application of various pretreatment methods to enhance ...1311501/fulltext01.pdfchicken feathers...

This thesis comprises 30 ECTS credits and is a compulsory part in the Master of Science with a Major in Environmental Engineering, 120 ECTS credits

No. 8/2009

Application of Various Pretreatment Methods to Enhance Biogas Potential

of Waste Chicken Feathers

Azar Khorshidi Kashani

Application of various pretreatment methods to Enhance Biogas Potential of Waste Chicken feathers

AZAR KHORSHIDI KASHANI, [email protected]

Master thesis

Subject Category: Environmental Engineering

University College of Borås School of Engineering SE-501 90 BORÅS Telephone +46 033 435 4640

Examiner: Ilona Sárvári Horváth

Supervisor,name: Ilona Sárvári Horváth

Supervisor,address: University of Borås, School of Engineering

SE-501 90 Borås

Date: 2009-09-21

Keywords: Chicken feathers, Keratin protein, Biogas potential, Lime treatment, Enzymatic treatment, Chemo-enzymatic treatment

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ACKNOWLEDGEMENTS

This thesis work has been performed at Department of Chemical Engineering at Faculty

of Engineering, University of Borås, Sweden. I would like to thank the supervisor of the

thesis Dr. Ilona Saravari Horvath for her guidance and assistance during this thesis work.

I'm also grateful to Dr. Dag Henriksson, Gergely Forgacs, Jonas Hanson and all other

enthusiastic people involved in helping me and supporting this work at the Department of Chemical Engineering.

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ABSTRACT Chicken feathers are the most abundant keratinous biomass in the world. Disposal of the

huge and increasing volume of waste feathers presents as a major concern for poultry

industry. On the other hand, energy and material recovery of this valuable protein source

is an important issue for organic solid waste treatment and bioenergy generation.

Anaerobic digestion is an environmentally and economically promising alternative

process for biogas production of waste feathers.

In this study in order to enhance the methane potential of batch anaerobic digestion of

chicken feathers this waste was treated by various kinds of pretreatments including

thermal, thermo-chemical, enzymatic, thermo-enzymatic and chemo-enzymatic methods.

Also the effect of different treatment conditions on the methane yield was investigated.

As a whole, thermo-chemical pretreatment with lime (Ca(OH)2) rendered the most

significant effect on enhancement of the chicken feathers methane potential. In particular

lime treated triplicate samples under treatment condition of 40g TS feather/l water, 0.1g

Ca (OH)2 /g TS feather, 100°C and 30 min produced the highest amount of methane (an

average maximum volume of 480 Nml/g VS, which is about 96.8% of the theoretical

methane potential of protein), during 50 days of anaerobic incubation. Increasing the

operational parameters such as feather concentration, lime loading, temperature and

reaction time improved the feathers solublisation resulting in a higher soluble chemical

oxygen demand (SCOD) concentration of the samples but inserted negative impacts on

the anaerobic digestion performance. Although other pretreatment methods improved the

SCOD concentrations of the feathers too, compared to the lime treatment those methods

didn’t show considerable effects on the enhancement of methane yield from the chicken

feathers. Thermo-enzymatic, enzymatic, and thermal pretreated triplicate samples

produced an average maximum of 185 Nml/g VS, 154 Nml/g VS, and 143 Nml/g VS

(37.3%, 31%, 28.8% of the theoretical methane potential) respectively, during 33 days of

50 days of anaerobic incubation. Especially, chemo-enzymatic pretreated sample showed

negative methane potential of only 41 Nml/g VS, i.e. 8% of the theoretical methane

potential. Consequently, lime pretreatment under the above recommended conditions can

be suggested for hydrolysis of chicken feathers to achieve significant enhancement of its

methane potential.

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TABLE OF CONTENTS

Acknowledgement ......……………………………………………………………………3

Abstract………….………………………………………………………………………...4

Table of content…………………………………………………………………………5-6

List of tables……………………………………………………………………………….7

List of figures……………………………………………………………………………8-9

Abbreviations…………………………………………………………………………….10 Chapter1. Introduction….………………………………………………………………..11

1.1 Background…………………………………………………………………………..11

1.1.1 Renewable Energy for a Sustainable Future……………………………………11-12

1.1.2 Biomass…………………………………….…………………………………..13-14

1.2. Biogas………………………...……………………………………………………..14

1.2.1 Biogas applications and benefits……………………………………………….14-16 1.2.2 Anaerobic Digestion Process……………………………………..…………….16-17

1.2.3 Environmental and operational parameters…………………………………….17-19

Chapter 2: Chicken Feather……………………………………………………………...20

2.1 Chicken Feather Waste Treatment……………………………………………….20-21

2.2 Anaerobic Digestion Process of Solid Poultry Slaughterhouse Waste…………..21-23

2.3 Specific Characteristic of Chicken Feathers and Keratin Protein …………………..23

2.4 Pretreatments methods for hydrolysis of poultry feathers…………………………...24

2.4.1 Hydrothermal pretreatments……………………………………………………26-26

2.4.2 Biological pretreatment………………………………………………………...26-27

2.4.3 Chemical-Biological pretreatment……………………………………………........27

2.5 Research Objectives…………………………………………………………….........28

Chapter 3: Materials and methods……………………………………………………….29

3.1 Equipments and apparatus………………………………….......................................28

3.2 Materials…………………………………………………...................................29-30

3.3 Methods………………………………………………………………………………30

3.3.1. Preparation of Waste Chicken Feathers…………………………………………...30

3.3.2. Inoculum ………………………………………………………….........................30

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3.3.3. Total Solids (TS%) and Volatile Solids (VS%) measurement………………...30-32

3.4 Pretreatment Methods………………………………………………………………..32

3.4.1 Thermo-Chemical Lime Pretreatment (Experiments 1, 2).. …………………...32-34

3.4.2 Biological Pretreatments (Experiment 3)…..…………………………………..34-35

3.5 Anaerobic Digestion Processes………………………………………………………36

3.5.1 Batch digestion process set-up for pretreated samples……................................36-38

Chapter 4: Calculation and Data Treatment………………………………………….39-40

Chapter 5: Results and discussion……………………………………………………….41

5.1 Effect of lime treatment on SCOD concentration (Exp.1, 2)…………………… 41-44

5.2 Effect of lime treatment on Anaerobic digestion performance (Exp. 1, 2) ….......44-51

5.3 Effect of biological treatments on SCOD concentration (Exp.3)...……………... 51-52

5.4 Effect of biological treatments on anaerobic digestion performance (Exp.3)……52-54

5.5 Conclusion……………………………………………………………………......55-56

5.6 Future work………………………………………………………………….........56-57

Reference……………………………………………………………………………..58-66

Appendices……………………………………………………………………………….67

Appendix A: Data Figures and Tables for the Results of TS% & VS% Measurement…67

Appendix B:

B.1 Data Figures and Tables for the Results of GC Measurements for Lime Treated

Samples………………………………………………………………………………..68-69

B.2 Data Figures and Tables for the Results of GC measurements for Biological and Combined Biological treated samples………………………………………………..69-70

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LIST OF TABLES Table page

1. Typical composition of biogas…………………………………………………...14

2. Some biogas equivalents………………………………………………………....14

3. Temperature ranges and optima for various anaerobic populations……………..17

4. Calculation of general theoretical methane potential for fat, protein and

carbohydrate using average chemical formulas ………........................................23

5. Thermo-chemical treated samples and treatment conditions…………………….33

6. Results of SCOD and average maximum methane yields of triplicate lime

treated samples of Exp.1, during 50 days of incubation…………………………44

7. Results of SCOD and average maximum methane yield of triplicate lime treated

samples 4 and 5 of Exp. 1, during 15 days of incubation, (liquid phase).……….47

8. Results of SCOD and average maximum methane yields of

triplicate lime treated samples of Exp.2 , during 15 days of incubation………..48

9. Results of SCOD and average maximum methane yields of

triplicate thermal, enzymatic and combined enzymatic samples

of Exp.3, during 50 days of incubation……………………………………….53

10. The recorded weighs during TS measurement and the results

for the TS% of the samples……………………………………………………...67

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

of the samples. ………………………………………………………………..67

12. Results of average methane yields for lime treated samples containing 40g TS

feather/l liquid, during 50 days incubation under thermophilic condition…...68-69

