Crit. Rev. Environ. Control 21, 411 490 (1991) [45] Dolfing J., Acetogenesis. In: Zehnder, J.B. (Ed.), Biology of Anaerobic Microorganisms. Wiley, New York, pp. 417468 (1988) [46] Vogels G.D., Keltjens J.T., Van der Drift C., Biochemistry of methane production. In: Zehnder, J.B. (Ed.), Biology of Anaerobic Microorganisms. Wiley, New York, pp. 707770 (1988) [47] Widdel, F., Microbiology and ecology of sulfate- and sulfur reducing bacteria. In: Zehnder, J.B. (Ed.), Biology of Anaerobic Microorganisms. Wiley, New York, pp. 469585 (1988) [48] Angelidaki I, Ahring BK. Thermophilic anaerobic digestion of livestock waste: effect of ammonia. Appl Microbiol Biotechnol 1993;38:5604. [49] De Baere LA, Devocht M, van Assche P. Influence of high NaCl and NH4Cl salt levels on methanogenic associations. Water Res 1984;18:5438. [50] Hansen KH, Angelidaki I, Ahring BK. Anaerobic digestion of swine manure: inhibition by ammonia. Water Res 1998;32:512. [50] Broughton MJ, Thiele JH, Birch EJ, Cohen A. Anaerobic batch digestion of sheep tallow. Water Res 1998;32: 14238. [51] Hanaki K, Matsuo T, Nagase M. Mechanism of inhibition caused by long-chain fatty acids in anaerobic digestion process. Biotech Bioeng 1981;23:1591610.

61

[52] Rinzema A. Anaerobic treatment of wastewater with high concentrations of lipids or sulfate. Ph.D. thesis, Wageningen Agricultural University, Wageningen, The Netherlands (1988) [53] Sayed SKI. Anaerobic treatment of slaughterhouse wastewater using UASB process. Ph.D. thesis, Wageningen Agricultural University, Wageningen,The Netherlands (1987) [54] Novak JT, Carlson DA. The kinetics of anaerobic long chain fatty acid degradation. J Water Pollut Control Fed 1970;42:193243. [55] Mackie RI, White BA, Bryant MP. Lipid metabolism in anaerobic ecosystems. Crit Rev Microbiol 1991;17: 44978. [56] Galbraith H, Miller TB. Physicochemical effects of long chain fatty acids on bacterial cells and their protoplasts. J Appl Bacteriol 1973;36:64758. [57] Galbraith H, Miller TB, Paton AM, Thompson JK. Antibacterial activity of long chain fatty acids and thereversal with calcium, magnesium, ergocalciferol and cholesterol. J Appl Bacteriol 1971;34:80313. [58] Hwu C-S, Donlon B, Lettinga G. Comparative toxicity of long-chain fatty acid to anaerobic sludges from various origins. Water Sci Technol 1996;34(56):3518. [59] Koster IW, Cramer A. Inhibition of methanogenesis from acetate in granular sludge by long-chain fatty acids. Appl Environ Microbiol 1987;53:4039. [60] Roy F, Albagnac G, Samain E. Influence of calcium addition on growth of highly purified syntrophic cultures degrading long-chain fatty acids. Appl Environ Microbiol 1985;49:7025. [61] Fukuzaki S, Nishio N, Shobayashi M, Nagai S. Inhibition of the fermentation of propionate to methane by hydrogen, acetate, and propionate. Appl Environ Microbiol 1990;56:71923. [62] Lin C-Y, Sato K, Noike T, Matsumoto J. Methanogenic digestion using mixed substrate of acetic, propionic and butyric acids. Water Res 1986;20:38594. [62] Buswell, E.G. and Neave, S.L. (1930). Laboratory studies of sludge digestion, Illinois Division of State Water Survey, Bulletin no. 30. [63] Christensen, T.H., Jansen, J. la Cour and Jrgensen, O. (2003). Datarapport om sammanstning og biogaspotentiale i organisk dagernovation. Milijproject Nr. 815, Milijstyrelsen. (In Danish)

