1

OPTIMIZATION OF BIOGAS PRODUCTION USING COMBINATIONS OF SAW DUST AND COW DUNG IN A BATCH ANAEROBIC DIGESTION BIOREACTOR.

A PROJECT REPORT SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES IN PATIAL

FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF DEGREE OF MASTER OF SCIENCE IN INDUSTIAL BIOCHEMISTRY/ BIOTECHNOLOGY

BY

UKONU, CHRISTIAN UGOCHUKWU

PG/M.Sc/09/51940

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA,

NSUKKA.

DECEMBER, 2011.

2

CERTIFICATION

Ukonu, Christian Ugochukwu, a postgraduate student with registration number

PG/M.Sc/09/51940 in the Department of Biochemistry, University of Nigeria, Nsukka has

satisfactorily completed the requirement for course work and research for the degree of Masters

of Science (M.Sc) in Industrial Biochemistry/ Biotechnology. The work embodied in this report

is original and has not been submitted in part or full for any other diploma or degree of this or

any other University.

_____________________ ___________________

Prof. I.N.E. Onwurah Dr. B.C. Nwanguma Supervisor Supervisor Date:__________________ Date:_______________

______________________ ___________________

Prof. L.U.S Ezeanyika Prof. L. S. Bilbis Head of Department External Examiner Date:__________________ Date:_______________

08/08/2012

3

DEDICATION

This work is dedicated to God Almighty for His underserved love, care, protection, provision,

sustenance, favour, blessings, grace and mercies all through the period I spent in this school.

4

ACKNOWLEDGEMENT

My unlimited praise still goes to God Almighty for His faithfulness.

My unlimited gratitude goes to my supervisors’ Prof. I.N.E Onwurah and Dr. B.C Nwanguma

for their fatherly advice, encouragement, and meticulous guidance throughout this research.

This legacy I will ever pursue.

My thanks also go to all the lecturers in the Department of Biochemistry for all their effort,

care and advice that had made me who I am today. I will not fail to mention the names of those

whose lives and teachings have come the wisdom of the ages. These include, Prof. L.U.S

Ezeanyika (Head of Department of Biochemistry), Prof. O.F.C Nwodo, Prof. Obidoa, Prof. O.U

Njoku, Prof. P.N Uzoegwu, Prof. Alumanah, Dr. S.O.O Eze, Dr. V. Ogugua, Dr. P.E Joshua,

Dr. H.A Onwubiko, Mr. C. Ubani, Dr.(Mrs). C.A. Anosike, Prof. I.C Ononogbu, Prof. F.C

Chilika and all other staff of the Department that are not mentioned. I have learned from your

legacies and will keep it rocketing.

I gratefully acknowledge and express deep appreciation to my wonderful parents, Nze and lolo

A.O Ukonu, to my brother, Emmanuel Ukonu, to my sisters, Mrs. onyinye , Amaka , Uchechi ,

Ogechi Ukonu, my cousin Izuchukwu Chukwu, my uncles Mr. F.I Ukonu, Dr. U. Nzekwe and

to all my relations unmentioned; for all their supports and prayers. I am indebted to Engr. O.

Onyejekwe, Mr. Duke Nwokoro, Mr. Chris Eke, Mr. C. Asumugha, Ms. G. Okoro and

Rev.(Mrs) B. Nzekwe; for their moral and financial support towards the success of this

programme. May your purse never get dry in Jesus name. With deep joy I express my

appreciation to my “fiancée” Dr. Ijeoma Chiedu for her love, encouragement, care, support and

prayers. I am indebted to you.

I am also grateful to my colleagues and friends, Ebubechukwu, Benjamine, Paul, Oje, Mrs.

Florence, Uche, Emmaculata, Wallace, Okechukwu, Victor etc. whose deep sharing and

synergy have moved me many levels beyond my thinking.

Finally, I am greatly indebted to the Department of Biochemistry, for giving me the opportunity

to develop my potentials at the University of Nigeria, Nsukka.

5

Ukonu, C.U

ABSTRACT

Optimization of biogas production by blending saw dust and cow dung (CD:SD) in the ratio 1:1

and addition of additives such as boric acid, NiSO4, CoSO4, Zn and Zeolite was carried out

using untreated saw dust +cow dung only as negative control and treated saw dust +cow dung

only as the positive control. The experiments were carried out in seven 50 litres metal prototype

bioreactors containing water and waste in the ratio 1:4. The reactions were monitored for 28

days (retention time) within the ambient temperature range of 22oC-35oC and pH range of 6.5-

9.5. The result of this investigation shows that lag phase of 18 and 21 were obtained with

cumulative biogas yield between 10.4 L/TS - 23.7 L/TS. The above values were lower than that

obtained for the negative control reactor which generated biogas of 29.82 L/TS with a time lag

of 24. Pretreatment of saw dust and addition of zeolite increased the biogas yield (54.5 L/TS)

and the onset of biogas flammability, in the case treated saw dust/ cow dung blend, and with a

lag time at the 13th day. The bioreactor having of a blend of saw dust+ cow dung+ zeolite has a

time lag of 15 and cumulative biogas yield of 30.1 L/TS, also relative to control. However,

some additive (Zn, CoSO4, Boric acid and NiSO4) only reduced the time lag of flammable gas

production but no effect on the biogas yield when compared with the negative control.

The overall result shows that a blend of saw dust and cow dung is a stable waste combination

for biogas production and it could be optimized by pretreatment of the saw dust with zeolite

before charging.

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TABLE OF CONTENT

Title page - - - - - - - - - - i

Certification - - - - - - - - - - ii

Dedication - - - - - - - - - - iii

Acknowledgement - - - - - - - - - iv

Abstract - - - - - - - - - - v

Table contents - - - - - - - - - - vi

List of table - - - - - - - - - - xiii

List of figure - - - - - - - - - - xiv

Abbreviations - - - - - - - - - - xv

CHAPTER ONE - - - - - - - - 1

1.0 Introduction and literature review - - - - - - 1

1.1 General Introduction - - - - - - - - 1

1.2 Literature Review - - - - - - - - 3

1.2.1 Biogas - - - - - - - - 3

1.2.2 Biogas composition - - - - - - - - 3

1.2.3 Chemical characteristics of biogas - - - - - - 3

1.2.3.1 Methane as a Component of Biogas - - - - - 4

1.2.4 Physical characteristics of Biogas - - - - - - 4

1.2.5 Purification of biogas - - - - - - - - 4

1.2.6 Collection of biogas - - - - - - - - 5

7

1.2.7 Types of Biogas plants - - - - - - - 5

1.2.7.1 Balloon plants - - - - - - - - 5

1.2.7.2 Fixed Dome plants - - - - - - - - 6

1.2.7.3 Floating Drum plant - - - - - - - 8

1.2.7.4 Prototype plant - - - - - - - - 9

1.2.8 Type of fermentation/culture plant - - - - - - 9

1.2.8.1 Continuous feeding - - - - - - - - 10

1.2.8.2 Batch feeding - - - - - - - - 10

1.2.9.0 Feed stocks (substrates) cow dung - - - - - - 11

1.2.9.1 Composition of cow dung - - - - - - - 11

1.2.9.2 Microbial floral present in the various types of the cow dung - 11

1.2.10 Saw dust - - - - - - - - - 12

1.2.11 Other substrates - - - - - - - - 12

1.2.12 Microbiology and Biochemistry of biogas production - - - 13

1.2.12.1 Hydrolysis - - - - - - - - 14

1.2.12.2 Acidification/Acetogenesis - - - - - - 14

1.2.12.3 Methane formation - - - - - - - 15

1.2.13 Interaction between the various microbial groups - - - - 16

1.2.14 Kinetics of Anaerobic fermentation - - - - - - 16

1.2.15 Factors influencing Biogas production from manure - - - 17

1.2.15.1 Temperature - - - - - - - - 17

8

1.2.15.2 pH - - - - - - - - - - 18

1.2.15.3 Retention time - - - - - - - - 18

1.2.15.4 Substrate (quality and characteristic of manure) - - - 19

1.2.15.5 Loading rate - - - - - - - - 20

1.2.15.6 Design of digester - - - - - - - 20

1.2.15.7 Volatile acid - - - - - - - - 20

1.2.15.8 Nutrient requirement (Additives) - - - - - - 21

1.2.15.8.1 Cobalt - - - - - - - - - 21

1.2.15.8.2 Nickel sulphate - - - - - - - - 22

1.2.15.8.3 Boric acid - - - - - - - - - 23

1.2.15.8.4 Zinc - - - - - - - - - 23

1.2.15.5 Zeolite - - - - - - - - - 23

1.2.16 Inhibitory factors of biogas production(s) - - - - 24

1.2.16.1 C/N ration (carbon/Nitrogen ration) - - - - - 26

1.2.16.2 Nitrogen inhibition - - - - - - - 26

1.2.16.3 Others inhibitors - - - - - - - - 26

1.2.17 Benefits of Biogas technology - - - - - - 26

1.2.17.1 Agriculture - - - - - - - - 27

1.2.17.2 Domestic and industrial uses - - - - - - 27

1.2.17.3 Other uses of biogas - - - - - - - 28

1.3.0 Aims and objectives - - - - - - - 28

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CHAPTER TWO - - - - - - - 29

2.0 Material - - - - - - - - - 29

2.1 Chemicals uses - - - - - - - - 29

2.2 Apparatus - - - - - - - - - 29

2.3 Collection and processing of sample - - - - - - 29

2.3.1 Collection of soil sample - - - - - - - 30

2.4 Preparation of waste - - - - - - - - 30

2.4.1 Treatment (pretreatment) of sawdust - - - - - - 30

2.5 Experimental design - - - - - - - - 30

2.5.1 Phase I - - - - - - - - - - 30

2.5.2 Phase II - - - - - - - - - 31

2.5.3 Phase III - - - - - - - - - 31

2.6.0 Methods - - - - - - - - 32

2.6.1 Charging of bioreactor - - - - - - - 32

2.6.2 Determination of quality of biogas produced - - - - 32

2.6.3 Determination pH of the bioreactor (slurry) - - - - - 32

2.6.4 Determination of pH of soil sample - - - - - - 32

2.6.5 Determination of the ambient and slurry temperature of the bioreactor - 33

2.6.6 Determination of biogas flammability - - - - - 33

2.6.7 Determination of the composition of biogas produce - - - 33

2.6.8 Determination of the total microbial count - - - - - 33

10

2.6.9 Determination of Ash content using the method of AOAC (1990) - - 34

2.6.10 Determination of moisture contents using the method of AOAC (1990) - 35

2.6.11 Determination of fibre content using the method of AOAC (1990) - - 35

2.6.12 Determination of fat content using soxhlet extraction method described

by pearson (1976) - - - - - - - - 36

2.6.13 Determination of crude nitrogen /Ammonium Nitrogen using micro-

kjedhal method described by pearson (1976) - - - - 37

2.6.14 Determination of carbon content using the method of

walkley and black (1934) - - - - - - - 38

2.6.15 Determination of carbon contents in soil by wet oxidation method of

walkley and black (1943) - - - - - - - 39

2.6.16 Determination of the particle size distribution - - - - 39

2.6.17 Determination of exchangeable acidity (EA = Exchangeable Aluminum

and Hydrogen) as described by Chapmen (1965) - - - - 41

2.6.17.1 Titration for exchangeable aluminum - - - - 41

2.6.18 Determination of the exchangeable bases in soil samples using the

Ammonium Acetate method as described by Chapman (1965) - - 41

2.6.18.1 Titration for Ca++ + Ma++ - - - - - - - 42

2.6.18.2 For ca++ only - - - - - - - - - 42

2.6.18.3 For Cation Exchange Charges (CEC) - - - - - 42

2.7.0 Preparation of reagents - - - - - - - 43

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2.7.1 Preparation of 0.5m Na2Cr2 07 – 2H2O in 5M H2 SO4 (Digestion solution) - 43

2.7.2 Preparation of 0.167M K2 Cr2 O7 - - - - - - 43

2.7.3 Preparation of 0.5M Fe2+ solution - - - - - - 43

2.7.4 Preparation of ferron indicator - - - - - - 43

2.7.5 Preparation of methyleted –methyl blue - - - - - 43

2.7.6 Preparation of nutrient Agar - - - - - - - 44

2.7.7 Preparation of maconkey Agar - - - - - - 44

2.8 Statistical Analysis - - - - - - - - 44

CHAPTER THREE - - - - - - - 45

3.0 Result - - - - - - - - - - 45

3.1 Proximate analysis of cow dung, untreated saw dust

and treated saw dust - - - - - - - - 45

3.2 Physical and chemical properties of soil sample - - - - 46

3.3 Time lag, cumulative biogas yield, mean±SEM of biogas yield, Temperature

and pH from cow dung. Phase I - - - - - - 47

3.4 The quantity of biogas produced daily in bioreactor I. Phase I - - 48

3.5 The quality of biogas produced daily in bioreactor I. Phase I - - 49

3.6 The quality of biogas produced daily in bioreactor 3. Phase I - - 50

3.7 The quality of biogas produced daily in bioreactor 4. Phase I - - 51

3.8 The quality of biogas produced daily in bioreactor 5. Phase I - - 52

3.9 The quality of biogas produced daily in bioreactor 6. Phase I - - 53

12

3.10 The quality of biogas produced daily in bioreactor 1-6. Phase I - - 54

3.11 Time lag, cumulative gas yield, mean±SEM of biogas yield, pH and

temperature in bioreactors. (Phase II) - - - - - - 55

3.12 Total viable count of the microbes in the reactors (CFU/ml). Phase II - 56

3.13 Composition of biogas produced in the bioreactors. Phase II - - 57

3.14 Time lag, cumulative biogas yield, mean±SEM of biogas yield,

pH and temperature of the bioreactor. Phase III - - - - 58

3.15 Total viable count of bacteria population in the bioreactors measured in

cfu/ml. Phase III - - - - - - - - 59

3.16 Composition of biogas produced in the bioreactors. Phase III - - 60

CHAPTER FOUR - - - - - - - - - 61

4.0 Discussion - - - - - - - - - 61

4.1 Conclusion - - - - - - - - - 65

4.2 Recommendation - - - - - - - - 65

REFERENCES - - - - - - - - - 66

Appendix one - - - - - - - - - - 78

Appendix two - - - - - - - - - - 84

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LIST OF TABLE

Table 1: Limiting concentration of various inhibitor of biomethane - - - 25

Table 2: Proximate analysis of the cow dung, untreated saw dust and

treated saw dust. - - - - - - - - 45

Table 3: Physical and chemical properties of soil sample - - - - 46

Table 4: Time lag, cumulative biogas yield, mean±SEM of biogas yield, temperature

and pH from cow dung. Phase I - - - - - - 47

Table 5: Time lag, cumulative biogas yield, mean±SEM of biogas yield, temperature

and pH in bioreactors. Phase II. - - - - - - 55

Table 6: Total viable count for the mixture in the bioreactors (cfu/ml).