13. Results of average maximum methane yields for thermal, enzymatic

and combined enzymatic samples under thermophilic condition,

during 50 days incubation…………………………………………………….69-70

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LIST OF FIGURES Figure Page

1. Global energy consumption from 1965 to 2030………………………………..11

2. Global energy consumption by fuel type from 1965 to 2030……………………..12

3. Potential pathway for biofuel production………………………………………13

4. Deployment of anaerobic digestion in the EU and the world…………………..15

5. Degradation of carbon in the anaerobic digestion process described by

4 steps: Hydrolysis, Acidogenesis, Acetogenesis and methanogenesis………...17

6. Chicken feathers image………………………………………………………......20

7. Degradation pathways during anaerobic digestion.……………………………...22

8. Keratin molecular structure………………………………………………………24

9. Protein hydrolysis during thermo-chemical treatment…………………………...25

10. COD Reactor with Direct Reading Spectrophotometer for SCOD

measurement of pre-treated samples……………………………………………..35

11. Samples maintained in the incubator at 55°C for anaerobic digestion process….37

12. Autosystem Gas Chromatograph with TCD for measurement of produced

methane and carbon-dioxide..................................................................................38

13. Results of SCOD measurement for lime treated samples containing 40gTS F/l

initial concentration (Exp.1) under various treatment conditions………………..41


initial concentration (Exp.1) with higher lime loadings at 120°C and for 2h....... 42


initial concentration (Exp. 2) under various treatment conditions.……………... 43

16. Results of SCOD measurement for lime treated samples of Exp. 2 with higher

lime loadings at 120°C and for 2h..……………...................................................43

17. Results of SCOD measurement for lime treated samples of Exp.1

selected for anaerobic digestion process………………………………………..44

18. Average maximum methane production curves for triplicate lime

treated samples of Exp.1, during 50 days of incubation………………………...45

19. Average maximum methane production curves for triplicate lime treated

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samples 4 and 5 of Exp. 1, during 50 days of incubation (liquid phase)… …...47

20. Average maximum methane production curves for triplicate lime

treated samples of Exp. 2, during 15 days of incubation………………………..49

21. Enzymatic, chemo-enzymatic and thermo-enzymatic pretreated samples

(Exp. 3)…………………………………………………………………………..51

22. Results of SCOD measurement for enzymatic and combined

enzymatic samples of Exp.3 containing …….…………………………………...52

23. Average maximum methane production curves for triplicate

thermal, enzymatic and combined enzymatic treated samples of Exp.3,

during 50 days of incubation……………………………………………………...53

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ABBREVIATIONS

F…………………………...Feathers

VS…………………………Volatile Solids

TS…………………………Total Solids

Std.………………………..Standard

R…………………………..Ideal Gas Constant

P(atm)…………………….Atmospheric Pressure

T…………………………..Temperature

K…………………………..Kelvin (Standard Temperature Unit)

COD………………………Chemical oxygen demand

SCOD……………………. Soluble Chemical Oxygen Demand AD…………………………Anaerobic Digestion

SSOFMSW………………..Source-Sorted Organic Fraction of Municipal Solid Waste OECD……………………..Countries that are members of the Organization for

Economic Co-operation and Development

TCD………………………..Thermal Conductivity Detector

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Chapter 1: Introduction

1.1 Background

The Rapid growth of the world population combined with concomitant economic

development exerts drastic increase in global energy demand. World energy consumption

is projected to expand by 50 percent from 2005 to 2030. Although in general developed

(OECD) countries consume the most energy, demand for energy is increasing faster in

developing and emerging (Non-OECD) countries, resulted from their robust economic

progress and expanding populations. Fig. 1 illustrates world total energy consumption

and contribution of OECD and Non-OECD in world energy consumption from 1965 to

2030 [1].

Fig. 1. Global energy consumption from 1965 to 2030, [1].

1.1.1 Renewable Energy for a Sustainable Future

Currently the global mix of fuels comes from fossil (78%), renewable (18%) and nuclear

(4%) energy sources [2]. Fig. 2 demonstrates the global energy consumption by fuel

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type from 1965 to 2030. As indicated in Fig.2 conventional fossil fuels appropriate the

significant and highest portion of the global fuel consumption, likewise. However, these

fuels are non-renewable and finite resources releasing the highest amount of carbon

dioxide (CO2) and other greenhouse gases into the atmosphere and realized as the main

cause of global warming and climate change [3]. Fossil fuel combustion accounts for

62% of the global warming potential of all anthropogenic greenhouse gases [1].

Fig. 2.Global energy consumption by fuel type from 1965 to 2030, [1].

The above rising concerns beside the economical considerations such as increasing oil

price, reducing reliance on fossil fuels and worldwide potential economic development,

are potent incentives to incite global efforts and investments in promotion of sustainable

renewable and clean energy resources and technologies. Renewable energies including

geothermal, solar, wind, biomass, hydropower, ocean thermal, wave action, and tidal

action are utilizing in many energy fields such as electricity generation, transportation

fuels, industrial processes, heating , cooling and process steam. Although renewables

currently provide less than 10% of the world's energy, renewable energy sources have the

potential to exceed current global energy demands even with existing technologies [1].

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1.1.2 Biomass

Biomass as a major source of renewable energy accounts for about 14% of primary

energy consumption, and following oil, coal and natural gas is the fourth world-wide

energy resource. The world production of biomass is estimated at 146 billion metric tons

a year, mostly coming from wild plant growth [4,5].

The major resources of biomass are agricultural crops, plants and forestry residues,

organic components of municipal and industrial wastes and even the fumes from

landfills. Biomass can be converted to non-solid fuels form including liquid biofuel

(bioethanol and biodiesel) and gaseous biofuels (biogas, syngas,…). Fig. 3 Indicated

potential pathway for biofuel production.

Fig. 3. Potential pathway for biofuel production [6].

1.2 Biogas Biogas is the gaseous biofuel made through anaerobic digestion process or fermentation

of organic fraction of biomaterials. Biogas can be also captured from landfills. Almost all

kinds of organic and biodegradable materials such as municipal and industrial organic

wastes, sludge from sewage treatment plants and process water from the food industry,

energy crops and crop residues can be utilized as the resources for biogas production.

Biogas comprises from methane (CH4), carbon dioxide (CO2) and trace amounts of some

other components. Table 1 shows the typical composition of biogas.

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compound Percentage (%)

Methane, CH4 50-75

Carbon dioxide, CO2 25-50

Nitrogen, N2 0-10

Hydrogen, H2 0-1

Hydrogen sulfide, H2S 0-3

Oxygen, O2 0-2

Table 1.Typical composition of biogas [7]. 1.2.1 Biogas applications and benefits

Biogas is an environmentally friendly, clean, cheap and versatile fuel. Anaerobic

digestion substrate for biogas production can be obtained from almost all kinds of bio-

wastes and non-food based biomasses. Therefore biogas has no potential negative impact

on food chain products and prices, changes in land use and deforestation [8,9]. Combustion of biogas has less dangerous and neutral carbon dioxide emissions [10].

Moreover methane is a potent greenhouse gas, and hence capturing and burning it helps

environment from the global warming point of view. Biogas has a wide range of

applications e.g. in transportation, electricity production, cooking, space heating, water

heating and industrial process heating or even as a renewable feedstock to produce

hydrogen [8]. Table 2 shows some typical applications for one cubic meter of biogas.

Application 1m3 biogas equivalent

Lighting Cooking Fuel replacement Shaft power Electricity generation

equal to 60 -100 watt bulb for 6 hours can cook 3 meals for a family of 5 - 6 0.7 kg of petrol can run a one horse power motor for 2 hours can generate 1.25 kilowatt hours of electricity

Table 2. Some biogas equivalents [11,12].

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Europe seems to be the leader in the global production and use of biogas [10]. Fig. 4 shows the deployment of anaerobic digestion in the EU and the world from 1995 to 2010.

Fig. 4. Deployment of anaerobic digestion in the EU and the world [9].

UK studies have shown that biogas is much cleaner and more efficient than biofuels for

use in transport. According to an EU well-to-wheel study of more than 70 different (fossil

and renewable) fuels and energy paths, biogas is the cleanest and most climate-neutral

transport fuel of all [10]. “A natural gas vehicle reduces CO2 over a gasoline car by 20-

30%. A car running on bio-methane reduces CO2 on a well-to-wheel basis by more than

100%over a petroleum-fuelled car [8].”

Biogas along with fossil natural gas is currently fuelling over 800,000 cars, truck and

buses in Europe and nearly 8 million vehicles worldwide [8]. Compressed biogas

is becoming widely used in vehicles in Sweden, Switzerland and Germany [7]. “Sweden

has led the world in the usage of biogas in transportation since 1996. Biogas producers

are operating a fleet of city buses in Sweden. Strong government support is important, it

includes 30 percent investment support, zero tax, reduced income tax for company car

users, and no congestion fees in the capital city of Stockholm [1].”