62

[64] Angelidaki, I. (2002). Course literature at Environmental Biotechnology 12133, Environment & Resources, DTU, Technical University of Denmark. [65] Pencho Dalev, An enzyme-alkaline hydrolysis of feather keratin for obtaining a protein concentrate for fodder., Biotechnology Letters Vol 12 No 1 71.-72 (1990) [66] Dalev P.G., Utilization of waste feathers from poultry slaughterhouse for production of a protein concentrate. Biores. Technol., 48, 265-267 (1994). [67] Morris, A.L., MacArthur, M.W., Hutchinson, E.G., Thornton, J.M.,1992. Stereochemical quality of protein structures. Proteins 12,345364. [68] Acamemic Technology, Itech Server, itech.dickinson.edu, http://images.google.com/images?hl=en&q=keratin+structure&um=1&ie=UTF-8 [69] Bourne, T.F., 1993. Biodegradation of keratins and phenolic compounds. Ph.D. Thesis, Georgia Institute of Technology. [70] Barret, G.C., 1985. Chemistry and Biochemistry of the Amino acids. Chapman & Hall, New York. [71] Penaud, V., Delgenes, J.P., Moletta, R., 1999. Thermo-chemical pretreatment of a microbial biomass: influence of sodium hydroxide addition on solubilisation and anaerobic biodegradability. Enzyme and Microbial Technology 25 (35), 258263. [72] Hyung Joo Suh1,3 and Hyo Ku Lee2, Characterization of a Keratinolytic Serine Protease from Bacillus subtilis KS-1., Journal of Protein Chemistry, Vol. 20, No. 2 (2001) [73] Cheng-gang CAI1, Bing-gan LOU2, Xiao-dong ZHENG1, Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis, Cai et 60 al. / J Zhejiang Univ Sci B 2008 9(1):60-67 [74] Onifade, A.A., Al-Sane, N.A., Al-Musallam, A.A., Al-Zarban, S., 1998. A review, potential for biotechnological applications ofkeratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins of livestock feed resources. Bioresour. Technol. 66, 111. [75] Wang, X., Parsons, C.M., 1997. Effects of processing systems on protein quality of feather meals and hog hair meal. Poult. Sci. 74, 491496. [76] Eggum, B.O., 1970. Evaluation of protein quality of feather meal under different treatments. Acta Agricul. Scand. 20, 230234. [77] Latshaw, J.D., Musharaf, N., Retrum, R., 1994. Processing of feather meal to maximize its nutritional value for poultry. Anim. Feed Sci. Technol. 47, 179188.

63

[78] Papadopoulos, M.C., El Boushy, A.R., Ketelaars, E.H., 1985. Effect of different processing conditions on amino acid digestibility of feather meal determined by chicken assay. Poult. Sci. 64, 17291741. [79] Barret, G.C., 1985. Chemistry and Biochemistry of the Amino acids. Chapman & Hall, New York. [80] Coward-Kelly, G, Frank K. Agbogbo a, Mark T. Holtzapple Lime treatment of keratinous materials for the generation of highly digestible animal feed: 2. Animal hair Bioresource Technology 97 (2006) 13441352 [81] Savitha G. Joshi, M.M. Tejashwini, N. Revati, R. Sridevi and D. Roma Isolation, Identification and Characterization of a Feather Degrading Bacterium., International Journal of Poultry Science 6 (9): 689-693, 2007 [82] Sangali, S. and Brandelli, A. (2000). Appl. Biochem. Biotechnol. 87, 1724. [83] Bockle, B., Galunsky, B., and Muller, R. (1995). Appl. Environ. Microbiol. 63, 37053710. [84] Zerdani, I., M. Faid and A. Malki, 2004. Feather wastes digestion by new isolated strains Bacillus sp. In Morocco. Afr. J. Biotechnol., 3: 67-70. [85] Ramnani, P., R. Singh and R. Gupta, 2005. Keratinolytic potential of Bacillus licheniformis RG1: structural and Biochemical mechanism of feather degradation. Can. J. Microbiol., 51: 191-196. [86] Korkmaz, H., H. Hur and S. Diyncer, 2004. Characterization of alkaline keratinase of Bacillus licheniformis strain HK-1 from poultry waste. Annals Microbiol., 54: 201211. [87] Manczinger, L., M. Rozs, V.G. Lgyi and F. Kevei, 2003. Isolation and characterization of a new keratinolytic Bacillus licheniformis strain World J. Microbiol. Biotechnol., 19: 35-39. [88] Williams, C.M., C.S. Richter, J.M. Mackenzie, J.R. Jason and C.H. Shih, 1990. Isolation, Identification and Characterization of a Feather-Degrading Bacterium. Appl. Environ. Microbiol., 56: 1509-1515. [89] Brandelli, A. and A. Riffel, 2006. Keratinolytic bacteria isolated from feather. Brazilian J. Microbiol., 37: 395-399. [90] Brandelli, A., 2005. Production of an extracellular keratinase Chryseobacterium sp. growing onraw feathers. Electronic J. Biotechnol., 8: 07173458. from