Phase II. - - - - - - - - - 56

Table 7: Composition of biogas produced in bioreactors. Phase II. - - - 57

Table 8: The time lag, cumulative biogas yield, mean±SEM of biogas yield,

temperature and pH in the bioreactors. Phase III - - - - 58

Table 9: Total viable count of bacteria population in the bioreactors

measured in cfu/ml. Phase III. - - - - - - 59

Table 10: Composition of biogas produced in the bioreactors. Phase III. - - 60

14

LIST OF FIGURES

Figure 1: Balloon Plants - - - - - - - - 6

Figure 2a and b: Fixed dome plant Nicarao design - - - - - 7

Figure 3a and b: Floating-drum plant in Mauretania - - - - - 8

Figure 4a and b: Prototype Plants - - - - - - - 9

Figure 5: Conversion of fermentation substrate to biogas. - - - - 15

Figure 6: The quantity of biogas produced daily in bioreactor 1 (1:1). - - 48

Figure 7: The quantity of biogas produced daily in bioreactor 2 (1:2). - - 49

Figure 8: The quantity of biogas produced in bioreactor 3 (1:0). - - 50

Figure 9: The quantity of biogas produced in bioreactor 4 (1:3). - - 51

Figure 10: The quantity of biogas produced daily in bioreactor 5 (1:4). - - 52

Figure 11: The quantity of biogas produced daily in bioreactor 6 (1:5). - - 53

Figure 12: The quantity of biogas produced daily in bioreactors 1-6 against the

Retention time. - - - - - - - - 54

15

ABREVIATIONS

CEC: Cation Exchange Charges.

CD: Cow dung.

SD: Saw dust.

BIOR: Bioreactor.

MEQ: Miliequivilent.

L: Litre.

TS: Total Solid.

AMB: Ambient

TEMP: Temperature

BA: Boric Acid

NiSO4: Nickel Sulphate

CoSO4: Cobalt Sulphate

Zn: Zinc

Kg: Kilogramme

g: gramme

BTU: British thermal unit.

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CHAPTER ONE

1.0 INTRODUCTION AND LITERATURE REVIEW

1.1 General Introduction

The anaerobic fermentation of organic materials has long been used to generate useful

resources which have been harnessed for the use of mankind (Uri, 1992; US Environmental

Protection Agency, 2001). As early as the 18th century, anaerobic process of decomposing

organic matter was known, and in the middle of the 19th century, it became clear that anaerobic

bacteria are involved in the decomposition process.

Anaerobic digestion provides some exciting possibilities and solutions to such global concerns

as alternative energy production, handling human, animal, municipal and industrial wastes

safely, controlling environmental pollution, and expanding food supplies (Uri 1992; Ofoefule

and Uzodinma, 2006). As demand for energy is increasing astronomically, and the fossil based

fuels become scarce and more expensive, and carbon dioxide emission levels become of greater

concern; Biogas a by-product of anaerobic fermentation and a renewable energy source have

currently been recognized globally as a means of solving the problem of rising energy prices,

waste treatment /management and creating sustainable development (Rao and Seenayya, 1994;

Ofoefule and Uzodima, 2004). Biogas is a colorless, flammable gas produced via anaerobic

digestion (fermentation) of animal, plant, human, industrial and municipal waste to produce

methane (50-70%), Carbon dioxide (20-40%) and traces of other gases such as nitrogen,

hydrogen, ammonia, hydrogen sulphide, water vapour etc. (Ofoefule and Ibeto, 2010).

However, the composition of the mixture depends on the source of biological waste and

management of digestion process (Yadar and Hesse, 1981). The natural generation of biogas is

an important part of biochemical reaction which takes place under anaerobic condition in the

presence of highly pH sensitive microbiological catalyst that are mainly bacterial (Uzodinma

and Ofoefule, 2009). Biogas production comprises of three biochemical process, which

includes; hydrolysis, acidogenesis/acetogenesis, and methanogenesis (Nagamani and

Ramasamy, 1999). Complex molecules (carbohydrate, protein, fats) are broken down into a

broad spectrum of end products (.i.e. acetic acid, H2/CO2, monocarbon compounds and organic

17

fatty acids larger than acetic) by fermentative bacterial ((Uri, 1992; US Environmental

Protection Agency, 2001).

Fatty acids longer than acetate are metabolized to acetate by obligate hydrogen-producing

acetogenic bacteria. Hydrogen and carbon dioxide can be converted to acetate by hydrogen-

oxidizing acetogen or methane by aceticlastic methanogens (methanogenesis). (Nagamani and

Ramasamy, 1999). At pH between 6.0-8.0 and ambient temperature between 280C-40oC in a

bioreactor (digester) under anaerobic condition. (Ntengwe et al, 2010).

Ntengwe et al, (2010), reported that the production of biogas from biomass is dependent on the

amount of acid formed which depends on the types of biomass (feedstock) used. (The biogas

production rate was found to be different for different biomass).

Cow dung has been established by researchers as being superior in quality biogas production

over other wastes (Odeyemi, 1987). Contrary to this, Kasisira and Muyiiya (2009) reported that

100% pig manure produces more gas per unit weight as compared to 100% cow dung. Other

research on biogas production has been investigated using poultry dung, grasses, swine dung,

Bambara nut etc. In the quest to improve and optimize the production of quantity and quality of

biogas from organic waste, Uzodinma and Ofoefule (2009) have shown that the biogas yield of

field grass could be optimized by combining it with rabbit, cow, swine and poultry wastes.

Ofoefule and Uzodinma, (2006) have also shown that blending cow dung with poultry waste

significantly reduces the time lag between gas production and onset of gas flammability.

Nevertheless, addition of required chemicals and inoculate to blend prior to digestion may

improve the quantity and quality of biogas from the blended wastes. To this effect, this work

was undertaken to investigate the optimization of biogas production from cow dung by

combining it with sawdust and addition of other mineral elements which serve as

micronutrients to improve the activity of the methanogenic bacteria, reduce the time lag and

optimize the quality and quantity of biogas produced.

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1.2.0 Literature Review

1.2.1 Biogas

Biogas is a biological gas which originates from bacteria in the process of biodegradation

(fermentation) of organic material (from plants, animals and sometimes human origins) under

anaerobic (oxygen free) conditions (Mahin, 1982). Biogas is also an alternative and renewable

energy source produced through anaerobic conversion of organic matter into a combustible

biogas rich in methane (CH4) and liquid effluent (Ogejo et al., 2009). Methanogens (methane-

producing bacteria) are the last link in a chain of microorganisms which degrade organic

material and return the decomposition products to the environment.

Each year some 59-880 million tons of methane are released worldwide into the atmosphere

through microbial activity. About 90% of the admitted methane is derived from biogenic source

(.i.e. from the decomposition of biomass). The remainder is of fossil origin (e.g. petrochemical

processes). (GATE and GZT, 2007).

1.2.2 Biogas Composition

Biogas obtained after fermentation is a mixture of gases among which methane (CH4) is useful.

However, depending on the source of the organic matter and the management of the anaerobic

digestion process, small amounts of other gases such as ammonia (NH3), hydrogen sulfide

(H2S) and water vapour (H2O) may be present (Ogejo et al., 2009). In general, biogas consists

of 55-80% methane and 20-45% carbon dioxide (CO2), with other gases such as hydrogen

sulfide (H2S) 0-3%, 0-1% hydrogen, nitrogen and ammonia (Uzodinma et al., 2011). It is also

characterized based on its chemical and physical characteristics (Uri, 1992).

1.2.3 Chemical Characteristics of Biogas

Different sources of organic matter lead to different specific composition, the presence of H2S,

CO2 and water vapour make biogas very corrosive and request the use of adapted materials.

(Pohland and Ghosh, 1971).

19

1.2.3.1 Methane as a Component of Biogas

Methane is a simple chemical molecule, with the chemical formula CH4. It is the principal

component of biogas (natural gas). (Reay et al., 2011). Methane occurs naturally as a

component of natural gas, it is odourless, lighter than air and highly flammable. Methane can

form mixtures with air that are explosives at concentration of 5-15%. Methane is not toxic, but

can cause death due to asphyxiation by displacing oxygen in confined environments or spaces.

The heating value of pure methane is 1,000 BTU per cubic foot (Ogejo et al., 2009).

Additionally, methane is considered a powerful greenhouse gas that can remain in the

atmosphere for up to 15 years, with a global warming potential (GWP) of 30. (I.e. every kg of

methane emitted to the atmosphere has the equivalent forcing effect on the earth’s climate of 30

times that of carbon dioxide over a two-year period) ( Ogejo, et al., 2009).

Other gases, such as CO2, H2S and NH3, found in biogas are not useful because CO2 limits the

combustion power of methane and lead to bad quality of flames. In addition, H2S and NH3 are

toxic, corrosive and have an irritating smell (Uzodinma et al., 2011).

1.2.4 Physical Characteristics of Biogas

According to its composition, biogas is a gas appreciably lighter than air and is colorless; it

produces twice as less energy by combustion with equal volume of natural gas. Like those of

any pure gas, the characteristic properties of biogas are dependent on pressure and temperature.

They are also affected by the moisture content (Uri, 1992; Ogejo et al., 2009).

1.2.5 Purification of Biogas

CO2 can be separated from biogas by the use of quicklime. Ammonia is also separated or

retained by charcoal; while hydrogen sulfide (H2S) is removed by a layer of iron filings. This

purification gives a pure biogas (biomethane) which burn with a high production of heat.

(Young, 1982; Ogejo et al., 2009).

20

1.2.6 Collection of Biogas

The biogas is collected in an inverted drum, which extended down into the slurry (to provide

seal) and moves freely to accommodate more or less biogas as needed. The weight of the drum

creates pressure on the slurry to provide free flow of biogas through the small hole at the top of

the reactor. The valve at the top of the drum prevents the inflow of air into the reactor which

will reduce the activities of the bacteria and eventually lead to explosion of the reactor. The

maintenance of the pressure in the reactor was done using drums slightly smaller than the tank

to prevent loss of biogas and tipping of the drum (Young and Yang, 1989).

1.2.7 Types of Biogas Plants

Anaerobic bioreactors are engineered containment vessels designed to exclude air and promote

the growth of methane bacteria. Several different types of anaerobic bioreactors are used

worldwide for municipal, industrial-food, and agricultural waste treatment (Ogejo et al., 2009).

The types of simple biogas plants include: balloon plant, fixed dome plant, floating-drum

plants and prototype bioreactor plant made with galvanized metal pan.

1.2.7.1 Balloon Plants

The balloon plant consists of a bioreactor bag (e.g. PVC) in the upper part of the plant (figure

1). The inlet and outlet are attached directly to the plastic skin of the balloon. The gas pressure

is achieved through the elasticity of the balloon and by added weights placed on the balloon.

The cost of construction of a balloon plant is low and it is easy to transport. Other advantages

are low construction sophistication; high bioreactor temperatures; uncomplicated cleaning,

emptying and maintenance. The disadvantage can be the relatively short life span, high

susceptibility to damage, and little creation of local employment, with limited self help

potential. A variation of balloon plant is the channel type bioreactor (digester). Balloon plants

can be recommended wherever the balloon skin is not likely to be damaged and where the

temperature is even and high (Sasse, 1988; Nagamani and Ramasamy, 1999).

21

Figure 1: Balloon Plants. (Source: Sasse, 1988).

1.2.7.2 Fixed Dome Plants

Fixed dome plants have been (Figure 2a) tested and widely employed in digestion

(fermentation) of organic waste. However, fixed dome bioreactor show higher microbial

distribution of all tropic levels than floating drum type biogas bioreactors (Omer and

Fadalla, 2003).

The fixed-dome plant consists of a bioreactor with a fixed, non-movable gas holder, which

sits on top of the reactor when gas production starts. The slurry is displaced into the

compensation tanks, gas pressure increases with the volume of gas stored at the height

difference between the slurry level in the reactor and the slurry level in the compensation

tank. Fixed-dome plant is relatively low in its cost of construction because of the absence of

moving parts and rusting steel parts (Fullford, 1985). If well constructed, fixed-dome plants

22

has a long life span. The underground construction saves space and protects the reactor

from temperature changes. The construction also provides opportunities for skilled local

employment. The disadvantages of fixed-dome plant are mainly the frequent problems with

gas-tightness of the brickwork gas holder (a small crack in the upper brickwork can cause

heavy losses of biogas); fixed-dome plant are, therefore, recommended only where

construction can be supervised by experienced biogas technicians. The gas pressure

fluctuates substantially depending on the volume of the stored gas. (Omer and Fadalla,

2003).

Figure2a: Fixed dome plant Nicarao design. (Source: Amaratunga,

1986).Figure 2b: Fixed dome plant Nicarao design: 1. Mixing tank with inlet pipe and sand trap. 2. Digester. 3. Compensation and removal tank. 4. Gasholder. 5. Gas pipe. 6. Entry hatch, with gastight seal. 7. Accumulation of thick sludge. 8. Outlet pipe. 9. Reference level. 10. Supernatant scum, broken up by varying level. (Source: GATE and GTZ, 2007).

23

1.2.7.3 Floating Drum Plant

Floating gas holder plant and fixed dome type are the two tested designs that are being widely

employed. Though economics of the model and their suitability are well documented, not much

work is down on the microbiological aspects of these bioreactors (Nagamani and Ramasamy,

1999).