Among biomass sub-sectors, solid biomas (72.5% biomass electricity) has increased by

an avarage of 5.8% per year from 1997 to 2007. However, growth in biogas electricity

has been much more considerable (an average of + 12.9% per year) [14]. European

Biogas electricity production in 2006 was 17272GWh per year, of which 7338GWh was

produced by Germany alone [15]. Beside biogas, anaerobic digestion produces high

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nutrient content fertilizers to use in agriculture [11]. Furthermore, biogas production has

no geographical limitations and doesn’t need sophisticated technology [16]. Biogas can

be produced even by a very basic construction using mostly used materials providing a

few simple design rules are followed. Moreover, biogas production is possible in small

scale sites, to obtain for outlying areas [17]. Accordingly, biogas is a 100% sustainable

fuel playing also a very important role in environmental friendly waste management and

organic waste disposal [8].

1.2.2 Anaerobic Digestion Process

Anaerobic digestion process for generation biogas occurs in four steps: Hydrolysis,

Acidogenesis, Acetogenesis and Metanogenesis. In the first step, hydrolysis, insoluble

and complex organic compounds such as lipids, polysaccharides, proteins, fats, nucleic

acids, etc. transform into soluble and simpler organic materials such as amino acids,

sugars and fatty acids by strict anaerobic hydrolytic bacteria [18,19]. In the acidogenesis

step obligate and facultative anaerobic group of bacteria (acidogens) ferments and

breakdown soluble products from the first step into acetic acid, hydrogen, carbon dioxide,

some volatile fatty acids (VFA) and alcohols. In the third step, acetogenesis, long chain

fatty acids and volatile fatty acids will be converted to acetate, hydrogen and carbon

dioxide by obligate hydrogen-producing acetogens [18]. Finally in the methanogenesis

step strict anaerobic methanogens convert acetic acid, hydrogen, carbon dioxide,

methanol and other compounds into a mixture of methane and carbon dioxide and other

trace gases (Table 1), [18,19]. Fig. 5 shows anaerobic digestion process in four steps:

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Fig. 5. Degradation of carbon in the anaerobic digestion process described by four steps:

Hydrolysis, Acidogenesis, Acetogenesis and Methanogenesis [20,21,18].

1.2.3 Environmental and operational parameters

Governing parameters such as temperature, pH, C:N ratio, hydraulic retention time

(HTR), stirring, organic loading rate (OLR), pretreatment, particle size, the presence of

toxicants, etc. can affect and control the anaerobic digestion process. Some of these

parameters may differ between different processes and different plants with various

feedstocks [18].

Temperature

Temperature has a significant impact on the biogas production process. The range of the

temperature differs for diverse kinds of fermentative bacteria:

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

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

Particulate organic matter Protein Carbohydrates lipids

Soluble organic matter Amino acids Sugars Fatty acids

Intermediary products Alcohol and VFAs

Acetate H2, CO2

CH4, CO2

Hydrolysis

Acidogenesis

Acetogenesis

Aceticlastic methanogenesis

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Although anaerobic digestion can be carried out both in the mesophilic and thermophilic

temperature range, thermophilic digestion systems results in more and faster biogas

production, and better pathogen and virus kill [9].

pH and Buffering Capacity

pH is an essential factor affecting the growth of microbes during anaerobic digestion. “To

maintain a dynamic equilibrium in the anaerobic system a pH between 6.5 and 7.5 is

desirable. [18]” ( or between 7 and 8, according to another literature [23].) At PH

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methods such as gas recirculation, mechanically stirring by mixing devices such as mixer,

scraper, piston, etc [19].

Particle size

Reducing the particle size of the feedstock by a physical pretreatment such as grinding

and milling increases the surface area for the contact between the substrate and active

bacteria [25], reduces the volume of digester [26,27] and enhances biogas yield.

Moreover, too large particles may result in clogging of the digester and making digestion

process difficult for bacteria [19].

Pretreatment

Due to the complexity of organic material, hydrolysis can be the rate limiting step for

anaerobic digestion process in cases that the substrate is in particulate form [18].

Therefore in this step physical, chemical and biological pretreatment of feedstock are

required to break down high molecular mass organic compounds into the simple and

more susceptible monomers for biodegradation. Pretreatment of substrate in rate limiting

step optimizes digestion process and increases the methane yield [19]. Pretreatment

methods are usually classified in following ways [18]:

(a) Chemical or thermo-chemical pretreatment of the feedstock with alkali or acid

(b) Biological pretreatment of fresh substrate through bacterial hydrolysis or enzyme

addition.

(c) Physical methods such as thermal treatment, high pressure, ultrasonic treatment,

milling, etc.

Toxicants

During digestion process some toxicant materials can have inhibitory effects on

methanogenic bacteria and consequently reduce the biogas yield. Toxicant may be

originated from the substrate or be produced during microbial breakdown [107]. The

most common and important toxic materials are free ammonia, high level of volatile fatty

acids, hydrogen, hydrogen sulphide (H2S). Besides, salts and xenobiotics can also be

inhibitory [18].

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Chapter 2: Chicken Feathers 2.1 Chicken Feathers Waste Treatment Poultry industry is continuously producing increasing amount of poultry meat and

noticeable quantities of organic residues such as feather, bone meal, blood, offal and so

on. Chicken feathers, making up about 5% of the body weight of poultry, is a

considerable waste product of the poultry industry being produced about 4 million tons

per year world-wide [30,31]. Disposal of waste feathers is a major concern for poultry

industry and accumulation of this huge volume of the waste feathers results in

environmental pollution and protein wastage.

Fig.6. Chicken feathers image [29].

Currently a minor quantity of waste feathers is used in other industrial applications such

as clothing, insulation and bedding [32], producing biodegradeable polymers [33] and

enzymes [34] and also as a medium for culturing microbes.

A higher quantity of pretreated feather is utilized to produce a digestible dietary protein

feedstuff for poultry and livestock [35-39]. However, to decrease the risk of disease

transmission via feed and food chain legislation on the recovery of organic materials for

animal feed is becoming tighter (Commission of the European Communities, 2000),

[40,110].

Hence development of other alternative methods to utilize enormous amount of feathers

and practical processes to fulfill these usages is inevitable [37]. Anaerobic digestion is an

environmentally and economically promising process to recover feather waste and other

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solid organic wastes to valuable materials such as biogas and fertilizers [40]. However,

slaughterhouse wastes are in general considered as difficult substrates for anaerobic

digestion because of their high protein and lipid content leading in production of some

by-products such as unionised ammonia, floating scum and accumulated log chain fatty

acids (LCFA) during anaerobic degradation, which are toxic and inhibitory to anaerobic

microorganisms in high concentrations [6–8]. Such practical difficulties have limited and

hindered the successful efforts on anaerobic digestion of feathers and other solid poultry

slaughterhouse wastes [31].

2.2 Anaerobic Digestion Process of Solid Poultry Slaughterhouse Waste

Solid poultry slaughterhouse waste is a complex substrate containing high quantity of

different proteins and lipids. Various bacteria take part in different steps of anaerobic

digestion of this waste. “In the hydrolysis step fermentative bacteria, especially the

proteolytic clostridium species, solublise proteins to polypeptides and amino acids. Lipids

are hydrolyzed to long chain fatty acids (LCFA) by β-oxidation and glycerol [41-43] and

polycarbohydrates to sugars and alcohols (Fig. 7), [41,44,43]. In the second step

“fermentative bacteria convert the intermediates to volatile fatty acids (VFAs), hydrogen

(H2), and carbon dioxide (CO2). Ammonia and sulphide are the by-products of amino

acid fermentation [41-43]. Hydrogen- producing acetogenic bacteria metabolize LCFAs,

VFAs with three or more carbons and neutral compounds larger than methanol to acetate,

H2, and CO2 (Fig. 7). As these reactions require an H2 partial pressure of ca. 10-3 atm,

they are obligately linked with micro-organisms consuming H2, methanogens, and some

acetogenic bacteria [45,43]. Methanogens ultimately convert acetate, H2 and, CO2 to

methane and CO2 (Fig. 7) [46,43]. In the presence of high concentrations of sulphate, H2

consuming acetogenic bacteria and sulphate reducing bacteria compete with methanogens

for H2 [47,43,40].