64

[91] Riffel, A., F. Lucas, P. Heeb and A. Brandelli, 2003. Characterization of a new keratinolytic bacterium that completely degrades Native feather keratin, 179: 258-65. [92] Bressollier, P., F. Letourneau, M. Urdaci and B. Verneuili, 1999. Purification and Characterization of a Keratinolytic Serine Proteinase from Streptomyces albidoflavus. Appl. Environ. Microbiol., 65: 2570-2576. [93] Montero-Barrientos, M., Rivas R, Velazquez, E. Monte and M.G. Roig, 2005. Terrabacter terrae sp. nov., a novel actinomycete isolated from soil in Spain. Int. J. Systematic Evolutionary Microbiol., 55: 2491-2495. [94] Goddard, D.R., Michaelis, L., 1934. A study on keratin. J. Biol. Chem. 106, 604614. [95] Priya Ramnani Rani Gupta Keratinases vis-a`-vis conventional proteases and feather Degradation, World J Microbiol Biotechnol (2007) 23:15371540 [96] Swan, J.M (1961) Austr. LChem., 14, 69 [97] Bockle B, Muller R (1997) Reduction of disulfide bonds by Streptomyces pactum during growth on chicken feathers. Appl Environ Microbiol 63:790792 [98] Standard Method for Determination of Total Solid in Biomass, Chemical Analysis and Testing Task, Laboratory Analytical Procedure (LAP-001). [99] Standard Method for Determination of Ash in Biomass, Chemical Analysis and Testing Task, Laboratory Analytical Procedure (LAP-005). (1994) [100] Raymond P. W. Scott, External Standard Method from Quantitative Chromatographic Analysis, part of the Chrom-Ed SeriesChromatography Book Series (online), Library 4 Science, Chromatography -Online.org http://www.chromatography-online.org/quant/Reference-Standards/GC-and-LC/ExternalStandard-Method.html [101] Hansena T. L., Schmidta J. E, Angelidakia I., Emilia Marcaa,Jes la Cour Jansenb, Mosbka H., Christensen T. H., Method for determination of methane potentials of solid organic waste., Waste Management 24 (2004) 393400 [102] Papadopoulos, M.C., 1989. Effect of processing on high-protein feedstuffs-A review. Biol. Wastes 29 (2), 123138. [103] Lopez, M., Espinosa, M.C., Escobedo, R., 2005. Estudio comparativo del pretratamiento qumico para mejorar la digestion anaerobia de residuos so lidos (Comparative study of chemical pretreatment to improve the anaerobic digestion of solid wastes). Revista CNIC. vol. 36, Numero Especial. La Habana. Cuba.

65

[104] Lopez, M., Espinosa, M.C., Effect of alkaline pretreatment on anaerobic digestion of solid wastes, Waste Management 28 (2008) 22292234 [105] Glossary of Energy Terms [106] Wang T-J. and Chen T-L., Lipase Production by Acinetobacter radioresistens in a Batch Fill-and-Draw Culture., Department of Chemical Engineering, National Cheng Kung University, 70101, Taiwan (1997) [107] Hobson, P.N. (1988). Problems and solution in full-scale biogas plant concerned with feedstocks and effluents. Anaerobic digestion 1988, 5th International Symposium on anaerobic digestion, eds Hall, E.R. and Hobson, P.N. [108] Asquith, R.S., 1977. Chemistry of Natural Protein Fibers. Plenum Press, New York, USA. [109] Mandal B., VVeeranki V.D., Alkaline Protease: a Tool to Clean Environment., Shampa Sen, Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati, India (2009) [110] Commission of the European Communities, 2000 Commission of the European Communities, 2000. White paper on food safety (presented by the Commission). Official Journal, No. C 076, 11.3.1997, pp. 14.