Floating drum plants (Bioreactors) consist of an underground reactor and a moving gas holder

(Figure 3). The gas holders float either directly on the fermentation slurry or in a water jacket of

its own. The gas is collected in the gas drum, which rises or moves down, according to the

amount of gas stored. The gas drum is prevented from tilting by a guiding frame. If the drum

floats in a water jacket, it cannot get stuck, even in substrate with high solid content. The

advantages are the simple and easily understood operation (Van Buren and Crook, 1985). The

volume of stored gas is directly visible. The gas pressure is constant, determined by the weight

of the gas holder. The construction is relatively easy, construction mistakes do not lead to major

problems in operation and gas yield. The disadvantages are high material costs of the steel

drum, the susceptibility of steel parts to corrosion. Because of this, floating drum plants have a

shorter life span than fixed dome plant (Uri, 1992).

[a] and [b] Figure 3a and b: (a) Floating-drum plant in Mauretania. (b) Water-jacket plant with external guide frame. 1 Mixing pit, 11 Fill pipe, 2 Digester, 3 Gasholder, 31 Guide frame, 4 Slurry store, 5 Gas Pipe. Source: Sasse (1988).

1.2.7.4 Prototype Plants

24

Prototype plant (bioreactor) is a Chinese metallic model biogas reactor designed and fabricated

with gauge 16 galvanized metals (Figure 4b). However, they are made specifically for batch

digestion of substrates. Its primary aim is to use the plant for popularization of the technology,

through exhibitions, workshops, conferences, etc. Hence the system comprises of fermentation

chamber, biogas storage chamber, the inlet pipe (made of a galvanized pipe), the outlet pipe

(made of two galvanized pipe) and a draining tap for discharging the slurry after a fermentation

batch. On top of the bioreactor is the stirrer and the gas pipe. The gas flow is regulated with the

aid of galvanized valve; the pressure is monitored with a low pressure guage. Prototype plants

are very cheap, save during transportation, are easy to operate and maintain (Eze, et al., 2003).

[a] [b] Photo: Ukonu, C.U

Figure 4a and b: Prototype Plants. (Source: Ofoefule and Uzodima, 2009).

1.2.8 Types of Fermentation/Culture Plant

There are two types of feeding plants in digestion (fermentation) process of organic matters

namely continuous and batch feeding plants.

1.2.8.1 Continuous Feeding

Gas production can be accelerated and made more consistent by continuously feeding the

digester with small amounts of waste daily. This will also preserve the nitrogen level in the

25

slurry for use as fertilizer (Houghton et al., 2002). In this type of feeding plant, it is

essential to ensure that the reactor is large enough to contain all the materials that will be

fed through in a whole digestion cycle. One solution is to use a double bioreactor,

consuming the waste in two stages with main part of the biogas (methane) being produced

in the first stage and the second stage finishing the digestion at a slower rate, while

producing another 20% or so of the total biogas (US Environmental Protection Agency,

2001).

1.2.8.2 Batch Feeding

In the batch system, digesters are filled once with fresh feedstock, with or without addition

of inocula, and sealed for the complete retention time, after which it is opened and the

effluent removed (Nayono, 2009). Batch systems are often considered as “accelerated

landfill boxes”, although in fact they achieve much higher biogas production rates than that

observed in landfills. This is largely because of two basic features. The first feature is that

the continuous recirculation of leachate not only allows the dispersion of inoculants,

nutrients and acids, but also improves the mixing condition. The second is that batch system

is run at higher temperatures than that normally observed in landfills.

One technical shortcoming of batch system is the risk of blockage of the leaching process

caused by clogging of the perforated floor. This problem is alleviated by mixing the

feedstock with bulking material (e.g. wood chips) and by limiting the thickness of the

fermenting wastes in order to limit compaction (Vandevivere et al., 2003). Although batch

systems have not succeeded in taking a substantial market share, especially in more

developed countries, the system is attractive to developing countries. The reason is that the

process offers several advantages as it does not require fine shredding of waste,

sophisticated mixing or agitation equipments, or expensive, high-pressure vessels, which

consequently lower the investment costs (Vandevivere et al., 2003; Koppar and

Pullammanappallil, 2008).

1.2.9.0 Feed Stocks (Substrates): Cow dung

Cow dung (fresh) is obtained in abattoirs where cows are slaughtered for human

consumption. The dung is obtained after the evacuation of the dung from the intestine of the

26

cow (slaugthered). Cow dung is of two types; the intestinal dung and the excrement

(excreted) dung from cows: The intestinal dung is the type removed from the intestine of

cows slaughtered in the abattoirs for human consumption. The intestinal cow dung consists

of the undigested residues of consumed matters. They are very fresh and contains the

normal microbial floral as found in the rumen of cow. Excreted dung is the dung excreted

by cow species, which are herbivores. It consists of digested residues of consumed matter

which has passed through the cow’s gastrointestinal system (Teo and Tech, 2011).

1.2.9.1 Composition of Cow Dung

Cow dung is composed of 1.8-2.4% nitrogen (N2), 1.0-1.2% phosphorus (P205), 0.6-0.8%

potassium (K2O) and 50-75% organic humus, carbon 39.17%, oxygen 53.10% , fat content

11% (Ofoefule and Uzodinma, 2006).

1.2.9.2 Microbial Floral Present in the various types of the Cow Dung

Research reveals that Aspergillus niger, Trichoderma harzianm, Bacillus cereus and

Bacillus subtilis have been isolated from cow dung which reduces the growth of Sclerotium

rolfsii, Fusarium oxysporum, Pythium aphanidermatum, Helminllosporuim mydis and

Rhizoctonia solani with inhibitory zones of up to 50% (Teo and Teoh, 2011). Furthermore,

it is the nature of the substrate that determines the types and extent of the fermentative

bacteria present in the reactor (Ogejo et al., 2009). Ramasamy et al., (1990), reported higher

presence of proteolytic organisms in cow dung fed-reactors and other animal waste fed-

reactors. Research has also shown higher amylolytic microorganisms in cow dung-fed

reactors. Ruminococcus flavefaciens, Eubacterium cellulosolvens, Clostridium

cellulosolvens, Clostristium cellulovorans, Clostridium thermocellum, Bacteroides

cellulosolvens and Acetivibrio cellulolyticus have been shown to be the predominant

fermentative bacteria present in cow dung fed-reactors (Ramasamy, 1997).

1.2.10 Sawdust

Sawdust is a by-product of cutting lumber with a saw, composed of fine particles of wood.

Economical disposal of sawdust and shavings is a problem of growing concern to the wood

industries. Enormous quantities of sawdust are produced annually by sawmills. The sawdust

27

produced in cutting a thousand board feet of 1 inch hardwood lumber with a saw cutting a

¼ inch kerf is at least (20.8) cubic feet of solid wood (12% moisture content) (Harkin,

1969). This large production of sawdust is of great challenge to the environments and has

aroused so much interest on how to control and channel the sawdust into a valuable or

profitable product.

The use of sawdust in combination with cow dung to produce biogas as carried out in this

investigation is one of the ways to control and utilize sawdust produced by cutting lumber

into a valuable product which will serve as a renewable energy. Vinodhini and Das (2009)

reported the use of neem sawdust as a biosorbant, which is employed in the uptake of

chromium (VI). Sawdust can also be used based on special physical qualities, which include

absorbant, abrasive, bulky and fibrous, nonconductive and granular for textured surfaces.

They can also be used as fuel, fiber and wood-based board and for chemical uses (Harkin,

1969).

The main constituents of sawdust include cellulose and lignin, and many hydroxyl groups,

such as tannins or other phenolic compounds (Vinodhiini and Das, 2009).

1.2.11 Other Substrates

Animal wastes are generally used as feed stock in biogas plants, but the availability of these

substrates is one of the major problems hindering the successful operation of biogas

reactors (Ogejo et al., 2009). Many workers have thus explored various other substrates for

biogas production. For biogas productions, the two most important parameters in the

selection of a particular plant feed stocks are the economic consideration and the yield of

methane from fermentation of that specific feed stock (Nagamani and Ramasamy, 1999).

Dhevagi et al. (1992) used cow dung, buffalo dung, dry animal waste, stray cattle dung,

goat waste, and poultry droppings for their biomethanation potential and observed that

poultry dropping showed higher gas production. Kasisira and Muyiija (2009), has shown

that 100% pig manure produced more gas per unit weight when compared to 100% cow

dung. This concurs with the finding of Hobson et al. (1981).

28

The combination of cow dung and poultry droppings has been shown to produce high yield

of biogas and reduction in the time lag between biogas production and gas flammability

(Ofoefule and Uzodinma, 2006). Combination of cow dung, pig and chicken manure and

water hyacinth has also been reported to increase the biogas yield and HRT (hydraulic

retention time) up to 15 days (Ntengwe et al., 2010). The combination with agricultural

waste and house hold waste has also been shown to increase biogas production (Ogejo et

al., 2009).

1.2.12. Microbiology and Biochemistry of Biogas Production

The degradation of organic matter to produce methane relies on the complex interaction of

several different groups of bacteria. Stable digester operation requires that these bacterial

groups be in dynamic and harmonious equilibrium. Changes in environmental conditions

can affect this equilibrium and result in the buildup of intermediates which may inhibit the

overall process. It is of utmost importance to understand the basic microbiological and

biochemical pathways, in order to master the biogas digestion system (Uri, 1992).

Biogas microbes consist of a large group of complex and differently acting microbial

species notably the methane-producing bacteria. The microbial diversity in biogas reactors

is as great as that of rumen, wherein seventeen fermentative bacterial species have been

reported to play important role for production of biogas (Wolin, 1979; (Ramasamy and

Nagamani, 1991). Furthermore, it is the nature of the substance that determines the type and

extents of the fermentative bacteria present in the reactor. Ramasamy et al.. (1990) reported

higher presence of proteolytic organisms in cow dung-fed reactors and other animal waste-

fed reactors. However, Rao and Seenayya (1993) observed that while cow dung-fed reactors

supported higher amylolytic microorganisms, poultry waste-fed digester showed higher

proteolytic population.

Effective biodegradation of organic wastes into methane requires the coordinated metabolic

activities of different microbial populations. The four metabolic groups which function in

anaerobic digestion include (a) the hydrolytic and fermenting bacteria, which convert a

variety of complex organic molecules (i.e., polysaccharides, lipid and proteins) into a broad

29

spectrum of end products (i.e., acetic acid, H2/CO2, monocarbon compounds, organic fatty

acids larger than acetic, and neutral compounds larger than methanol); (b) the hydrogen-

producing acetogenic bacteria, which include both obligate and facultative species that can

convert the products of the first group; the organic acids larger than acetic acid (e.g.

butyrate, propionate) and neutral compounds larger than methanol (e.g. ethanol, propanol)

to hydrogen and acetate; (c) the homoacetogenic bacteria which can convert very wide

spectrum of monocarbon compounds to acetic acid; (d) the methanogenic bacteria which

convert H2/CO2, monocarbon compounds (i.e. methanol, CO, methylamine) and acetate into

methane, or can form methane from decarboxylation of acetate (. Hao et al., 2011)

These diverse microbes found in the reactors undergo three concerted biochemical steps or

processes, which include hydrolysis, acidification/acetogenesis and methane formation

(methenogenesis).

1.2.12.1 Hydrolysis

In the hydrolysis step, the organic matter is hydrolyzed externally by exoenzyme (cellulase,

amylase, protease and lipase) produced by microorganisms (Figure 5).

The fermentative bacteria (extracellular enzymes) decompose the long chains of the

complex carbohydrates, proteins and lipids into a broad spectrum of simple end products

(i.e., acetic acid, H2/CO2, monocarbon compounds, organic fatty acids larger than acetic,

and neutral compounds larger than methanol) (Karakashev et al., 2006).

1.2.12.2 Acidification/Acetogenesis

Acidification is a fermentative process where acid-forming bacteria, also known as

acidogens, convert the products of hydrolysis into simple organic acids, alcohol, carbon

dioxide, and hydrogen gas. The hydrogen-producing acetogenic bacteria, which include

both obligate and facultative species, convert the products of the first group, that is, the

organic acids larger than acetic acid (e.g. butyrate, propionate) and neutral compounds

larger than methanol (e.g. ethanol, propanol) to hydrogen and acetate ( Hao et al., 2011).

30

1.2.12.3 Methane Formation

Methane-forming bacteria (Methanogens) utilize hydrogen, carbon dioxide and acetic acid

to form methane and carbon dioxide. Methane forming bacteria are obligate anaerobes

which reproduce slowly and are much more sensitive to environmental conditions. In

contrast to the acidogenic and acetogenic bacteria, the methanogenic bacteria belong to the

archaebacter genus i.e. to a group of bacteria with a very heterogenous morphology and a

number of common biochemical and molecular-biological properties that distinguish them

from all other bacteria generally (Karakashev et al., 2006; Qi et al., 2003).

Figure 1: Conversion of fermentation substrate to biogas. (Source: Uri, 1992)

31

1.2.13 Interaction between the Various Microbial Groups

Methane and acid-producing bacteria act symbiotically. On the one hand, acid-producing

bacteria create an atmosphere with ideal parameter for methane-producing bacteria

(anaerobic condition). On the other hand, methane-producing microorganisms use the

intermediates of the acid-producing bacteria to produce methane (U.S Environment

Protection Agency, 2001).

1.2.14 Kinetics of Anaerobic Fermentation

Several kinetic models have been developed to describe the anaerobic fermentation process

(Nagamani and Ramasamy, 1999). The mechanism of the reaction is first; the fermentative

action of acid forming microbes on the substrate (S) to produce alcohol, hydrogen (H2),

acids and carbon dioxide (CO2) and Second the action of methane-forming bacteria

(methanogensis) to produce methane (CH4) and C02 as indicated below (where SE is the

intermediate product, E is the enzyme, K1 and K2 are rate constant (s-1))

S + E SE E + CH4 + H2 +CO2 …………. (1)

The bacteria responsible for the reaction belong to the genera bacteriodes and clostridium.

The rate of reaction is assumed to depend on the concentration of SE, temperature, pH, and

the geometric of the bioreactor. The breakdown of S has been reported to follow the

Michaelis Menten mechanism (Levenspiel, 1962). The rate of reaction of S can be given as

below, which yield first order kinetics

R1 = K1 [S][E] = R-1 + R2 = K-1 [SE] + K2[S.E] ………………… (2)

ds/dt = K2SE/(K-1 + K2)/K1 + S = K2SE/KM + S ……………….. (3)

In S1/S2 = K2E/KM + S Xdt =Kdt………………………………….. (4)

Where T is the time, K-1 is the rate constant (S-1), R1, R2 R-1 are the rates of reaction

(Kmols-1-) R1, R2 are the initial and final substrate concentration (Kmols-1), km is a constant,

32

equation (4) can be used to evaluate the first order kinetic of biogas production at a given

conditions of temperature and pH (Ntengwe et al., 2010).