During this process produced ammonium from protein degradation dissociates to

unionised ammonia which is toxic and inhibitory to anaerobic microorganisms in high

concentrations [48-50]. Meanwhile Lipid degradation produces floating scum and

accumulated long-chain fatty acids (LCFA) [50-53]. “LCFA degradation (β-oxidation) is

22

considered a limiting step in the anaerobic degradation of complex organic substrates

[50-52,54] because LCFA oxidizing bacteria are slow growers [55] and because as

syntrophic substrates, like volatile fatty acids (VFA), their anaerobic microbial

degradation is limited by high hydrogen (H2) partial pressure [55, 43]. H2 is produced in

several steps in the anaerobic degradation of complex organic substrates and removed

from the process mainly by hydrogen-consuming methanogens and some acetogenic

bacteria [43]. Furthermore, in high concentrations LCFA [52,6-60] and unionized VFA

[61,62] are inhibitory to anaerobic microorganisms.” Consequently, to successfully

prevent LCFA and VFA accumulation in the anaerobic digestion of slaughterhouse

wastes determination the effect of the substrate loading and hydraulic retention time

(HRT) is in particular important [31]. Fig.7 illustrates degradation pathways in anaerobic

digestion process:

Hydrolysis Acidogenic Ammonia fermentation

Homoacetogenesis Acetotrophic mathanogenesis

Fig.7. Degradation pathways during anaerobic digestion. [41,44,43,40]

The theoretical methane potential for proteins, fat and carbohydrates can be calculated

using their component composition in Buswell’s formula [62] as shown in Table 4:

Carbohydrates Protein lipids

Sugars Amino acids Long-chain Fatty acids

Volatile fatty acids

other than acetic acid Beta oxidation

Hydrogen Acetic acid

Acetogenic oxidation

Hydrogenotrophic methanogenesis

Methane

23

Table 4. Calculation of general theoretical methane potential for fat, protein and

carbohydrate using average chemical formulas [63,64,18].

2.3 Specific Characteristic of Chicken Feathers and Keratin Protein

Chicken feathers are composed of over 90% of keratin protein, small amounts of lipids

and water. Feathers keratin consists of high quantities of small and essential amino acid

residues such as glycyl, alanyl and seryl as well as cysteinyl and valyl [65,66,30].

Keratin is also the main protein components of hair, wool, nails, horn, and hoofs. Animal

hair, hoofs, horns and wool contain α-keratin, and bird’s feather contains β-keratin. The

polypeptides in α-keratin are closely associated pairs of α helices, whereas β-keratin has

high proportion of β pleated sheets. “This conformation confers an axial distance between

adjacent residues of 0.35 nm in β -sheets, compared to 0.15 nm in a-helices. The β sheets

have a far more extended conformation than the α –helices” [67,108, 80].

Keratins are insoluble macromolecule comprises a tight packing of supercoiled long

polypeptide chains with a molecular weight of approximately 10 kDa. High degree of

cross linked cystin disulphide bonds between contiguous chains in keratinous material

imparts high stability and resistance to degradation [35-37,33]. Hence, a keratinous

material is a tough, fibrous matrix being mechanically firm, chemically unreactive, water-

insoluble and protease-resistant [80]. Such a molecular structure makes feathers poorly

degradable under anaerobic digestion condition [31,37]. Fig. 8 shows keratin molecular

structure:

Component Chemical formula Theoretical biogas potential (Nm3 CH4 per ton VS) Fat C57H104O6 1014 Protein C5H7NO2 496 Carbohydrate (C6H10O5)n 415

24

Fig. 8. Keratin molecular structure [68]. 2.4 Pretreatments methods for hydrolysis of poultry feathers

Because of the complex, rigid and fibrous structure of keratin, poultry feather is a

challenge to anaerobic digestion. It’s poorly degradable under anaerobic conditions.

[69,33] However, application of appropriate pretreatments methods hydrolyzes feather

and breaks down its tough structure to corresponding amino acids and small peptides

[70,35].

For more than half a century many studies have been performed and various pre-

treatment methods have been applied to improve the digestibility of feather meal as well

as development of its nutritional value for production of a dietary protein feedstuff for

animals [30,72,75]. These pretreatments methods may also enhance feather biogas

potential. However, only a few studies have been reported on this subject [30]. Feather

meal treatment methods are usually categorized into two groups: hydrothermal treatments

and microbial keratinolysis [74,35].

25

2.4.1 Hydrothermal pretreatments

Hydrothermal pre-treatment includes thermo-chemical treatment methods (such as acidic

hydrolysis and alkali hydrolysis), and also steam pressure cooking [35,73]. These

methods usually need high temperatures [75] or high pressure [76,77] with addition of

diluted acids such as hydrochloric acid [76] or alkali such as sodium hydroxide [78,35].

“Acidic solutions promote the loss of some amino acids such as tryptophan. [79]”

Although alkaline reactions are sometimes slower and may not go to completion,

degradation of some amino acids with hydroxide is less. Hence the use of bases is

recommended. A stepwise diagram for the hydrolysis of protein rich material under

alkaline condition is indicated in Fig. 9 [80].

Fig. 9. Protein hydrolysis during thermo-chemical treatment [80].

As a whole, hydrothermal hydrolysis usually consumes high amount of energy and

employs expensive equipment during lengthy processes (8 to 12 hrs), [65,37].

PROTEIN α-keratin (hair), β-keratin, animal tissue, plant matter.

HYDROLYSIS Peptide bond is broken.

Smaller peptides and free amino acids are generated.

DEAMIDATION GLN and ASN residue in protein

react and form GLU and ASP residues, with ammonia as a

product.

SMALLER PEPTIDES & FREE AMINO ACIDS Smaller peptides with a higher digestibility (structure) and

free amino acids are dissolved in the liquid phase.

DEGRADATION Several amino acids are not stable under alkaline conditions and

undergo reactions that generate different products (e.g. other amino acids, ammonia)

26

Thus, optimization of the treatment conditions is an important issue from technological

and economical points of view when applying this method.

2.4.2 Biological pretreatment

Biodegradation of feathers is another alternative method. Some bacterial strains can

produce keratinase proteases which have keratinolytic activity and are capable to

keratinolyse feather β-keratin. These enzymes help the bacteria to obtain carbon, sulfur

and energy for their growth and maintenance from the degradation of β-keratin [81].

Various keratinases from different microorganisms such as Bacillus sp. [84] Bacillus

licheniformis [85-88] Burkholderia, Chryseobacterium, Pseudomonas, Microbacterium

sp. [89] Chryseobacterium sp. [90,91] Streptomyces sp. [92,93] has been isolated and

studied to date [72, 81-83].

Microbial proteases are classified into acidic, neutral, or alkaline groups, depends on the

required conditions for their activity and on the characteristics of the active site group of

the enzyme, i.e. metallo-, aspartic- , cysteine- or sulphydryl- or serine-type. Alkaline

proteases which are active in a neutral to alkaline pH, especially serine-types, are the

most important group of enzymes used in protein hydrolysis, waste treatment and many

other industrial applications. Alkaline protease from Bacillus subtilis was used for the

keratinolysis of waste feathers [109].

Subtilisins are extracellular alkaline serine proteases, which catalyse the hydrolysis of

proteins and peptide amides. Savinase is one of these enzymes; Alcalase, Esperase and

Maxatase are others. These enzymes are all produced using species of Bacillus. Maxatase

and Alcalase come from B. licheniformis, Esperase from an alkalophilic strain of a B.

licheniformis, and Savinase from an alkalophilic strain of B. amyloliquefaciens [109]. An

important advantage of enzyme treatment method is fully biodegradability of enzymes by

themselves as proteins. Hence, unlike other remediation methods, there is no buildup of

unrecovered enzymes or chemicals that must be removed from the system at the end of

degradation process. Although enzymatic treatment is a promising technology; it has

some limitations and disadvantages, as well. Currently, the main disadvantage of using

alkaline proteases is the high cost of the enzymes production. Much of the cost of

27

producing enzymes is related to high purification of enzymes solutions to avoid the side

effects and side activities of the crude enzyme solution which is cheaper. Furthermore, in

contrast with microbes which can reproduce themselves and increase their population to

be able to consume a large quantity of substrate and survive in harsh environments,

extracellular enzymes like alkaline protease do not have reproducibility. Namely,

increasing the enzyme population must be done through adding new enzymes from

outside into the system. On the other hand, these alkaline proteases lose some reactivity

after they interact with pollutants and could eventually become completely inactive.

Hence they do not have the adaptability to the harsh environment even though they can

survive in a wide range of environmental conditions. This means that the enzyme

concentrations must be monitored and controlled during the process in order to optimize

enzyme kinetics for site-specific conditions [109].