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APPENDICES
APPENDIX A: Tables and Data Figures for the Results of TS% & VS% Measurement:
Weight of oven dried (105C) crucibles (g) Total weight of air dried sample and dried(105C) crucible (g) Total weight of oven dried sample and crucible (105C) (g) Weight of dried sample (105C) (g)

Sample

TS%

48.78

51.25

51.04

2.2593

91.40

47.67

50.21

49.98

2.3120 91.12

48.90

51.37

51.15

2.2535 91.35

Table 10. The recorded weighs during TS measurement and the results for the TS% of the samples.
Weight of oven dried (550C) crucibles (g) Total weight of oven dried (105C) sample and dried (550C) crucible (g) Total weight of burned sample and crucible (550C) (g) Weight of burned sample (g) VS% of TS%

Sample

VS%

44.84

45.62

44.67

0.0066

99.15

90.62

44.67

45.41

44.85

0.0041

99.44

91.06

45.00

45.74

45.00

0.0041

99.44

91.29

Table 11. The recorded weighs during VS measurement and the results for the VS% of the samples.

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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

12

15

Blank Untreated 0,1g/g 30min 100C 0,1g/g 30min 120C 0,2g/g 1h 120C 0,2g/g 2h 120C

0.061545110 -0.004482785 0.040896956 0.024326879 0.014278450 0.010159925

0.107782159 0.005514148 0.097728058 0.062333417 0.027579944 0.018492598

0.166422791 0.007283593 0.237959717 0.189068178 0.149519133 0.122586108

0.254385275 0.011975649 0.270855041 0.206595321 0.148542139 0.127801511

SAMPLES

DAYS

18

21

25

28

Blank Untreated 0,1g/g 30min 100C 0,1g/g 30min 120C 0,2g/g 1h 120C 0,2g/g 2h 120C

0.295161441 0.008422816 0.315416032 0.237553138 0.140260389 0.132080761

0.355546697 0.004018749 0.323729309 0.227847347 0.107803545 0.093856274

0.368813312 0.011778914 0.350435115 0.278952618 0.157972583 0.149245047

0.378083595 0.025134312 0.371622572 0.278944583 0.145736381 0.120585316

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SAMPLES

DAYS

32

39

47

50

Blank Untreated 0,1g/g 30min 100C 0,1g/g 30min 120C 0,2g/g 1h 120C 0,2g/g 2h 120C

0.411118003 0.047009350 0.367779852 0.299425868 0.121931338 0.094253524

0.433809509 0.007942775 0.403629101 0.296685305 0.173767625 0.123459981

0.463857722 0.027654587 0.456906043 0.316122637 0.209098577 0.158209156

0.510750945 0.047366399 0.480531477 0.338409710 0.230633498 0.122153496

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:
DAYS 18 22 29 33

SAMPLES

Blank
Untreated Thermal Enzymatic Thermo-Enzymatic Chemo-Enzymatic

0.009014246 0.006225877 0.007200646 0.020873279 0.022282808 0.030937420

0.014617669 0.009859087 0.011673398 0.037026724 0.044268167 0.040294133

0.021896883 0.022840392 0.023989006 0.062039793 0.079669038 0.041024846

0.035878597 0.040670314 0.040484616 0.089158365 0.117232248 0.032207047

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SAMPLES

DAYS

18

22

29

33

Blank
Untreated Thermal Enzymatic Thermo-Enzymatic Chemo-Enzymatic

0.044965396 0.057649437 0.059607799 0.112290231 0.145019907 0.022700818

0.058197332 0.087810093 0.086200427 0.135679498 0.16877803 0.015066339

0.076712876 0.120080353 0.1239123 0.1482558 0.179538023 0.005187397

0.085083086 0.135370181 0.14380032 0.153980185 0.185265871 0.015728094

SAMPLES

DAYS

38

43

50

Blank Untreated Thermal Enzymatic Thermo-Enzyma Chemo-Enzymat

0.102888315 0.134891874 0.141166233 0.150328111 0.180084479 -0.028445423

0.117800289 0.12598066 0.13913784 0.147217018 0.178922111 0.044213761

0.135924701 0.116355492 0.132497512 0.134521632 0.164682812 -0.059780228

Table 13. Results of average maximum methane yields for thermal, enzymatic and combined enzymatic samples under thermophilic condition, during 50 days incubation.

70