Hashimoto (1981) developed an equation, which attempts to describe kinetics of methane

fermentation in terms of several parameters. According to this equation, given below for a

given loading rate So/q daily volume of methane per volume of digester depended on the

biodegradability of the material (B0) and kinetic parameters UM and K.

Yv = (B0 .So/q) {1-(k/q µm-1+k)}………………………….. (5)

Where

Yv = is volumetric methane production rate, 1CH4l-1 digester d -1

So is influent total volatile solid (VS) concentration ( gl-1). Bo is ultimate methane yield,

CH4 (g-1 VS) added.

- q is hydraulic retention time d-1

- µm is maximum specific growth of microorganisms d-1

- K. is kinetic parameter, dimensionless

1.2.15 Factors Influencing Biogas Production From Manure

1.2.15.1 Temperature

Anaerobic microorganisms are temperature sensitive and temperature specific. In general,

methane production increases with increase in temperature. The three temperature regimes

used in anaerobic digesters are psychrophilic, mesophilic, and thermophilic with optimums

temperature ranges for the growth of methane forming bacteria of 41 to 77%, 860F to 1040F

and 1220F to 144oF, respectively (Hao et al., 2011). The U.S Environmental Protection

Agency (2001) reported that two temperature ranges are of interest for anaerobic treatment;

33

the mesophilic range and the thermophilic range, the mesophilic range extends from 85oF to

1000F (29 0C to 38 0C) with 950F (350C) usually chosen as the optimum temperature. The

commonly accepted bounds for the thermophilic range are 1200F to 1350F (490C to 570C).

Uzodinma et al. (2011) shows that steady increase in biogas production can be achieved

under the optimum mesophilic temperature of 400C when agro-based wastes are blended in

different proportion. Different bacteria dominate at different temperatures. Imbalance

between different bacteria groups may develop, causing methane production to be reduced

and other gases to be given off. So it is important to maintain the temperature within these

range.

1.2.15.2 pH

Maintaining an acceptable pH in the digester is important for the system to work well.

Acid-forming bacteria prefer a pH above 6.2 (Ntengwe et al., 2010). While methane-

forming bacteria live best under neutral to slightly alkaline conditions. Once the process of

fermentation has stabilized under anaerobic conditions, the pH will normally take on a

value of between 7.0 and 8.5 (Uzodinma and Ofoefule, 2009). Due to the buffer effect of

carbon dioxide-bicarbonate (CO2 = HCO3) and ammonia –ammonium (NH3-NH4+), the pH

level is rarely taken as a measure of substrate acids and/or potential biogas yield. According

to Ogejo et al., (2009), most anaerobic bacteria will perform well in the pH range of 6.8 to

7.2. The pH of the substrate decreases initially when organic material is first loaded into the

digester and volatile acid are produced. However, the pH of the digester will increase and

then stabilize, when methane producing bacteria consume the acids (due to alkalinity

produced).

1.2.15.3 Retention Time

The retention time is the number of days the organic material stays in the digester (Rao and

Seenayya, 1993). There are two significant retention times in anaerobic digester, solid

retention time (SRT) and hydraulic retention time (HRT). The SRT is the average time the

bacteria (solid) are left in the anaerobic digester. The HRT is the time the liquid is in the

anaerobic digester. SRT is the most important retention time, and should be determined

34

correctly because it indicates the potential of bacteria washout. If a significant washout of

bacteria occurs, the digester fails (Igoni et al., 2007).

However, the effective retention time may vary widely for the individual substrate

constituents. Biomethanation potential of market waste has been reported to be stable at 20

days HRT with 48% reduction in VS and with biogas production of 35 liters Kg-1d-1

(Bouallagui et al., 2003). Liquid cow manure, liquid pig manure, liquid chicken manure and

animal manure mixed with plant material have also shown to have HRT of 20-30days, 15-

25days, 20-40 days and 50-80 days respectively (Angelidaki, I. and Ellegaard, 2003). Short

retention time indicates that bacteria are washout faster than they can reproduce.

1.2.15.4 Substrate (Quality and Characteristics of Manure)

The characteristics of solid wastes determine the successful anaerobic digestion process

(e.g. high biogas production potential and degradability). In municipal solid waste, substrate

characteristics may vary due to the method of collection, weather season and cultural habits

of the community (Hartmann and Ahring, 2006). The composition of waste also determines

the relative amounts of organic carbon and nitrogen present in the waste substrate (C/N

ratio). A solid waste substrate with high C/N ratio is not suitable for bacterial growth due to

deficiency of nitrogen. As a result the gas production rate and solid degradability will be

low. On the other hand, if the C/N ratio is very low, the degradation process leads to

ammonia accumulation which is toxic to the bacteria (Hartmann and Ahring, 2006).

Kayhanian and Hardy (1994) found that a C/N ratio (based on biodegradable organic

carbon and nitrogen) within the range of 25–30 is considered to be optimum for an

anaerobic digester. To maintain the C/N level of the digester material at optimum levels,

substrates with high C/N ratio can be co-digested with nutrient-rich organic wastes (low

C/N ratio) like animal manure or food waste (Zaher et al.,2009).

The quality of the substrate is also affected by animal diet, manure handling, and storage

method (Uzodimnma and Ofoefule, 2009). Substrate from animal fed with higher energy

feed (e.g. Grain-based diets) has the potential to yield more methane gas compared to

substrate from animals fed with roughage diet (Ramasamy et al., 1990). However, materials

35

with high cellulose do not digest well. They act as filler and reduce the capacity of the

digester to produce gas. Materials that float to the top of the digester or sink to the bottom

of the digester are undesirable. They form scum and those that sink may clog the bottom of

the reactor (Ogejo et al., 2009). Currently, materials that are highly degradable produce

more gas.

1.2.15.5 Loading Rate

The ability of a digester to convert organic material into methane is related to its loading

rate. Loading rate is commonly defined as the amount of volatile solids fed to the digester

per day per unit volume of the digester. Volatile solid is the measure of the amount of

digestible organic material in a feedback. In general, materials with high volatile-matter

content produce more biogas if digested properly (Vartak et al., 1997a). Accor ding to

Mohanrao (1974), a daily loading rate of 16 kg VS/M3 of digester capacity produced 0.04

0.074 m3 of gas/kg of dung-fed. A lab-scale digester operating at different OLRs produced

a maximum yield of 0.36 M3/kg VS at an OLR of 2.91 kg VS/M3/day (Sundrarajan et al.,

1997).

1.2.15.6 Design of Digester

Floating gas holder type and fixed dome type are the two first designs that are been widely

employed, though economics use of the model and their suitability are well documented,

not much work have been done on the microbiological aspects of these digesters (Omer and

Fadalla, 2003). Generally, fixed dome digesters have shown higher microbial distribution of

all tropic levels than floating drum type biogas bioreactor (Eze, et al., 2003). Biogas plants

constructed above the ground (level) must be made of steel to withstand the pressure within;

maintenance is, however, much simpler for systems built above ground and a black coating

will help provide some solar heating.

1.2.15.7 Volatile Acid

The concentration of volatile fatty acid (VFA) serves as an indicator during anaerobic

digestion process. An increase in volatile acid concentration suggests that either the organic

36

loading to the system has been increased or that the methane farming bacteria are inhibited

from performing their normal functions. A decrease in volatile acids indicates either that the

loading has been reduced or that the system is acclimatizing (adjusting) to new conditions

(Uzodinma and Ofoefule, 2009). Products of the acid forming reaction are the acid salts of

short-chain fatty acids. Acetic acid is usually the most predominant volatile acid present,

but there are also lower concentrations of propionic and butyric acids. (Formic, Isobutyric,

volaric, Isovolaric, Isocaproic, caproic, and haptanoic) which may also be present in the

total make up (Igoni et al., 2007).

1.2.15.8 Nutrient Requirement (Additives)

Macronutrients have been shown to be important for good biomass growth and system

performance and should therefore be added daily in bioreactor. The subject of nutrients

needed by microorganisms involved with biological treatment of biomass usually focuses

on nitrogen (N) and phosphorus (P). These are the micronutrients required to satisfy the

needs of active biota (living organisms) in either aerobic or anaerobic systems (US

Environment Protection Agency, 2001). Potassium (K) is also considered a macronutrient

and its requirement is based on the nitrogen requirement (Marcato et al., 2007). Nutrient

considerations are not limited to nitrogen and phosphorus. Ramasamy and Nagamani (1999)

have shown that several inorganic nutrients are required for an anaerobic process to

function at optimal efficiency. The elements that produced the most notable results are

some metals which are required in trace amounts and are referred to as micronutrients.

These include iron, nickel, calcium, magnesium, sulfur, cobalt, selenium, mercury, silver

and molybdenum (US Environment Protection Agency, 2001). Micronutrients are generally

more important for the methane forming bacteria. The lack of micronutrients will be

evident by a gradual reduction in system performance as indicated by low COD reduction

and high effluent VFAs (Seenayya et al., 1992).

1.2.15.8.1 Cobalt

Cobalt is a naturally occurring element with one stable isotope (59Co) and 26 known

radioactive isotopes. They are dosed directly into the reactor feed line. Cobalt and copper

37

serve as link to overcome the difficulties in the attachment between the similarly charged

rumen bacteria and plant cell walls by providing free available divalent cations (Somers,

1983). Cobalt appears to be a better choice because it is also an essential trace element

(micronutrient) required exclusively by rumen microorganisms for the biosynthesis of

vitamine B12. Saxena and Ranjhan (1978a), reported that supplementation of cobalt above

minimal requirement, may be beneficial during rapid rumen fermentation for increased

growth and activity of microbes. Salsbury et al. (1956) has demonstrated increase in the in

vitro digestion of cellulose of Alfalfa leaf meal in the presence of urea, by feeding of cobalt

.This he attributed to the high level of protein content of the meal. Seenayya et al. (1992)

observed that addition of calcium (5mM), cobalt 50µgg-1TS), iron (50mM), magnesium

(7.5mM), molybdenium (10-20mM), nickel (10µgg-1TS) individually as well as in

combination, enhanced the biogas production and attributed this to the increased

methonogenic population in the digesters. In agreement to this, Jarvis et al. (1997) observed

that addition of cobalt (0.2mgl-1) improved the gas yield and methane content of gram

clover silage-fed reactor. US Environmental Protection Agency (2001) reported the

required range of cobalt for anaerobic digestion to be 0.01-0.03mg/l.

1.2.15.8.2 Nickel Sulphate

Nickel is a transition metal and has been reported by US Environmental Protection Agency

(2001) as a micronutrient required for the growth of methanogenic archaea and acetogenic

bacteria. Geetha et al. (1990) observed that addition of nickel at 2.5ppm increased the

biogas production from digesters fed with water hyacinth and cattle waste blend and

attributed this to higher activity of nickel dependent metalo-enzymes involved in biogas

production. Jain et al. (1981) also reported the increase in biogas production and methane

content by addition of Cd and Ni at 600 and 400µg-1of dry matter respectively. Takashima

and Speece (1989) investigated heavy metals in cells of ten methanogenic strains. They

showed the presence of the following heavy metals (in falling concentration): Fe >> Zn ≥

Ni > Co = Mo > Cu. A proper dosage of heavy metals is required for anaerobic processes.

Nickel ions at a concentration of 5mg.L-1 for instance will stimulate methane production by

methanobacterium thermautotrophicum to its optimum production (Oleszkiewicz and

38

Sharma, 1990). The recommended quantity of nickel required by microorganism or

anaerobic bacteria is between the ranges of 0.05-0.10mg/l (US Environmental Protection

Agency, 2001).

1.2.15.8.3 Boric Acid

Boric acid also known as boraic acid or ortho-boric acid is a naturally occurring compound

that contains the elements boron, oxygen and hydrogen (H3BO3) (WHO 1998). It is found

naturally in air, water (surface and ground water, soil and plants including food crops).

Boric acid is derived from boron, a naturally occurring element in rock, soil and water.

Boron is universal in the environment and typically found in the form of borates or

combined with other chemical (Woods, 1994). Boric acid is generally a white odourless and

generally stable under ambient condition. Boric acid is known as boron compound of

environment significant (Eisler, 1990). Boron has been shown to be an essential plant

micronutrient and some boric acids products are used to correct boron deficiencies in plants

(Woods, 1994). Singh et al, (1993) observed that addition of borax and diborane at 0.2g/l

increased the biogas production from digester fed with water hyacinth as the substrate.

1.2.15.8.4 Zinc

Heavy metal transfer and distribution have rarely been considered in anaerobic digestion

(Theis and Heyes, 1978). Nevertheless, feed pig is supplemented with Cu and Zn as

essential (Jondrovile et al., 2003). According to the previous work done by Theis and Heyes

(1978); Beline et al. (2004), metals were found to be associated with the organic matter in

liquid wastewater or manure. The recommended amount of Zn required for anaerobic

(methane) bacteria ranges from 0.1-0.3mg/l (US Environmental Protection Agency, 2001).

1.2.15.8.5 Zeolite

Zeolite is a crystalline, hydrated aluminosilicate of alkali and alkaline earth cations with an

infinite open three-dimensional structure (Gayoso and Gil, 1994). Natural zeolite is formed

by the alteration of volcanic ash, in lake and marine waters (Mumpton, 1999). These beds

were found to contain as much as 95% of single zeolite. They are generally flat-lying and

39

easily mined by surface methods. Zeolite has the property of losing and gaining water

reversibly and to exchange extra frame work cations, but without change of crystal

structure (Butterfield and Borgerding, 1981; Kello, 1995). Natural zeolite (those found in

volcanogenic sedimentary rocks) are used as building stones, lighter-weight aggregate and

pozzolans in cements and concretes, as filler in paper, in the taking-up of Cs and Sr from

nuclear waste and fallout, as soil amendment in agronomy and horticulture, in the removal

of ammonia in municipal, industrial and agricultural waste and drinking waters as

exchangers (Mumpton, 1999). Large-scale cation exchange processes using natural zeolite

were first developed by Ames, (1967) and Mercer et al. (1970) who demonstrated the

effectiveness of clinoptilolite for extracting NH4+ from municipal and agricultural waste

stream. In Tahoe- Truckee (Truckee, CA) sewage treatment plant, it has been shown that

>97% of NH4+ was removed from tertiary effluent (Butterfield and Borgerding, 1981).