2.4.3 Chemical-Biological pretreatment

Keratins are insoluble macromolecule comprises super coiled long polypeptide chains

with high degree of cross linked disulphide bonds between contiguous chains. According

to the literatures disulfide bonds in keratin significantly decrease protein digestibility

[94]. And “for complete easy degradation of feather all enzymatic keratinolysis from any

organism essentially needs to be assisted by a suitable redox [95].” therefore, it has been

suggested that some reductants, such as thioglycollate, copper sulphate , ammonia and

sodium sulphite [96] and others, might cleavage the disulfide bonds in keratin and allows

the proteases to have access to their peptide bond substrates [97], and consequently

improve the degradability of feathers [94,35,12,65]. For instance Ramnani et al., 2007

found that savinase is capable of near complete feather degradation (up to 96%) in the

presence of sodium sulfite [95].

28

2.5 Research Objectives

Considering the abundance and continual increase in the production of chicken feather

waste as a high value resource of protein and also the hard degrading structure of feather

keratin, the objective of this study was to investigate the feasibility and the effects of

various pretreatment methods on the hydrolysis of chicken feather for enhancement of its

methane potential.

For this purpose chicken feathers were pretreated by thermal, thermo-chemical,

enzymatic, thermo-enzymatic and chemo-enzymatic methods followed by anaerobic

digestion of pretreated feathers. Besides, the effects of the variation in treatment

conditions during thermo-chemical treatment on the methane yield of chicken feathers

and optimization of these conditions were studied.

29

Chapter 3: Materials and methods

3.1 Equipments and apparatus

The following equipments and supplies were applied for the experiments:

• 118 ml glass bottles (flasks) with rubber septum, as bioreactors

• 250µl gas tight glass syringe with a pressure lock to take fixed volume and

pressure samples from the reactors.

• Regulated incubator at 55°C for incubation the samples in a thermophilic

condition.

• Autosystem Gas Chromatograph equipped with thermal conductivity detector

(TCD), for the measurement of CH4 and CO2.

• COD Reactor with Direct Reading Spectrophotometer for SCOD measurement of

the pre-treated samples.

• Convection drying oven with temperature control of 105±3°C for TS

measurement of feather

• Muffle furnace with temperature control of 550°C for VS measurement of feather.

• Autoclave for thermal pre-treatments of samples

• Shaking water bath regulated at 37°C and 150 rpm for chemical pretreatment of

samples

• Centrifuge for separation the suspended solid and liquid phase of samples for

SCOD (soluble chemical oxygen demand) measurement of pre-treated samples.

• Digital pH meter to measure and adjustment of the pH of the pre-treated samples

for digestion and final pH of digested samples at the end of experiments.

3.2 Materials

• Waste Chicken Feather as a bioresource for biogas production.

• Inoculum from thermophilic Biogas Plant, Sobacken-Borås.

• Lime for thermo-chemical treatment.

30

• Sodium sulfate for chemo-enzymatic treatment.

• Savinase ®ClEA for enzymatic treatment.

• Gas mixture of 80% N2 and 20% CO2 for air removal of the samples head space.

• 100% CO2 gas as CO2 standard for gas chromatography and also carbonation of

lime treated samples.

• 100% CH4 gas as methane standard for gas chromatography

• Phosphate buffer to adjust the pH of the samples in experiment 3.

3.3 Methods

3.3.1 Preparation of Waste Chicken Feathers

Waste chicken feathers were cleaned and washed with lukewarm water a few times, and

then air-dried at room temperature followed by drying in the oven at 105°C±3.

After drying the feathers were grinded and stored in capped dishes in cooling room.

3.3.2 Inoculum

Active thermophilic inoculum was obtained from thermophilic Biogas Plant, Sobacken-

Borås and stored at 55°C in an incubator for 3 days in order to readapt the inoculum to

55°C, ensure degradation of easy degradable organic matters still present in the inoculum

and remove dissolved methane.

3.3.3 Total Solids (TS%) and Volatile Solids (VS%) measurement

Total Solids percentage (TS%) of the feathers was measured according to the

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

Total Solid in Biomass (LAP-001)” [98] as follows:

-Crucibles were dried in drying oven 105°C±3 over the night and were weighed

accurately to the nearest 0.1 mg and the weight was recorded.

31

-Air dried; milled feathers were weighed into the dried crucibles to the nearest 0.1 mg.

The total weight of the -each sample and crucible were recorded.

-Samples were placed into the convection oven at 105±3°C and were dried for overnight

to constant weight.

-Samples were removed from the oven and placed in a desiccator to cool to room

temperature.

-The total weight of the crucibles and oven dried samples were measured to the nearest

0.1 mg and recorded.

Total Solid (TS%) of the samples were calculated according to the following equation:

Total Solids percentage (TS %) = (W2 /W1) x 100

Where:

W1 = weight air dried sample

W2 = weight 105°C dried sample = weight 105°C dried sample plus dish – weight dish

Data figures for TS% measurement are shown in table 11 appendix A. And hence,

Average Measured TS% of Chicken Feather was:

Feather (TS%) = 91.29%

Volatile Solids percentage (VS%) of feather was measured according to the

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

Ash in Biomass” [99] as follows:

-Crucibles were heated at 550°C±10 for 4 hours and placed in a desiccator to cool to the

room temperature. Then crucibles were weighed accurately to the nearest 0.1 mg and the

weight was recorded.

-Oven dried (105°C) feathers were weighed into the dried crucibles to the nearest 0.1 mg.

The total weight of the each sample and crucible were recorded.

-Samples were placed into the muffle furnace at 550°C±10 for 3 hours, reheated and

reweighed to constant weight till varies by less than 0.3 mg.

32

-Samples were removed from the oven and placed in a desiccator to cool to the room

temperature.

-The total weight of the crucibles and burned residue were measured to the nearest 0.1

mg and recorded.

Volatile Solids (VS %) of the samples were calculated according to the following

equation:

%Volatile Solids (VS % of TS) = (W1-W2/W1) x 100

Where:

W1 = weight 105°C dried sample, and

W2 = weight of ash (burned residue) = weight burned residue plus dish – weight dish

Data figures for TS% measurement are shown in table 10 appendix A.

And hence, average Measured Volatile Solid% of TS Feather was:

Feather VS% (of TS) = 99.34% of TS

And Average Measured Volatile Solid% of Air Dried Feather was:

Feather (VS%) = 90.69%

3.4 Pretreatment Methods

3.4.1 Thermo-Chemical Lime Pretreatment (Experiments 1, 2)

Various concentrations of Lime (Ca (OH)2 g/g TS F) were added to the mixtures of 2

different concentrations (40 &100g TS/l water) of milled and 105°C dried chicken

feathers. 50 ml of each sample was prepared in duplicate. Afterward, samples were

closed with aluminum foil loosely and were heated in the autoclave at different

temperatures for different treatment times according to the Table 5:

33

Exp. Number

Feather Concentration (g TS F/l liquid)

Lime loading (g/g TS F)

Autoclave Temperature

(°C)

Time (min)

0.1 0.2

0.4 1 2

1

40

4

100, 110, 120

30,60,120

0.1 0.2 0.4 1

2

100

2

100, 110, 120

60,120

Table 5. Thermo-chemical treated samples and treatment conditions (Exps.1 and 2) After cooling the samples to the room temperature in a desiccator, pH measurement for

the samples was carried out. In general, due to the presence of the lime pH values of the

treated samples has been maintained around 11.5-12.5.

To adjust the pH of the samples to the suitable value for anaerobic digestion and also to

convert the existing lime in the samples to the water-soluble Ca(HCO3)2 (as much as

possible), samples were carbonated with pure CO2 gas while the pH were controlled

continuously. In this way the pH of the samples decreased to about 8-8.5 and major

amount of the lime was converted to water-soluble calcium bicarbonate (Ca(HCO3)2) and

also low soluble calcium carbonate (CaCO3) [35].

One of each duplicated samples were centrifuged and the liquid phase of them were used

for soluble chemical oxygen demand (SCOD) concentration measurement.

Considering the SCOD measurement results, the following uncentrifuged samples which

their centrifuged couples had revealed high SCOD concentration and also contented

much lower amount of the precipitated lime and calcium carbonate (CaCO3) were

selected to use for the anaerobic digestion process (samples had been made in 50ml

volume):

- For experiment 1, using 40 g TS feather/l concentration, the selected samples had been

treated under the following conditions:

34

1- 0.1g lime /g TS feather, 30 min, 100°C:

2g TS feather + 48 ml water + 0.2g lime

2- 0.1g lime /g TS feather, 30 min, 120°C:

2g TS feather + 48 ml water + 0.2g

3- 0.2g lime /g TS feather, 1 h, 120°C:


4- 0.2g lime /g TS feather, 2 h, 120°C:


- For experiment 2, using 100 g TS feather/l concentration, the selected samples had been

treated under the following conditions:

1- 0.1g lime /g TS feather, 2h, 120°C:


2- 0.2g lime /g TS feather, 2h, 120°C:

5g TS feather + 45 ml water + 1g lime

3- 1g lime /g TS feather, 2h, 120°C:


4- 2g lime /g TS feather, 2h, 120°C:


3.4.2 Biological Pretreatments (Experiment 3)

In this series of experiment the effect of thermal, enzymatic, combined thermo-enzymatic

and combined chemo-enzymatic pretreatments on hydrolysis of feather were examined.