Nitrification of sludge is accelerated by the use of clinoptilolite, which selectively

exchanges NH4+ from wastewater and provides an ideal growth medium for nitrifying

bacteria which oxidized NH4+ to nitrate (Liberti et al., 1995). Chabazite has also been used

to remove polar H2O, H2S and CO2 from low-BTU (British Thermal Unit) natural gas and

developed a zeolite adsorption process for purifying methane produced by decaying garbge

in a Los Angeles landfills (Ames,1967).

In animal waste treatment, natural zeolite are potentially capable of (i) reducing the malodor

and increasing the nitrogen retention of animal waste (ii) controlling the moisture content

for ease handling of excrement and (iii) purifying the methane gas produced by the

anaerobic digestion of manure. (Koelliker et al., 1980; Milian et al., 2001).

1.2.16 Inhibitory Factors Affecting Biogas Production (S)

Inhibition in anaerobic digestion process by the presence of toxic substances can occur to

varying degrees, causing upset of biogas production and organic removal or even digester

failure (Stronach, et al., 1986). These kinds of substances can be found as components of

the feeding substrate (organic solid waste) or as by-products of the metabolic activities of

bacteria consortium in the reactor. Previous publications on anaerobic digestion show a

wide variation in the inhibition/toxicity levels for most substances. The main reason for

40

these variations is the significant influence by microbiological mechanisms such as

acclimation, antagonism, and synergism (Chen, et al., 2008). Acclimation is the ability of

microorganism to rearrange their metabolic resources to overcome the metabolic block

produced by the inhibitory or toxic substances when the concentrations of these substances

are slowly increased within the environment. Antagonism is defined as a reduction of the

toxic effect of one substance by the presence of another, whereas synergism is an increase

in the toxic effect of one substance by the presence of another. Several substances with

inhibitory/toxic potential to anaerobic digestion, such as ammonia, sulfide, light metal

ions, heavy metals and organic substances and biotics (bacitracin, flavourycin, lasalocid,

monensin, spriamcin, etc.) and detergents used in livestock husbanding have been shown

to exhibit inhibiting effect on the process of biomethane production. The table below lists

the limit concentrations (mg/L), for various inhibitors.

Table 1: Limiting concentrations of various inhibitor of bio-methane.

Substances mg/L (limit conc)

Copper 10-250

Calcium 8000

Sodium 8000

Magnesium 3000

Nickel 100-1000

Zinc 350-1000

Chromium 200-2000

Sulfide 200

Cyanide 2

Source: (Kossmann et al., 1999).

41

1.2.16.1 C/N Ratio (Carbon/Nitrogen Ratio)

Microorganisms need both nitrogen and carbon for assimilation into their cell structures.

Various experiments have shown that the metabolic activity of methanogenic bacteria can

be optimized at C/N ratio of approximately 8-20, where by the optimum point varies from

case to case, depending on the nature of the substrate. The ratio of C/N (25-30:1) is

optimum for biogas production (Bardiya and Gaur, 1997). The reverse of the ratio between

the carbon and nitrogen inhibit the activities of the microorganisms in the bioreactor.

1.2.16.2 Nitrogen Inhibition

All substrates contain nitrogen for higher pH values, even a relatively low nitrogen

concentration may inhibit the process of fermentation. Noticeably, inhibition occurs at a

nitrogen concentration of approximately 1,700mg ammonium-nitrogen (NH4-N) per liter

substrate, nevertheless, with time, the methanogens are capable of adapting to NH4-N

concentrations in the range of 5000-7000mg/L substrate. The main prerequisite being that

the ammonia level (NH3) does not exceed 200-300mg NH3-N per liter substrate. The rate of

ammonia dissolution in water depends on the process temperature and pH value of the

slurry (Hajarnis and Ranade, 1992).

1.2.16.3 Other Inhibitors

Mallik et al. (1990) have reported the use of cannabis for biogas production, but observed

that use of fresh cannabis at 31% completely inhibited the biogas production due to the

higher presences of alkaloid.

1.2.17.0 Benefits of Biogas Technology

Anaerobic digestion provides some exciting possibilities and solutions to such global

concerns as alternative energy production, handling human, animal, municipal and industrial

wastes safely, controlling environmental pollution, and expanding food supplies (Batjes et

al., 1996). Anaerobic digestion converts organic waste into profitable by-product as well,

reduces their environmental pollution potential (Nayono, 2009). Digestion of organic waste

reduces groundwater and surface water contamination potential. It also reduces the odor of

42

the green house gas emission (Ogejo et al., 2009). Biogas can be used as a fuel in stationary

and mobile engines, to supply motive power, pump water, drive machinery (e.g., threshers,

grinders) or generate electricity (Brown, 2006).

1.2.17.1 Agriculture

Due to the decomposition and breakdown of parts of its organic content, digested sludge

provides fast-acting nutrients that easily enter into the soil solution, thus becoming

immediately available to the plants (Okoroigwe, 2007). They simultaneously serve as

primary nutrients for the development of microorganisms. They also nourish actinomycetes

(ray fungi) that act as organic digesting specialists in the digested sludge (Chinedu et al.,

2008).The humic matter and humic acids present in the sludge contribute to a more rapid

humification, which in turn help reduce the rate of erosion while increasing the nutrient

supply and hygroscopicity (Ogejo et al., 2009). The elevated ammonium content of

digested sludge helps reduce the rate of nitrogen washout as compared to fertilizers

containing substantial amount of more water-soluble nitrates and nitrites (Mercer et al.,

1970).

1.2.17.2 Domestic and Industrial Uses

The primary domestic uses of biogas are cooking and lighting. Because biogas has different

properties from other commonly used gases, such as propane and butane, and is only

available at low pressures (4 - 8 cm water), stoves capable of burning biogas efficiently

must be specially designed. To ensure that the flame does not "lift off," the ratio of the total

area of burner parts to the area of the injector orifice should be between 225 and 300:1

(FAO, 1981). Recent Indian designs have thermal efficiencies of around 60% (Mahin,

1982). In China the Beijing-4 design has a thermal efficiency of 59 - 62%, depending on the

pressure (Chan U, 1982). Lighting can be provided by means of a gas mantle, or by

generating electricity. Highest lamp efficiencies require gas pressures of 40 cm, which are

only possible with fixed dome reactors. Reported gas consumption for cooking and lighting

is 0.34- 0.41 m3 per capita/day and 0.15m3 per hour per 100 candle power respectively. A

typical family of six uses approximately 2.9 m3/day of biogas (Hossain, 2001).

43

1.2.17.3 Other Uses of Biogas

Biogas is a potential source of revenue which can be obtained from sales of digested manure

(liquid and solid), and excess electricity, or of reuse of digested solid as live stock bedding

(Brown, 2006).

1.3.0 Aim and Objectives

This study which aims at investigating the optimization of biogas production by blending

cow dung with saw dust. These were designed to achieve the following objectives:

To determine the pH at which biogas production is optimized.

To determine the temperature at which biogas is produced at optimum.

To optimize the quantity and quality of biogas produced from the anaerobic

fermentation of organic waste, and

To reduce the lag phase of the fermentation process.

44

CHAPTER TWO

2.0 MATERIALS

2.1 Chemical

All the chemicals used in the research were of analytical grade and were obtained from Merck

(Germany) or BDH chemicals Ltd (poole England). These includes, 95% sulphuric acid,

Sodium hydroxide, 1% boric acid-indicator mixture, 0.01N Hydrochloric acid, Sodium

sulfate/copper sulfate, boric acid, boric solution, 90% ethanol, oil-free petroleum spirit (boiling

range 40oC-60oC or 60oC-80oC, diethyl ether, carbon tetrachloride, nickel sulphate, cobalt

sulphate, zinc, nutrient agar, marconkey agar, distilled water etc.

2.2 Apparatus

Bioreactor(s) (prototype) of 50-liter capacity constructed at the National Centre for Energy

Research and Development, University of Nigeria, Nsukka were used; Speriam gas analyzer of

model number 66429, made in USA; Mercury in glass thermometer (0-100OC); pH meter

(SEARCHTECH) of model number PHS-3C, made in USA; Weighing balance (50kg capacity)

of model number Z051599; Gas burner; incubator, micro kjeldahl.

Other equipment used includes test-tubes, beakers, conical flasks; syringes; measuring cylinder

(Pyrex); crucible; Buchner funnel; oven; muffle furnace; hose pipe; water trough; graduated

(transparent) bucket, Soxhlet extractor.

2.3 Collection and Processing of Samples

Fresh cow dung (intestinal) were obtained from the abattoir in new community market Ikpa in

Nsukka, Enugu State of Nigeria, while the saw dust was obtained from Timber market Frame

Road Nsukka, Enugu State of Nigeria.

45

2.3.1 Collection of Soil Sample

Soil Sample was collected from Umunaga in Ubaha, Okigwe local Government Area, Imo

State.

2.4 Preparation of Waste

Varied quantities of both cow dung and saw dust were weighed out and thoroughly mixed in a

plastic water bath before charging into a 50-litre bioreactor. Appropriate quantities of water and

waste were used, which were determined by the moisture content of the waste used.

2.4.1 Pretreatment of Saw Dust:

The saw dust was pretreated using the method described by Cao et al. (1996).

Exactly 3.75kg of sawdust contained in a plastic water bath was soaked with 1.45N NH4OH

solution (1:1 ratio w/v) for 72 hours. Thereafter, the saw dust was thoroughly washed with

distilled water and then dried in an oven at 80oC for 72 hours. The dried pre-treated saw dust

was then mixed thoroughly with an appropriate quantity of cow dung before charging into the

bioreactor.

2.5 Experimental Design

2.5.1 Phase I

In the first phase (preliminary stage), a set of six batch bioreactors (labeled 1-6) of 50-litre

capacity was set up. Each was filled up to 75% (3/4) with a fixed amount of cow dung, varying

the quantity of water, to determine the best performed bioreactor mixture for the next phase of

the research, (the amount of water taken also depends on the moisture content of the cow dung)

as shown below

Bioreactor 1 (1:1): consisted of 18.75kg of cow dung to 18.75 litres of water.

Bioreactor 2 (1:2): consisted of 12.5kg of cow dung to 25 litres of water.

Bioreactor 3 (1:0): consisted of 37.5kg cow dung only.

Bioreactor 4 (1:3): consisted of 9.38kg of cow dung to 28.1 litres of water.

Bioreactor 5 (1:4): consisted of 7.5kg of cow dung to 30 litres of water.

46

Bioreactor 6 (1:5): consisted of 6.2kg of cow dung to 31.3 litres of water.

2.5.2 Phase II

In the second phase, the best performing mixture of cow dung to water ratio of the phase I was

used. In this stage, five batch bioreactors (labeled 1-5) of 50-liters capacity were set up and

were charged up to ¾ of the bioreactor volume, varying the amount of cow dung and sawdust

while the volume of water remained constant as shown below

Bioreactor 1 (1:0:4): consisted of 7.5kg of cow dung to 30 litres of water only.

Bioreactor 2 (0:1:4): consisted of 7.5kg of sawdust to 30 litres of water only.

Bioreactor 3 (3:2:4): consisted of 4.3kg of cow dung to 3kg of sawdust to 30 litres of water.

Bioreactor 4 (2:3:4): consisted of 3kg of cow dung to 4.3kg of sawdust to 30 litres of water.

Bioreactor 5 (1:1:4): consisted of 3.75kg of cow dung to 3.75kg of sawdust to 30 litres water.

These digestion processes were carried out for the period of 28 days retention time.

2.5.3 Phase III

In the third phase, the mixture of the best performed bioreactor in phase II was used .Measured

quantities of micronutrients, such as cobalt sulphate, nickel sulphate, boric acid, zinc and

zeolite were charged into the bioreactors, respectively. Pretreated sawdust was also charged

into a separate bioreactor. This was set-up using seven bioreactors as follow

Bioreactor 1: contained 3.8kg of cow dung, 3.8kg of sawdust (pretreated) and 30 litres of water.

Bioreactor 2: contained 3.8kg of cow dung, 3.8kg of sawdust (untreated), 30 litres of water.

Bioreactor 3: contained 3.8kg of cow dung, 3.8kg of sawdust, and 30 litres of water and 7.5g of

boric acid.

Bioreactor 4: contained 3.8kg of cow dung, 3.8kg of sawdust, 30 litres of water and 0.00375g

of nickel sulphate.

Bioreactor 5: contained 3.75kg of cow dung, 3.75kg of sawdust, and 30 litres of water and

0.00125g of cobalt sulphate.

Bioreactor 6: contained 3.8kg of cow dung, 3.8kg of sawdust, 30 litres of water and 0.01125g

of zinc.

47

Bioreactor 7: Contained 3.8kg of cow dung, 3.8kg of sawdust, and 30 litres of water and 345g

of zeolite.

2.6.0 Methods

2.6.1 Charging of Bioreactor(s)

The different variants were weighed and mixed thoroughly in a water trough. The mixtures

were charged into the 50-litres metal prototype batch bioreactor(s). The waste was charged up

to ¾ of the bioreactor volume, leaving ¼ head space for gas collection.

The bioreactors were properly tightened with the valve locked to exclude air. The bioreactor

contents were stirred adequately (50 periods per minute) on a daily basis throughout the

retention period to ensure homogenous dispersion of the substrate and microbes in the mixture.

2.6.2 Determination of Quantity of Biogas Produced

The quantity of biogas produced in litre/total solid was obtained by downward displacement of

water by the biogas on daily bases.

2.6.3 Determination of pH of the Slurry in the Bioreactor

The pH of the slurry were determine daily using pH meter (Search Tech, model PHS 3C).

Sample of the slurry were collected before and after stirring, and the pH were determined using

pH meter at 12 hours interval.

2.6.4 Determination of pH of Soil Sample

Ten (10g) grams of the soil sample was weighed into two 50-ml beakers set up simultaneously;

25ml of distilled water was then added to one of the beakers labeled water (H2O), while 25ml

of 0.1N potassium chloride was added to the other beaker labeled KCl. The content of each

beaker was stirred with a glass rod and was allowed to stand for 30 minutes with intermittent

stirring. The pH meter was calibrated with buffered solution of pH 4.0 and 7.0 and the pH of

the sample taken after 30 minutes.

48

2.6.5 Determination of the Ambient and Slurry Temperatures of the Bioreactor

The ambient and slurry temperatures of the bioreactor(s) were also monitored at 12 hours

interval throughout the retention period after charging of the bioreactors with mercury in glass

thermometer (0-100oC). The slurry temperature was determined by immersing the mercury bulb

into the slurry and it was held at the tip of the thermometer. The temperature was taken when

the mercury reading in the glass had been steady for one minute.

2.6.6 Determination of Biogas Flammability

The flammability of the biogas produced was determined using a fabricated gas burner. The

fabricated gas burner was connected to the bioreactor’s valve (tap); with a pipe hose, the valve

was then open to allow the flow of gas through the hose to the gas burner, and was ignited.

2.6.7 Determination of the Composition of Biogas Produced

The composition of the flammable biogas produced in each of the reactors was determined

using Speriam Gas Analyzer (model 66429 made in USA) which shows composition of

methane, carbon monoxide, hydrogen sulphide and oxygen. The inlet pot of the Speriam Gas

Analyser was taken close to the biogas outlet pipe and the gas was allowed to flow into the

analyzer which analyzes the quantity of methane and carbon dioxide produced in percentage,

while the quantities of hydrogen sulphide and oxygen are given in ppm.

2.6.8 Determination of the Total Microbial Count of the Slurry

Total viable counts (TVC) of the microbes for the digested slurry mixture were carried out to

determine the microbial load of the variant mixture using the modified method of Miles and

Misra (1938) as described by Okore (2004). This was carried out at four different periods

during the digestion; at the point of charging, flammability, peak of production and at the end

of the retention time.

49

The method consists of placing drops (0.02ml) of serial dilution on the surface of poured agar

plate and counting the colonies that develop on incubation of the plates. The method is useful

when the bacteria are best grown in surface culture or when an opaque medium is employed.

Procedure

Ten test-tubes and culture plates were thoroughly washed clean and allowed to dry.

Thereafter, 10ml of distilled water was pipetted into one test tube, while 9ml of the distilled

water was pipetted into the other nine test-tubes. These were incubated in the culture plate at

100oC for 30minutes. They were allowed to cool. Thereafter, 1ml of the sample was diluted

into the test-tubes serially.

About 0.01ml drops of ten-fold dilution selected was then dropped on the surface of the

medium agar from a height of 2.5cm, using a 2ml sterile string. Thereafter, the agar media was

poured on the culture plate and was swirled gently. The media was allowed to cool and gel at

room temperature with the lid closed. The cultured plate was incubated at 37oC for 24 hours.

Thereafter, the total count of the colonies of the selected dilution dropped was taken.

2.6.9 Determination of Ash Content of the substrates (cow dung and saw dust)

The residue remaining after the destruction of the organic matter of feeding stuff is referred to

as ash. This was determined by the Method of AOAC (1990).

Procedure

The crucible (silica dish) was heated at 600oC for 30 minutes and was cooled in a

desiccator and was weighed. Five (5g) of the sample was weighed into the crucible and the

weight of the weighed crucible + sample was taken. The crucible containing the sample was

then put into a heater in fume cupboard to burn off the less volatile organic material. Pre-ashing

was stopped when smoking stopped. The crucible was thereafter transferred into a cool muffled

furnace and the temperature was increased to 600oC and maintained until a whitish-grey (as

remains) was obtained. The crucible was then removed and cooled in a desiccator. The weight

of the crucible + ash was weighed and noted. The % ash content was obtained using the

calculation below,

50

% Ash = (weight of crucible + weight of ash- weight of crucible) x 100

Weight of sample

2.6.10 Determination of Moisture Content of the substrates using the Method of AOAC

(1990)

Procedure

A clean crucible was ignited and cooled in a desiccator and the weight taken. Exactly 2g

of the sample was placed in the crucible and the weight of the crucible + sample was taken. The

crucible was then dried in the oven at 100oC for 24 hours to constant weight (by reweighing

after every 4 hours then after 30 minutes until a constant weight was obtained). The weight was

taken and the % moisture content calculated as shown,

% moisture content = 100 x weight of sample - weight of crucible + sample after drying

Weight of sample taken

2.6.11 Determination of Fibre Content substrate using the Method of AOAC (1990)

The fibre contents of the samples were determined using the method described by AOAC

(1990).

Three (3.0g) gram quantity of the sample was weighed into a flask and the oil was removed by

grinding the sample to pass a 1-mm mesh sieve by soxhlet extraction and was dried. The air-

dried fat-free material was then transferred into a beaker. Exactly 200ml of 0.128M sulphuric

acid was added into the beaker at room temperature. The material was dispersed for 30 minutes

and brought to boiling within 1 minute. Excess foaming was reduced by adding 1ml of

antifoam solution. The mixture was then boiled for 30 minutes with the volume being

maintained at constant level, by addition of water. The container was being rotated every 5

minutes to mix and remove particles from the sides). Before the end of the 30 minutes, 11 cm

Whatman No. 451 filter paper was fitted into a Buchner funnel. Boiled water was poured into

the funnel and was allowed to stand until the funnel was hot. At the end of the 30 minutes

boiling period, the acid mixture was allowed to stand for 1 minute. Thereafter; it was poured

into a shallow lever of hot water (the suction was adjusted so that the filtration of the bulk of

51

200ml is completed within 10 minutes). The insoluble material was washed with boiling water

until the ash material turned neutral to litmus paper. These were repeated by washing the

residues back into the beaker using 200ml of 0.313N sodium hydroxide measured at room

temperature. This was boiled for 30 minutes as described above (allowed to stand for 1 minute

and filtered hot through a filter crucible with gentle suction).The insoluble material was then

transferred into a crucible with hot water and was washed with boiling water first, then with

0.1N hydrochloric acid, followed with water until the washing turned neutral to litmus paper;

(the washing was repeated twice with alcohol).The content in the crucible was then dried at

100OC and was cooled in a desiccator and the weight of the content was taken. Furthermore,

the crucible was placed in a cool muffle furnace and the temperature was increased and

maintained at 500OC, until ashing was completed. The crucible was then removed from the

muffle furnace, cooled in a desiccator and the weight taken.

Calculation

% Fibre = Change in weight (loss in wt in ignition in grammes) 0.03

2.6.12 Determination of Fat Content of the Substrate, using Soxhlet Extraction Method

(Pearson, 1976).

Procedure

A clean flask was dried in an oven at 100oC and was cooled in a desiccator before

weighing. Five (5g) gramme of the sample were transferred into the flask. The sample was then

ground to pass 1-mm sieve in a thimble, plugged with cotton wool and was placed into the

extractor. The extraction was done with petroleum spirit for 4 hours first. Thereafter, the

residue was transferred into a small mortar, ground lightly and was then returned to the

extraction apparatus. The mortar was washed and rinsed with small quantity of petroleum spirit,

and transferred into the flask. The extraction was continued for additional 1 hour (the thimble

was removed, if the flask was seen to contain insoluble matter) until most of the solvent had

distilled from the flask into the extractor. It was then placed in an oven for 2 hours. This was

cooled and weighed. The percentage oil was calculated as follow:

52

% oil (w/w) = initial weight of sample – final weight of sample after extraction x 100 (Initial) weight of sample taken.

2.6.13 Determination of Crude Nitrogen/Ammonium-Nitrogen of the substrate, using

Micro-Kjedhal Method as Described by Pearson (1976).

Nitrogen in sample is converted to ammonium-nitrogen by digestion with sulphuric acid using

a catalyst. The ammonia liberated when this digest is reacted with sodium hydroxide is

removed by steam distillation and collected with 1% boric acid-indicator mixture. This is then

titrated with 0.01N HCl to give % nitrogen in the sample.

Procedure:

Two grams (2g) of the dried sample (cow dung/soil) was weighed and transferred into a

Kjeldahl flask and 4g mixture of Na2SO4 and CuSO4 was then added. About 25ml of

concentrated sulpuric acid was also added to the flask, which was taken to the heater. After

swirling, the mixture was heated gently at first, until frothing stoop, then more strongly, until a

near clear solution resulted. The digest was cooled and transferred quantitatively into a 250ml

volumetric flask and made up to mark. The mixture was shaken properly and 5ml of the digest

was pipetted into the distillation unit. Exactly 10ml sodium hydroxide solution was added into

the sample chamber and the liberated ammonia was collected with 10ml boric acid-indicator

mixture in a conical flask placed at the condenser of the markham unit. The distillation of the

mixture was stopped 5minutes after the boric acid-indicator mixture turned green. Thereafter,

the conical flask was removed and was titrated with 0.01N HCl until the original colour of the

boric acid-indicator mixture was restored.

% N = 0.00014 X Titre value X 50 X 100 OR T x N x 14.01/1000 x 100/ws = % N

Weight of sample taken

% c.p = N X 6.25 Where: T = Sample Titre, N = Normality

Ws = weight of sample

53

2.6.14 Determination of Carbon Content of the Substrate, Using the Method of

Walkley and Black (1934)

Using the Walkley-Black method, the calculation of organic matter assumes that 77% of the

organic carbon is oxidized by the method and that soil organic matter contains 58% carbon.

Since both of these factors are averages from a range of values, it would be preferable to omit

them and simply report the results as "easily oxidizable organic carbon.

Procedure

Two gram (2g) dried organic waste was weighed and transferred to a 500-mL

Erlenmeyer flask. About 10ml of 0.167 M K2Cr2O7 was then added by means of a pipette and

20mL of concentrated H2SO4 was added by means of a dispenser and was swirlled gently to

mix thoroughly, (avoiding excessive swirling that would result in organic particles adhering to

the sides of the flask out of the solution).This mixture was allowed to stand for 30 minutes. The

flasks were placed on an insulation pad during this time to avoid rapid heat loss. The

suspension was diluted with 200mL of water to provide a clearer suspension for viewing the

endpoint. Then 10mL of 85% H3PO4 and 0.2g of NaF were added using a suitable dispenser,

(The H3PO4 and NaF were added to complex Fe3+ which would interfere with the titration

endpoint). Finally, 10 drops of ferroin indicator was added. (The indicator was added prior to

titration to avoid deactivation by adsorption). The mixture was then titrated with 0.5 M Fe2+ to

a burgundy end point. The colour of the solution at the beginning was yellow-orange but turned

to dark green at the endpoint (the change in colour depends on the amount of unreacted Cr2O7 2-

remaining, which shifts to a turbid grey before the endpoint and then changes sharply to a wine

red at the end point). Use of a magnetic stirrer with an incandescent light made the endpoint

easier to see in the turbid system (fluorescent lighting gives a different endpoint colour). The

reagent blank was also run using the above procedure without soil (the blank is used to

standardize the Fe2+ solution daily).

Calculation

% organic carbon:

%C = (B-S) x M of Fe2+ x 12 x 100

54

g of soil x 4000

Where:

B = mL of Fe2+ solution used to titrate blank

S = mL of Fe2+ solution used to titrate sample

12/4000 = milliequivalent weight of C in g.

2.6.15 Determination of Carbon Content in Soil by Wet Oxidation Method of Walkley and

Black (1934)

Two grammes of the sample was weighed into a 500ml conical flask and 10ml of 1.00N

K2Cr2O7 solution was added to the soil using the pipette. Exactly 20ml of concentrated H2S07

was added into the solution with gentle swirling. This was allowed to cool for 10 minutes.

Thereafter, 200ml of distilled water was added into the solution using a measuring cylinder.

About 0.2g of crystal sodium fluoride was weighed and thoroughly mixed in the mixture.

Exactly 1.00ml of a solution indicator was added into the mixture and was titrated against

1.00N Fe2SO4 solution in the burette. The blank contained the reagent as the standard without

soil sample.

Calculation:

(B-T) X N x 0.003 x 100/wt x 1.33 = % C

While

% C x 1.724 = % O. M

Where: B = Blank Titre T = Test Sample Titre N = Normality of Fe2SO4

2.6.16 Determination of the Particle Size Distribution of Soil Sample:

The particle size distribution was determined using the Bouyoucos hydrometer method

described by Day (1965).

Exactly 50g of soil sample were weighed into a 500ml shaker bottle with cover; thereafter, 0.1

N sodium hydroxide was pipetted into the bottle containing the weighed sample. About 200ml

distilled water was added and was thoroughly mixed with a glass rod and allowed to stand

55

overnight. The following day, the bottle was tightly closed and was shaken horizontally in a

reciprocal shaker for 30 minutes. This was removed and transferred into a 100ml graduated

measuring cylinder using a washing bottle with fine jet. The hydrometer was then gently placed

into the suspension when the cylinder was ¾ full and was made up to 1000 ml. The hydrometer

was carefully removed from the suspension; the cylinder was then inverted 4 times with the

hand-palms covering the mouth of the cylinder. This was then placed on a bench and the

hydrometer was immediately inserted carefully into the suspension and was allowed to stand

for 40 seconds. At the end of the time, the first hydrometer and temperature readings were

taken. The above inversion of the cylinder was repeated and allowed to stand for 2hours.

Thereafter, the second hydrometer and temperature readings of the suspension were taken. The

suspension was decanted and washed until a clear suspension was obtained, then the sediment

was transferred with a washed bottle into a 250ml beaker and was dried at 105oC. Thereafter,

the dried sample was sieved with 0.250ml sieve and the weight of the coarse sand which

remained on the sieve was taken.

Calculation:

NOTE: oF = 9/5 oC + 32.

% (Clay Fraction) = 2hr reading of hydrometer x 100 Wt of sample used 1 % silt (Fraction) = 1st hydrometer reading – 2nd hydrometer reading x 100 Wt of sample used Total Sand = 100 – % Clay - % Silt Coarse Sand = Obtained as above Fine Sand = % total Sand - % coarse Sand

Dispersion Ratio (% DR) = % Clay + % Silt in water % Clay + % Silt in calgon

56

2.6.17 Determination of Exchangeable Acidity in Soil (Ea = Exchangeable Aluminum and

Hydrogen) as Described by Chapman (1965).