Milled and oven dried feathers, 0.9g TS F/vial, (1g F/vial) were pre-treated in the small

flasks (118 ml), in triplicate and one excess sample for SCOD measurement. For the

enzymatic treatment an alkaline endopeptidase enzyme, Savinase, was used.

Furthermore, for chemo-enzymatic treatment sodium sulfite was also added as chemical

reductant agent to cleavage disulphide bonds. The pH of the samples was adjusted to

pH=8.0 using phosphate buffer. The total volume of each sample was 10 ml.

Pretreatments were conducted using the following conditions and materials:

35

1- Thermal treatment: autoclaving for 5min at 120°C

0.9g TS feather + 9.1 g potassium phosphate buffer solution

2- Enzymatic treatment: incubation for 2h at 55°C

0.9g TS feather + 9g potassium phosphate buffer solution + 100mg enzyme (1% w

enzyme/vial)

3- Thermal-Enzymatic treatment: autoclaving feather for 5min, at 120°C, followed

by buffer and enzyme addition and incubation for 24h at 55°C


enzyme/vial)

4- Chemical-Enzymatic treatment: water bath for 60h at 37°C 150 rpm


enzyme/vial or 100mg/10ml) + 0.0252g Na2SO3 (20 mM/l)

The extra pretreated samples were centrifuged and the liquid phase of them was used for

SCOD measurement (Fig. 10):

Fig. 10. COD Reactor with Direct Reading Spectrophotometer for SCOD

measurements of the pre-treated samples.

36

3.5 Anaerobic Digestion Processes

3.5.1 Batch digestion process set-up for pretreated samples

In this step for lime treated samples (Exps.1 and 2) 5g of each sample consisting of both

proportional liquid and solid phases were transferred to 3 small flasks (118 ml) to make

triplicate samples for anaerobic digestion process. Then, during stirring of the inoculum

20 ml of the inoculum was transferred to each of the flasks. Total volume of each sample

was 25 ml. Hence, the VS content of pretreated feathers in each flask for samples of

experiment 1 was 0.191g VS F/Vial (0.765%VS) and respectively, for pretreated samples

of experiment 2 it was 0.453g VS F/Vial (1.8% VS). 3 untreated samples (control

samples) and 3 blanks were also prepared with the following materials:

- For experiment 1:

Untreated samples:

0.191g oven dried (TS) feather + 4.8 ml water +20 ml inoculum

Blank samples:

5ml water + 20 ml inoculum

- For experiment 2:

Untreated samples:

0.453g oven dried (TS) feather + 4.55 ml water +20 ml inoculum

Blank samples:

5ml water + 20 ml inoculum

To evaluate the effect of the solid phase on the methane productivity of pretreated

samples with 40g TS F/l and 0.2g lime /g TS F which contained negligible amount of

insolublised substrate and more amount of precipitated lime and carbonate calcium in

their solid phase, anaerobic digestion was also performed using just liquid phase of those

samples (samples 4 and 5 in Table 6).

For biological pretreated samples (experiment 3) also during stirring of the inoculum 50

ml of the inoculum was transferred to each flask which contained 10ml pretreated

37

feathers. The total volume of each sample was 60 ml. 3 untreated samples and 3 blanks

were also prepared, as following:

Untreated samples:

0.9g TS feather /vial (1g F/vial) + 9.1 g phosphate buffer solution + 50 ml inoculum

Blank samples:

10 ml phosphate buffer solution + 50 ml inoculum

In the final step the sample flasks, prepared for the anaerobic digestion on the above

described ways, were closed with a rubber septum and an aluminum cap and were

flushed with a mixture of gas containing 8o% N2 and 20% CO2 for 2 minutes to provide

anaerobic condition in the headspace of the reactors and prevent pH-change in the water-

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

Fig. 11. Samples maintained in the incubator at 55°C for anaerobic digestion process.

Volume of the produced CH4 and CO2 were measured at least twice a week using a Gas

Chromatograph equipped with TCD (Fig. 12).

38

Gas samples of 250µl were taken from the headspace of the flasks through the septum

using a gas tight syringe equipped with a pressure lock, and then were injected directly

into the gas chromatograph (GC). Pure CH4 and CO2 gases were used as standard gases

in GC measurements. To avoid build-up of the gas over pressure in the flasks leading to

gas leakage, gas pressure inside of the flasks was usually kept below 2 bars and the over

pressure was released under a hood by inserting a hospital needle in the rubber septum.

After the release an additional gas sample was taken and measured in a similar way as

described previously. During the incubation period the samples were regularly shaken

and moved around in the incubator to compensate any minor temperature variations at the

different parts of the incubator. Samples were shaken also before each GC measurement.

Fig. 12. Autosystem Gas Chromatograph with TCD for measurement of produced

methane and carbon-dioxide.

39

Chapter 4: Calculation and Data Treatment

The produced amount of methane was determined according to the “GC External

Standard Method” [100]. In this standard, assuming, the response index of the detector is

unity, if the (p)th gas component in the mixture is at a concentration of (cp (s)) in the

sample and (cp(st)) in the standard gas, then:

cp(s) = (ap(s)/ap(st)) * cp(st)

Where:

cp(s) is the concentration of the component (p) in the sample,

(ap(s)) is the area of the peak for the component (p) in the sample chromatogram,

(ap(st)) is the area of the peak for the component (p) in the reference chromatogram,

And (cp(st)) is the concentration of the standard in the reference.

Assuming ideal gas mixtures and using the ideal gas law, from the mole numbers of each

gas components measured in the sample of known volume, the mole numbers of each gas

components in the head space can be calculated without measuring the actual pressure in

the flasks. Furthermore, t he amount of CH4 (or CO2) produced between two subsequent

sampling in the head space of each flask was calculated from the difference of mole

numbers of methane (or carbon-dioxide) determined after releasing the overpressure and

the mole numbers of methane (or carbon-dioxide) determined at next sampling time

before the release. To calculate the produced methane volumes the following

experimental conditions were considered:

T = 22°C = 295 K, Atmospheric Standard Pressure, Patm= 101325 Pa,

R (Ideal Standard Gas Constant) =8.314, Sample (syringe) Volume (Vs) = 250 µl,

40

Finally, Normal Volume of the produced methane per gram VS (Nm3 CH4/kg VS) was

calculated for each sample at standard conditions of 273 K and 101325 Pa and the data

are presented as produced methane (Nm3 CH4/kg VS) versus time (days). Calculations

for all triplicates were computed and analyzed using MS Excel-Sheet and the blank

samples performance (gas production of the inoculum) was subtracted from the

performance (gas production) of the other samples.

41

Chapter 5: Results and discussion

5.1 Effect of lime treatment on SCOD concentration (Experiments. 1, 2)

In this study thermo-chemical treatment with lime exerted the most significant effect on

solublisation of the complex and rigid structure of feather keratin and generated a rich

mixture of small peptides and free amino acids resulting in high concentrations of soluble

chemical oxygen demand (SCOD). The average values for SCOD of the samples under

various pretreatment conditions such as different feather concentration, lime loading,

temperature and reaction time are shown in the Figs. 13-16:

0

10000

20000

30000

40000

50000

60000

SCOD (mg/l)

0g/g(30m)

0,1g/g(30m)

0,2g/g(30m)

0g/g(1h)

0,1g/g(1h)

0,2g/g(1h)

0g/g(2h)

0,1g/g(2h)

0,2g/g(2h)

Lime conc., Time

SCOD Concentration

100°C

110°C

120°C

Fig. 13. Results of SCOD measurement for lime treated samples containing 40gTS F/l

initial concentration (Exp. 1), under various treatment conditions.

42

45000460004700048000

49000500005100052000

53000

SCOD (mg/l)

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

Lime Conc. (g/g)

SCOD concentration (120°C, 2h)

0,4 g/g

1 g/g

2 g/g

4 g/g


initial concentration (Exp. 1) with higher lime loadings at 120°C for 2h.