Five grammes of soil sample was weighed into a funnel containing filter paper No. 1 fitted to a

leaching rack with 100ml volumetric flask to collect the filtrate. The soil was leached with

1.00N potassium chloride (KCl) solution and 100ml of the filtrate was collected. The solution

was thoroughly mixed and 25ml of it pipetted into a 250ml conical flask. Distilled water

(100ml) was later added into the 250ml conical flask and 3 drops of phenolphthalein were

added as indicator. This was titrated against 0.05N sodium hydroxide (previously standardized).

Calculation:

T x N x Vol/Aliq. X 100/ws = Meg. EA/100g Soil.

2.6.17.1 Titration for Exchangeable Aluminum:

To the flask titrated for EA, 1 drop of 0.05N HCl was added to bring the solution back to

colourless condition. The pinkish colour of the solution was returned by the addition of 10ml of

4% sodium fluoride. The solution was titrated with HCl, standardized till the solution became

colourless. The amount of HCl used was calculated using the equation below:

T x N x Vol/Aliq. x 100/ws = Meg. Al2+/100g soil.

H+ Hydrogen (H) was obtained by substitution of Meg.

2.6.18 Determination of the Exchangeable Bases in Soil Samples Using the Ammonium

Acetate Method as Described by Chapman (1965):

Exactly 5g of the soil sample was weighed into a No. 1 filter paper fitted into a funnel on a

leaching stand with 100ml volumetric flask to collect the leaches. The sample was leached with

1.00N ammonium acetate (NH4 OAC) solution and 100ml of the filtrate collected with the

volumetric flask. The filtrate was removed from the stand and was labeled “A” solution (to

determine Ca, Mg, Na, and K. The soil on the filter stand was washed with 35ml of methanol

and the washed solution was discarded. Thereafter, the soil was leached again with 0.1N

potassium chloride (KCl) and 100ml of the filtrate was collected in another flask and was

labeled “B” solution (to determine the amount of CEC).

57

2.6.18.1 Titration for Ca++ + Mg++

Ten (10ml) of the solution labeled “A” was pipetted into 100ml conical flask, and 10ml of

ammonium chloride/ammonium buffer 10 solution was then added to the flask and was mixed

thoroughly. About 10.01g of Eriochrome black T indicator was added to the mixture and was

titrated immediately with 0.01N EDTA in the burette.

Calculation:

T x N x Vol/Aliq. x 100/ws = Meg. Mg / 100g Soil.

Where:

T = Sample titre

Vol = Volume of leachate collected

Aliq. = ml aliquot titrated.

Wt = Weight of Sample leached.

2.6.18.2 For Ca++ Only:

Ten (10ml) of the solution “A” was pipetted into a 100ml clean conical flask and to it;

20ml of 20% potassium hydroxide was added. After thorough mixing, 0.01g calcium indicator

was added, and the mixture was titrated against 0.01N EDTA in the burette.

Calculation:

T x N x 100/ws x Vol/Aliq. = Meg. Ca++/100g Soil.

Where:

T = Sample titre

Vol = Volume of leachate collected

Aliq. = ml aliquot titrated.

Wt = Weight of Sample leached.

NOTE: Mg (Magnesium) is gotten by difference.

.e.g. Meg. Ca + Mg – Meg. Ca = Meg. Mg/100g Soil.

2.6.18.3 FOR CEC:

From the solution labelled “B”, 50ml of the aliquot was pipetted into a 250ml conical flask and

to it; 20ml of neutralized formalin (pH 7.0) was added. Thereafter, 2 drops of phenolphthalein

58

indicator was added and the mixture was titrated with 0.1N sodium hydroxide (standardized) in

the burette.

Calculation:

T x N x Vol/Aliq x 100/wt = Meg. CEC/100g Soil.

Na++ and K++ were determined colorimetrically using a flame-photometer with 1.00N

NH4 OAC leachate.

2.7.0 Preparation of Reagents:

2.7.1 Preparation of 0.5M Na2Cr2O7 •2H2O in 5M H2SO4 (Digestion Solution):

Exactly 140 g of Na2Cr2O7•2H2O was dissolved in 600 ml of distilled water. Slowly, 278 ml

of

concentrated H2SO4 was added, allowed to cool and was diluted to 1 liter with deionized water.

2.7.2 Preparation of 0.167M K2Cr2O7 :

Exactly 49.04 g of dried K2Cr2O7 was dissolved in 400ml of water and thereafter, made up to 1 L. 2.7.3 Preparation of 0.5 M Fe2+ Solution: Exactly 196.1 g of Fe (NH4)2(SO4)•6H2O was dissolved in 800mL of water containing 20mL of concentrated H2SO4 and diluted to 1 L. The Fe2+ in this solution oxidizes slowly on exposure to air so it was standardized against the dichromate daily. 2.7.4 Preparation of Ferroin Indicator: Exactly 3.71 g of O-phenanthroline and 1.74 g of FeSO4•7H2O were slowly dissolved in 250mL of water. 2.7.5 Preparation of Methyl Red-Methyl Blue: Methyl red- methyl blue was prepared by dissolving 1.25g of methyl red and 0.825g of methyl blue in 1 litre of ethanol (90%).

59

2.7.6 Preparation of Nutrient Agar: Exactly 2.8g of nutrient agar was weighed and dissolved in 100ml of water. The solution was then autoclaved at 100oC for 30 minutes to sterilize and homogenize the solution. 2.7.7 Preparation of Maconkey Agar: Mackonkey agar was prepared by dissolving 5.15g in 100ml of water. The solution was then autoclaved at 100oC for 30 minutes to sterilize and homogenize the solution.

2.8 Statistical Analysis

The data obtained in the experiment were analyzed statistically for mean and standard deviation

and regression using Statistical Package for Social Sciences (SPSS) version 19.

60

CHAPTER THREE

3.0 RESULT

3.1 Proximate Analysis of Cow Dung, Untreated Sawdust and Treated Sawdust.

Table 2 shows the results of proximate analysis of cow dung, untreated saw dust, and treated

saw dust. The treated saw dust contained less moisture, ash, fibre, C:N and fat while there was

a slight increase in protein, total solids, carbon and volatile solids when compared with the

untreated saw dust. The cow dung contained higher moisture content due to the nature of the

waste.

Table 2: Proximate Analysis of the cow dung, untreated sawdust and treated sawdust.

PARAMETERS COW DUNG UNTREATED SAWDUST

TREATED SAWDUST

Moisture (%) 83.55 27.23 26.80 Ash (%) 2.7 1.95 1.60 Fibre (%) 0.04 4.84 4.79 Crude nitrogen (%) 0.25 0.38 0.64 Crude protein (%) 1.62 2.34 4.03 Fat content (%) 0.15 5.23 4.13 Total solids (%) 15.32 72.65 73.20 Carbon content (%) 97.3 31.91 37.12 Volatile solids (%) 12.68 70.70 71.6 Carbohydrate (%) 15.32 58.30 58.65 C:N 29.20 155.46 91.08 Energy (kj/g) 12.98 - -

61

3.2 Physical and Chemical Properties of Soil Sample.

Table 3 shows the physical and chemical properties of soil sample (zeolite) which includes the

particle size of the soil sample, the exchangeable acidity, bases, CEC, pH, base saturation,

phosphorus, nitrogen and organic matter. The particle size showed higher percentage of clay,

which was 35%, followed with fine sand which was 28%. The pH of the soil using water and

potassium chloride as solvents respectively was acidic, the organic matter (0.47%) was higher

than carbon content (0.2%). The concentration of calcium ion of 1.4 me/100g among the

exchangeable bases, hydrogen ion of 19.2 me/100g among the exchangeable acids. This gave

the sample the property of zeolite when compared with other types of zeolite.

Table 3: Physical and Chemical properties of soil sample.

Parameters Soil Sample Test Class SC/SCL Particle Size (%) Clay Silt Fine Sand C. Sand

35 19 28 18

pH Value H2O KCl

4.6 3.4

Organic Matter (%) Carbon Organic matter

0.2 0.47

Nitrogen (%) 0.042 Exch. Bases (me/100g) Na+ K+ Ca+ Mg2+

0.27 0.23 1.4 0.8

CEC 18.4 Base Saturation (%) 14.67 Exch. Acidity (me/100g) Al3+ H+

1.0 19.2

Phosphorus (ppm) Trace Microbial Respiration (Mg Co3) Trace

62

3.3 Time Lag, Cumulative Biogas Yield, Mean±SEM of Biogas Yield, Temperature and pH from Cow dung. Phase 1.

The data in Table 4 below show the lag period, cumulative gas yield (l/Ts) and means volume

of biogas yield. The data show that the lag period of the mixtures in the bioreactors was at the

fifth day of production. Bioreactor 4 shows a higher yield of biogas which is 147 litre/TS

followed by bioreactor 6 which is 145.8 litre/TS while the bioreactor 3 shows the lowest

production at 68.8 litres/TS with mean gas yield of 2.64 ± 1.95.

Table 4: Time Lag, cumulative, Mean±SEM of biogas yield, temperature and pH from cow dung. Phase I

Parameters Bior1 (1:1)

Bior2 (1:2)

Bior3 (1:0)

Bior4 (1:3)

Bior5 (1:4)

Bior6 (1:5)

Time Lag (days)

5 5 5 5 5 5

Cumulative gas yield(l/TS)

74.3 86.8 71.1 147 133.1 152.8

Mean volume of gas yield

2.8±1.9 3.1±2.0 2.6±1.1 5.3±2.0 4.8±2.2 5.5±1.6

Mean±SEM of pH

7.94±0.69 7.88±0.64 8.21±0.68 7.72±0.41 7.64±0.46 7.48±0.37

Mean±SEM of Temp.

31.9±3.5 32.4±3.9 32.2±4.0 31.3±3.6 30.5±2.8 30.6±1.58

Bior = Bioreactor.

The quantity of cow dung used was kept constant while the quantity of water was varied as

above.

63

3.4 The Quantity of Biogas Produced Daily in Bioreactor 1. Phase 1.

Figure 2 shows the quantity of biogas produced daily in bioreactor 1 (1:1). From the graph, the

regression line shows a shallow slope which was accompanied by a large change in X and a

small change in Y. The trend indicates co-vary relationship between the day and the biogas

produced; the regression line shows a downward displacement which shows that as the day

progresses, the quantity of biogas produced reduces.

64

3.5 The Quantity of Biogas Produced Daily in Bioreactor 2. Phase 1.

Figure 3 shows the quantity of biogas produced in bioreactor 2 (made up of 3.45kg of cow dung

and 7.0kg of water). The line of regression shows a downward trend (decrease) in the amount

of biogas produced across the retention time. That is, as the day went by, the quantity of biogas

produced decreased.

65

3.6 The Quantity of Biogas Produced Daily in Bioreactor 3. Phase 1.

Figure 4 shows the quantity of biogas produced daily in bioreactor 3 which contains 7kg of the

substrate without the addition of water. The regression line also showed a decrease in biogas

produced as the day progressed from day 1 to day 28. Initially, in day 2, there was an increase

in gas produced. The gas produced is made mainly of CO2 and these decreased as the day

progressed. The increase in the production of CO2 initially is due to the presence of other

aerobic organisms which also play a role in degradation, but they die as the day progresses.

66

3.7 The Quantity of Biogas Produced Daily in Bioreactor 4. Phase 1.

Figure 5 shows the quantity of biogas produced daily in bioreactor 4 (1:3). The volume of water

is three times the quantity of cow dung in the bioreactor. The daily production shows higher

quantity of biogas on daily basis but still maintain a downward trend with a shallow slope (.i.e.

decreases in production of biogas as the day progressed across the retention time

67

3.8 The Quantity of Biogas Produced Daily in Bioreactor 5. Phase 1.

Figure 6 shows the quantity of gas produced on daily basis in bioreactor 5 (1:4).The amount of

biogas produced increased. i.e the regression line has an upward trend (steep slope) as the day

progressed. The biogas produced in reactor 5 has a cumulative gas yield of 124.8 l/TS, and the

mean value of 4.62 ± 2.1 l/TS.

68

3.9 The Quantity of Biogas Produced Daily in Bioreactor 6. Phase 1.

Figure 7 shows the quantity of biogas produced in bioreactor 6 on daily basis. The figure shows

an upward trend of the production of gas as the day progressed in bioreactor 6 (1:5). This could

be as a result of the hydrogen and carbon dioxide ratio in the bioreactor. The cumulative gas

yield was 145.8 l/TS with mean value of 5.39±1.57 l/TS.

69

3.10 The Quantity of Biogas Produced Daily in Bioreactor 1-6. Phase 1.

Figure 8 shows the quantity of biogas produced against the retention time; the result shows an

increase in production of gas which was mainly carbon dioxide (CO2). This was observed in all

the bioreactors. The peak of production of the biogas (methane) was observed at the 16Th day.

Thereafter, the rate of production started decreasing (decline phase).The bioreactor 6 (1:5) had

the highest rate of production of biogas.

70

3.11 Time Lag, Cumulative Gas Yield, Mean±SEM of Biogas Yield, pH and Temperature in Bioreactors. (Phase II).

In phase II, bioreactors 1 and 3 had very short time lag of 13 day each, followed by bioreactor 5 with time Lag at the 15th day and with highest cumulative biogas yield of 36.3 l/TS, while bioreactor 2 (saw dust only) had the longest time lag of 23 with least biogas yield. The ambient temperature showed a positive correlation with the daily biogas yield and with the slurry temperature.

Table 5: Time lag, cumulative gas yield, mean volume of biogas yield, pH, and Temperature in bioreactors. (Phase II)

Parameter Bior1 (1:0)CD

Bior2 (0:1)SD

Bior3 (3:2)CD:SD

Bior4 (2:3)CD:SD

Bior5 (1:1)CD:SD

Time Lag 13 23 13 21 15

Cumulative gas yield(l/TS)

19.5 8.5 35.5 12.3 36.3

Mean±SEM of biogas produced

0.6±0.8* 0.3±0.3* 1.1±0.9* 0.4±0.6* 1.1±1.7*

Mean±SEM of pH

8.42±0.35 6.22±0.44 7.16±0.56 6.41±0.29 7.00±0.6

Mean±SEM of Temperature

31.8±3.4* 32.0±3.7* 32.5±3.7* 33.3±3.8* 32.6±3.6*

Ambient Temperature = 27.3±2.6oC; Retention Time = 1-33 Day.