As seen in Figs. 13 and 14 for the samples of experiment 1, containing 40gTS F/l liquid

concentration, SCOD concentration increased drastically from a minimum of 850 mg/l

under 0g Ca(OH)2/g TS F, 100°C, 30min treatment conditions i.e. with no lime addition

to a maximum of 59450 mg/l under 0.2g Ca(OH)2/g TS F, 120°C, 2h treatment

conditions. However, further increase in the lime loading to 0.4, 1.0, 2.0, and 4.0g

Ca(OH)2/g TS F at 120°C with a reaction time of 2h reduced the SCOD concentration of

the samples, comparatively. The lowest value of 47675 mg/l SCOD was obtained with

addition of the highest amount of lime (4g Ca(OH)2/g TS F). Increasing some other

pretreatment conditions such as reaction time and temperature didn’t change SCOD

concentration significantly. Previously, Coward-Kelly et al. 2005 [35], studied

pretreatment of feather with lime to generate an amino acid rich foodstuff for animals.

They found that feather solublisation significantly increases from 0 to 0.1 g Ca(OH)2/g

TS F, but does not change considerably for higher lime loadings. Hence, lime loading

shows a critical value below which the digestibility greatly declines and above which the

digestibility does not change substantially [35]. However, as expected, increasing feather

concentration from 40 to 100g TS F/l liquid in experiment 2 increased the SCOD

concentration. (Figs. 15 and 16)

43

0

20000

40000

60000

80000

100000

120000

140000

160000

SCOD (mg/l)

0g/g(1h)

0,1g/g(1h)

0,2g/g(1h)

0g/g(2h)

0,1g/g(2h)

0,2g/g(2h)

Lime conc., Time

SCOD Concentration

110°C

120°C


initial concentration (Exp. 2) under various treatment conditions.

125000130000135000140000145000150000155000160000165000170000

SCOD (mg/l)

0,4 g/g 1 g/g 2 g/g Lime conc. (g/g TS)

SCOD Concentration (120°C, 2h)

0,4 g/g

1 g/g

2 g/g

Fig. 16. Results of SCOD measurement for lime treated samples of Exp. 2 with

higher lime loadings at 120°C and for 2h.

For instance, as indicated in Figs.16and 14, sample with 100 g TS F/l concentration under

2g Ca(OH)2/g TS F, 120°C, and 2h treatment conditions, revealed the highest SCOD

concentration, of 168500 mg/l, while for the sample with 40 g TS F/l concentration

treated at the same conditions the SCOD concentration was 52000 mg/l respectively i.e.

the relative SCOD releases for these samples were 1685 and 1300mg SCOD/g TS F.

44

Therefore we can conclude that the relative SCOD release could be increased by about

30% when higher concentration of feathers was used for the treatment.

Meanwhile, the effects of the variation of other pretreatment conditions on 100 g TS F/l

concentrated samples (experiment 2) were similar to those of 40 g TS F/l samples

(experiment 1). i.e. increasing the lime loading from 0 g/g TS F to 0.2 g/g TS F improved

SCOD concentration drastically but further increase in the lime loading (from 0.2g Ca

(OH)2/g TS F to 2 g Ca (OH)2/g TS F) could improve SCOD only slightly . And the same

as in the experiment 1, increasing the other pretreatment conditions such as temperature

and reaction time didn’t exert noticeable positive effect on increasing of SCOD

concentration.

5.2 Effect of lime treatment on Anaerobic digestion performance

(Experiments 1, 2)

Regarding the objectives of this study and the results obtained by SCOD measurements,

the best pretreated samples containing high SCOD concentrations, optimal pretreatment

conditions and the least content of precipitated lime and calcium carbonate had been

selected for anaerobic digestion process. Table 6 and Figs. 17, 18 illustrate the SCOD

concentration (after treatment) and maximum methane yield of the selected samples

containing 40 g TS F/l concentration, during 50 days of anaerobic incubation:

Table 6. Results of SCOD and average maximum methane yields of triplicate lime treated

samples of Exp.1 during 50 days of incubation.

Sample pretreatment Feathers Concentration (g TS/l liquid)

Pretreatment Conditions

SCOD (mg/L)

Concentration of substrate in vials (g VS/Vial)

Maximum Methane yield (Nml/g VS)

Percentage of theoretical methane potential

1 Control, untreated

--- 47.4 9.6%

2

0.1g lime/gTS, 100°C, 30 min

41600 480 96.8%

3


55400 338 68.1%

4


63100 230 46.4%

5

40

0.2g lime/gTS, 120°C,120 min

67200

0.191 (0.765%)

123 24.8%

45

0

10000

20000

30000

40000

50000

60000

70000

SCOD (mg/l)

0.1g/g,100°C,30min

0.1g/g,120°C,30min

0.2g/g, 120°C,

1h

0.2g/g, 120°C,

2h

Lime conc., temp., time

SCOD Concentration

0.1g/g,100°C, 30min0.1g/g,120°C, 30min0.2g/g, 120°C, 1h0.2g/g, 120°C, 2h

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

for anaerobic digestion process.

Methane Normal Volume

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0 10 20 30 40 50 60

Time (days)

N VO

L (m

3/kg

VS

)

Untreated

0,1g/g30min100°C0,1g/g30min120°C0,2g/g 1h120°C

0,2g/g 2h120°C

Fig. 18. Average maximum methane production curves for triplicate lime treated

samples of Exp. 1, during 50 days of incubation.

According to these results, beside the considerable improvement of SCOD concentration,

lime treatment showed the most significant effect on increasing the methane productivity

of chicken feathers. In particular for sample 2 of this experiment, pretreatment under

(0.1g Ca(OH)2 /g TS F, 100°C and 30 minutes) conditions demonstrated the highest

46

increase in the methane yield of 480 N ml CH4/g VS which is about 96.8 % of the

theoretical methane potential. General theoretical methane potentials for fat, protein and

carbohydrates were illustrated in Table 4.

Although increasing the pretreatment conditions such as feather concentration, lime

loading, reaction time and temperature showed an overall positive effect on SCOD

enhancement, exert negative effect on the methane yield. For instance increasing the lime

loading from 0.1 g to 0.2 g/g TS feather for samples 4 and 5 also increased the SCOD to

some extent, but resulted in the highly increased amount of precipitated lime and

carbonate calcium, unstable anaerobic digestion performance and much less efficiency in

the methane productivity of those samples (Figs. 17,18 and Table 6). According to the Coward-Kelly et al. (2006), protein and amino acid degradation are associated with

ammonia production which is the most important toxicant for anaerobic digestion of

proteins (e.g., deamidation of asparagine and glutamine, generating asparatate and

glutamate and ammonia) (Figure 9) [80,35].

Therefore shorter reaction time and lower temperatures (approximately 100°C) in

treatment of chicken feathers are preferred because the degradation of susceptible amino

acids and ammonia production may be reduced to a minimum (35,80,102). It means that

increasing the treatment temperature and time in this experiment has led in more feathers

solublisation. The increased solublised feathers, which compared to the sample 1 were

observable in the lime treated samples of 2-5, have increased the SCOD concentration

and also overloaded these samples with amino acids. Meanwhile, increasing the treatment

temperature and time has resulted in more amino acid degradation associated with

accumulated ammonia. This accumulated ammonia has inhibited the methane

productivity of the samples 2-5.

To evaluate the effect of the precipitated lime and carbonate calcium in the solid phase of

these samples on the methane productivity, extra anaerobic digestion assay was done for

samples 4 and 5 using just liquid phase of these samples which contained negligible

amount of insolublised substrate and high amount of precipitated lime and carbonate

calcium in their solid phase. As seen in table 7 and Fig. 19 bellow, some improvements in

47

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

and 51.2% for sample 5.

Table 7. Results of SCOD and average maximum methane yield of triplicate lime treated

samples 4 and 5 of Exp. 1, during 15 days of incubation, (liquid phase).


-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0 10 20 30 40 50 60

Time (days)

N V

OL

(m3/

kg V

S)

Untreated

0,2g/g 1h120°C

0,2g/g 2h120°C

Fig. 19. Average maximum methane production curves for triplicate lime treated samples 4 and 5 of Exp. 1, during 50 days of incubation (liquid phase).

Sample Pretreatment

Feathers

Concentration

(g TS/l liquid)

Pretreatment

Conditions

SCOD

(Mg/L)

Concentration

of substrate in

vials

g VS/Vial

Maximum

Methane

yield

Nml/gVS

Percentage

of theoretical

methane

potential

1 Control,

untreated

--- 47.4 9.6%

2 0.2g lime/g TS,

120°C, 60 min

63100

266

53.6%

3

40

0.2g lime /g TS,

120°C, 120min

67200

0.191 (0.765%)

186 37.5%

48

However, for samples 2 and 3 of experiment 1, because of the presence of more

insolublised substrate in the solid phase, using both solid and liquid phase of the sample

is inevitable. Meanwhile, for these samples almost no visible precipitated lime and

calcium carbonate were observed to be separated.

Increasing the feather concentration to 100g TS F/l in experiment 2, which also resulted

in increasing of SCOD (Figs. 15, 16), led in much lower and even depressed methane

productivity of the most samples during 15 days of anaerobic incubation. Table 8 and

Fig. 20 illustrate the SCOD concentration (after treatment) and maximum methane yield

of the selected samples containing 100 g TS F/l concentration, during 15 days of

anaerobic incubation:

Table 8. Results of SCOD and average maximum methane yields of triplicate lime treated

samples of Exp. 2, during 15 days of incubation.

Sample Pretreatment

Feathers

Concentration

(g TS/l liquid)

Pretreatment

Conditions

SCOD

(mg/L)

Concentration of substrate in vials(g VS/Vial)

Maximum

Methane

yield

( Nml/g VS)

Percentage

of theoretical

methane

potential

1 Control,

untreated

--- 118 23.8%

2

0.1g lime /g TS,

120°C, 60 min

114200 139 28%

3

0.2g lime/g TS,

120°C, 60 min

153800 53 10.7%

4

1g lime /g TS,

120°C, 60 min

162500 23 4.6%

5

100

2g lime /g TS,

120°C, 60min

168500

0.453

(1.8%)

20 4.0%

49


0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 5 10 15 20Time (days)

N V

OL

(m3/

kg V

S)

Untreated

0,0g/g 1h120°C 0,2g/g 1h120°C 0,1g/g 1h120°C1g/g 1h120°C2g/g 1h120°C

Fig. 20. Average maximum methane production curves for triplicate lime treated

sample of Exp. 2, during 15 days of incubation.

The increased SCOD concentration and meanwhile decreased methane yield of these

samples probably reflect the effect of the overloading of the system with organic

substrate leading in accumulation of ammonia which inhibited CH4 productivity of the

protein material during anaerobic digestion process. Accordingly, less feathers loading

may result in more efficient anaerobic digestion process.

It is mentionable that the same considerations i.e. high SCOD content, least precipitation

of lime and calcium carbonate (CaCO3) and optimal conditions had been applied in

selection of the samples of experiment 2 for anaerobic digestion process.

After lime treatment the measured pH for the samples was around 11.5-12.5. Carbonating

samples with CO2 gas before digestion process decreased the pH to about 8-8.5. Although

no buffer was used to adjust the pH during the digestion process, final measurement of

the pH indicated that the pH of the samples had been maintained almost at the same level

of the starting of the AD process (pH of 8-8.5). According to the literatures “Calcium

hydroxide is an alkaline material poorly soluble in water that maintains a relatively

constant pH (~12), provided enough lime is in suspension. This low solubility ensures a

constant pH during the thermo-chemical treatment and relatively weaker conditions

(compared to sodium hydroxide and other strong bases) that helps in reducing the

50

degradation of susceptible amino acids. The new carboxylic acid ends react in the

alkaline medium to generate carboxylate ions, consuming lime in the process [35].”

Lo´pez Torres et al.,2007 [104] found also the similar point and reported that in contrast

with the other AD systems the digesters fed with lime pretreated waste maintained its

alkalinity and neutral pH during digestion process without necessity of continuously

addition of alkali.

Samples 2 and 3 of experiment 1 had a fast onset in methane production but samples 4

and 5 had a one week lag phase. However, in repetition of the AD process using liquid

phase of samples 4 and 5 no delay was observed in the start of the methane production.

The ammonia production of in vitro rumen digested lime soluble chicken feather keratin

was also previously studied by Coward-Kelly et al. (2005) [35]. They found that

ammonia production from soluble keratin in rumen fluid was similar to that of soybean

and cottonseed meals and was greatly less than that of urea. Soybean and cottonseed

meals are the most popular protein sources for cattle which do not result in ammonia

toxicity. Therefore, soluble feather keratin is likely more readily digested than the other

proteins and no ammonia toxicity will result from cattle being fed soluble keratin [35].

Similar performance might be expected from lime treated samples during anaerobic

digestion of feather for biogas production, namely no ammonia toxicity is produced and

inhibits the anaerobic microorganisms for the recommended condition.

According to Lo´pez Torres et al. 2007, Alkaline pretreatment of organic materials with

Ca(OH)2 not only increases the level of soluble COD but also surface area of complex

organic matter, due to fiber swelling. These facts make these materials more susceptible

to enzymatic attack by microorganisms and enhance anaerobic digestion processes [104].

Another significant advantage of alkaline treatment is disruption of the disulphide bonds

in feather which was previously noticed by Salminen et al. [30]. All of the above results support the positive effect of lime pretreatment on hydrolysis of

the chicken feather and other organic materials, and according to the achieved results in

the present study pretreatment of chicken feather under (40g TS feather/l, 0.1g Ca(OH)2/g

dry feather, 100°C, 30 min) condition is the optimum condition to exert the most

significant effect on increasing the methane yield of chicken feather. Coward-Kelly et

al., 2005 [35] found that pretreatment of feather under 0.1g Ca(OH)2/g dry F, 100°C and

51

300 min treatment conditions can solublise 80% of feather keratin to produce an amino

acid rich foodstuff for animals and in this study the pretreatment times was modified to

30 min for anaerobic digestion of feathers resulted in 96.8 % of the theoretical potential

methane productivity. This shorter treatment time is safer for AD process and more

profitable from economical point of view.

5.3 Effect of biological treatments on SCOD concentration (Exp.3)

In this series of the experiments the effect of the thermal, enzymatic, combined thermal-

enzymatic and combined chemical-enzymatic pretreatment on solublisation and methane

yield of chicken feather were investigated.

Fig. 21 shows the samples after enzymatic, chemo-enzymatic and thermo enzymatic

pretreatment.

Fig. 21. Enzymatic, chemo-enzymatic and thermo-enzymatic pretreated samples (Exp.3).

The average values for SCOD concentration of the pretreated samples are demonstrated

in the Fig. 22.

52

0

5000

10000

15000

20000

25000

30000

35000

40000

SCOD (mg/l)

Enzymatic Thermo-Enzymatic

Chemo-Enzymatic

Treated Samples

COD Concentration

Enzymatic

Thermo-Enzymatic

Chemo-Enzymatic

Fig. 22. Results of SCOD measurement for enzymatic and combined enzymatic

pretreated samples of Exp.3.

As seen in the Fig. 22 these methods of pretreatment solublised the feather and showed

positive effect on increasing the SCOD concentration of the samples. As seen in the

Fig.22 these methods of pretreatment solublised the feather and showed positive effect on

increasing the SCOD concentration of the samples. But in contrast to lime treatment,

where the highest relative SCOD release was around 1680 mg SCOD/g TS F here the

highest relative SCOD release value was 407 mg SCOD/g TS F produced by the chemo-

enzymatic treatments. It is still much lower than the relative SCOD release of 1040 mg

SCOD/g TS F for the recommended lime treatment conditions of 40g TS F/l liquid, 0.1g

Ca(OH)2/g TS F, 100°C and 30 min.

5.4 Effect of biological treatments on anaerobic digestion performance

Although combined enzymatic pretreatments could solublise feather and increase the SCOD concentration, methane yield enhancement by these methods were also much

lower than those of lime pretreatment. Table 9 and Fig. 23 illustrate maximum methane

productivity of these samples during 50 days of anaerobic incubation:

53

Table 9. Results of SCOD and average maximum methane yield of triplicate thermal, enzymatic and combined enzymatic pretreated samples of Exp.3.

Average Normal vol CH4 EXP6- Feather

-0.1

-0.05

0

0.05

0.1

0.15

0.2

0 10 20 30 40 50 60

Time (days)

N V

OL

(m3/

kg V

S) Untreated

Thermal

Enzymatic

Thermo-Enzymatic

Chemo-Enzymatic

Fig. 23. Average maximum methane production curves fort triplicate thermal, enzymatic

and combined enzymatic treated samples of Exp.3, during 50 days incubation.

samples

Feathers concentration

Treatment Conditions

SCOD Mg/L

Maximum Methane yield Nml/gVS

Percent of theoretical methane potential

1 Control, Untreated ---- 135 27.2% 2 Thermal,

120°C, 5 min ---- 143 28.8%

3

Enzymatic, 1%w enzyme/vial, 55°C, 2 h

18,640

154

31%

4

Thermal-Enzymatic, 120°C, 5min- 1%w enzyme/vial, 55°C, 24 h

32,760

185

37.3%

5

1g F/vial (1.5% VS)

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