Bior = Bioreactor; CD = Cow dung; SD = Sawdust

71

3.12 Total Viable Count for the Mixture in the Reactor (cfu/ml). Phase II.

Table 6 below shows the microbial population of the mixtures of cow dung and saw dust in

each of the bioreactors; measured in colony forming unit per ml (cfu/ml).The microbial

population of the reactors were determined at the point of charging, flammability, peak of

production and the end of the retention time. Between the point of charging and the point of

flammability, there was a high population of bacteria. This could be attributed to the presence

of other bacteria such as aerobic and pathogenic bacteria which are found at the early stages of

the digestion.

Table 6: Total Viable count for the mixtures in the reactors (cfu/ml). Phase II.

PERIOD Bior 1 CD Bior 2 SD Bior3CD:SD (3:2)

Bior4CD:SD (2:3)

Bior5CD:SD (1:1)

At the point of

charging.

2.71x102 2.12x102 6.1x102 1.17x102 1.02x102

At the point of

flammability.

5.4x103 2x103 2.73x103 5.2x103 2.20x103

At the peak of

production.

2.76x103 1.3x103 0 5x103 2.1x103

At the end of

retention period.

3.0x102 0 1.76x102 1.5x102 1.7x102

Bior = Bioreactor; CD = Cow dung; SD = Saw dust.

72

3.13 Composition of Biogas Produced in the Bioreactors. Phase II.

The result in table 7 shows the composition of biogas produced from the mixtures in the bioreactors, respectively. The mixture of cow dung (CD only) 1:0 ratio had a higher percentage of methane (72%) with the least quantity of carbon dioxide followed by bioreactor 3 3:2 (CD: SD) with 70% methane and bioreactor 5 with 70% methane. Bioreactor 4 2:3 (CD: SD), while bioreactor 2 (saw dust only) had the lowest methane composition of 18% and highest carbon dioxide composition of 90%.

Table 7: Composition of biogas produced in the bioreactors. Phase II.

Parameters Bior1CD Bior2SD Bior3CD:SD(3:2) Bior4CD:SD(2:3) Bior5CD:SD(1:1)

Methane (%)

72 18 70 65 70

Carbon (%) 27 90 28 30 28

Bior = Bioreactor; CD = Cow dung; SD = Sawdust

73

3.14 Time Lag, Cumulative Biogas Yield, Mean±SEM of Biogas Yield, pH and Temperature of the Bioreactors. Phase III.

Table 8 shows that bioreactor 1 (Treated) had the shortest time lag at the 12th day and higher cumulative biogas yield of 54.7 l/TS, followed by bioreactor 7 with time lag at 15 day and cumulative biogas yield of 30.1l/TS when compared to bioreactor 2 (untreated) with longer time lag at 24th day and biogas yield of 29.8 l/TS. Bioreactor 5, 6, 4, and 3 had a shorter time lag but with less biogas yield when compared with the bioreactor 2.The ambient temperature showed a positive correlation with the biogas yield and the slurry temperature which are significant.

Table 8: The Time lag, cumulative biogas yield and Mean±SEM of biogas, pH and Temperature of the bioreactors. Phase III. Parameter Bior1

Treateda Bior2 Untreatedb

Bior3 + Boric acid

Bior4 +NiSO4

Bior5 +CoSO4

Bior6 +Zn

Bior7 +Zeolite

Lag days 12 24 21 21 18 21 15 Cumulative gas yield(l/TS)

54.7 29.8 10.4 12.1 23.7 13.7 30.1

Mean±SEM of biogas produced

2.0±1.4* 1.1±1.1* 0.4±0.7* 0.4±0.7* 0.9±0.7* 0.5±0.6* 1.1±1.3*

Mean±SEM of pH

9.42±0.30 8.62±0.33 8.27±0.39 8.25±0.43 8.4±0.36 8.46±0.44 8.64±0.37

Mean±SEM of Temperature

29.5±3.0* 29.7±2.9* 29.5±3.0* 29.9±3.1* 30.1±3.0* 29.8±3.0* 29.1±2.9*

Mean±SEM of Ambient Temperature =25.3±2.6 *= Significant correlation. a = Positive control (Bioreactor 1 Treated); b = Negative control (Bioreactor 2 untreated); Day = 1-28 days. The ratio of cow dung, sawdust and water is 1:1:4 (3.75kg, 3.75kg and 30 litres) respectively.

74

3.15 Total Viable Count of Bacteria Population in the Bioreactors Measured in cfu/ml. Phase III

The table 9 below shows the microbial count of the mixture of cow dung, saw dust and

micronutrient in bioreactors. The microbial population was determined at the point of charging,

flammability, peak and at the end of the retention period .The population increased between the

point of charging and flammability; though it did not show a regular trend. This could be as a

result of physical and chemical factors including temperature and change in the pH of the

slurry.

Table 9: Total Viable Count of bacteria for the mixtures in the bioreactors (cfu/ml). Phase

III.

PARAMETER Bior1 Untreated

Bior2 treated

Bior3+ Boricacid

Bior4+ NiSO4

Bior5+ CoSO4

Bior6+ Zn

Bior7+ Zeolite

At the point of

charging.

4.7x104 6.2x104 4.1x104 5.8x104 4.4x104 5.8x104 7.4x104

At the point of

flammability.

6.4x105 5.2x103 6.2x105 5.3x103 4.5x103 2.96x103 2.28x103

At the peak of

production.

7x103 1.5x103 8x103 3.7x105 2x105 1x103 7x103

At the end of

retention

period.

7x103 3.1x103 6x103 8x103 1.4x105 3.7x103 5x103

75

3.16 Composition of Biogas Produced in the Bioreactors. Phase III.

Table 10 shows that bioreactor 5 had 72% of methane, followed by bioreactor 3, 4, and 7

which had methane value of 70%, respectively. These show an improvement in the quality of

biogas produced when compared with both the positive and negative (bioreactor1 and 2

respectively) with methane value of 68% and 63% respectively.

Table 10: Composition of biogas produced in the bioreactors. Phase III

PARAMETERS Bior1 Untreated

Bior2 Treated

Bior3 +Boric acid

Bior4+ NiSO4

Bior5+ CoSO4

Bior6+ Zn

Bior7+ Zeolite

Methane (%) 68 63 70 70 72 69 70 Carbon Dioxide (%) 30 35 29 28 27 30 29

76

CHAPTER FOUR 4.0 DISCUSSION

Phase I of this investigation is the preliminary stage of this research. This study was carried out

to investigate the best ratio of water to cow dung (based on the moisture content of the cow

dung) for biogas production. This study was monitored for 28 days (retention time) using six

Bioreactors numbered 1-6 with ratio as 1:1 (Bior1), 1:2 (Bior2), 1:0 (Bior3), 1:3 (Bior4), 1:4

(Bior5), 4:1 (Bior6) of CD:H2O (CD =cow dung; H2O = water.) respectively at ambient

temperature range of 22-33oC and slurry temperature range of 22 -40oC.

The production of flammable biogas commenced within 12 hours of charging. Table 4 shows

that Bioreactors 1, 2, 3, 4, 5 and 6 had the time lag on the 5th day, with Bioreactor 4 having the

highest cumulative gas yield of 147 l/TS, followed by bioreactor 2 with cumulative gas yield of

86.8 l/TS. This was followed by Bioreactor 1 with cumulative gas yield of 74.3 l/TS, Bioreactor

3 with cumulative biogas yield of 71.1 l/TS and Bioreactor 5 with the least cumulative gas yield

of 55.5 l/TS. The high production of biogas observed in Bioreactor 4 (1:3) could be attributed

to the water ratio added to the cow dung, which provided the medium for maximum activities

of the extracellular enzymes and mass transfer of the anaerobes within the reactor, when

compared with the control (Bioreactor 3) which had no water added to it. However, the result in

table 4 shows that as the water ratio increased, the biogas yield increased (Uri, 1992). Contrary

to the trend, Bioreactor 5 (1:4) and Bioreactor 6 (1:5) showed declines in the cumulative biogas

yield when compared with Bioreactor 4 (1:3). The decline could also be as a result of the

properties of the cow dung (such as the moisture content) as shown in table 2.

This implies that the mixture in Bioreactor 4 (1:3) with 9.4kg of cow dung and 28.1 litres of

water was the best mixture for flammable biogas production. However, further analysis of the

result obtained in Bioreactor 4 (1:3) as shown in figures 9, indicated a shallow slope with a

weak regression (R2) value of 0.162 which shows a downward trend as the day progressed. This

effect could be attributed to the short retention time observed in bioreactor 4 (1:3).

Furthermore, the graphical representation of Bioreactor 5 and 6 (Figure 10 and 11,

respectively), shows a steep slope with a daily increase in biogas production. This informed the

choice of Bioreactor 5(1:4) for further investigation.

77

Figure 12 shows an early production of non-combustible biogas made up of mainly carbon

dioxide (CO2) in all the bioreactors while the peak production of flammable gas was observed

at the 16th day.

In phase II of the study, retention time was monitored for 33 days, within the ambient

temperature range of 21-33oC and slurry temperature range of 23-40oC. In this phase, the

quantity of water used was kept constant while the quantities of the saw dust and cow dung

used were varied as shown in table 5.

The production of flammable gas commenced in the Bioreactors at different time lag (Table 5),

the result shows that Bioreactor 3 (3:2) blend with cumulative biogas yield of 35.5 l/TS and

bioreactor 1(1:0) cow dung only with cumulative biogas yield of 19.5 l/TS had the shortest

retention time lag of 13 day, followed by Bioreactor 5 (1:1) blend with time lag of 15 and

highest cumulative biogas yield of 36.3 l/TS. Bioreactor 4 (2:3) blend had the time lag of 21

and cumulative biogas yield of 12.3 l/TS while Bioreactor 2 (0:1) saw dust only, had the least

time lag of 23 and cumulative biogas yield of 8.5 l/TS (table 5). The least time lag and

cumulative biogas yield observed in Bioreactor 2 (saw dust only) could be attributed to less

microbial population (table 6), high fat and fibre content. This indicates that saw dust only

contained a lot of cellulose, hemi-cellulose, pectin, lignin, plant wax etc, which are very

difficult to degrade and could be a major rate-determining step in anaerobic digestion (Kozo et

al., 1996). The less microbial population and high fat and fibre content of saw dust in

Bioreactor 2 led to the reduction in the pH of the slurry to less than 6.5-8.0 as reported by

Ntegwe et al. (2010) as the pH range needed for maximum activity of methanogenic organisms

to covert the free fatty acid into acetate and acetate into methane.

The onset of flammable biogas as observed in Bioreactor 1(cow dung only) could also be as a

result of the less fat content of the waste (cow dung) as shown in table 2 and microbial

population of the waste (table 6). Bioreactor 4 (2:3) CD:SD with time lag of 21 and biogas

yield of 12.3 l/TS, has the influence of high fat and fibre content of the waste on the pH of the

slurry as also observed in Bioreactor 2. However, it showed an improvement both on the time

lag and the biogas yield when compared with Bioreactor 2. This was due to the addition of cow

dung which supplied the microbial population that degraded the fibre and converted the fatty

acid into acetate, thereby improving the pH of the slurry for methogenic activities. The higher

78

quantity of cow dung over the saw dust in Bioreactor 3 (3:2) contributed to the shorter time lag

and increase in the biogas yield when compared with Bioreactors 1, 2 and 3 (Table 2). Contrary

to this, Bioreactor 5 (1:1) CD: SD had the highest cumulative biogas yield when compared with

Bioreactors 1, 2, 3 and 4. These could be attributed to the equal quantity of the cow dung and

saw dust in the reactor, which encouraged the mass transfer and direct contact of the

microorganisms with wastes to enable easy digestion.

The average ambient temperature of 27.3±2.6oC shows a positive correlation with the slurry

temperature and the quantity of biogas produced in Bioreactors 1, 2, 3, 4, 5 which is significant

at p≤0.05.

The result of the investigation in phase II, implies that Bioreactor 5 (1:1) CD:SD with 3.8kg

cow dung and 3.8kg of saw dust is a better waste combination or blend for flammable biogas

production if there is no urgent need for biogas utilization, whereas, the mixture (3:2)

(Bioreactor 3) would be preferred if the biogas is required urgently.

The Phase III of this investigation was monitored for 28 days (retention time) at ambient

temperature range of 22oC - 35oC and within the anaerobic pH range of 6.5 - 9.5. Daily biogas

production commenced within 24hrs of charging the respective bioreactors while the onset of

flammable biogas production in the bioreactors commenced on different time lag. The results in

Table 8 showed that Bioreactor 1(treated saw dust; positive control) produced flammable

biogas at the shortest time lag at 12 day with the highest cumulative biogas yield of 54.7 l/TS

when compared with the negative control (Bioreactor 2), followed by bioreactor 7 (zeolite) with

time lag at 15 day and cumulative biogas yield of 30.1 l/TS. Bioreactors 5, 4 and 3 had the time

lag of 18, 21, 21 days respectively, while Bioreactor 2 (untreated saw dust) had the longest time

lag at 24 day and cumulative biogas yield of 29.8 l/TS. The shortest time lag and higher

cumulative biogas yield observed in bioreactor 1 (treated) could be attributed to the property of

the treated saw dust (table 2) which had slight increase in the percentage of carbon,

carbohydrate, volatile solid, protein, total solid and less percentage of fibre (liberated by the

action of 1.45N NH4OH) as shown in table 2 when compared with the untreated saw dust.

Addition of zeolite has also been shown to significantly increase the cumulative biogas yield

and the time lag when compared with the negative control (Bioreactor 2). This agreed with the

work done by Milian et al. (2001) who showed that zeolite improve biogas production. This

79

could be as a result of the presence of calcium ions in the zeolite (table 3), which have been

reported to antagonize the ammonium (NH4+) ion produced in the bioreactor by absorption on

the active area of the material (Milian et al 2001). Hansen et al., (1998) reported that

ammonium ions inhibit the activity of methanogenic organisms at 200mg concentration. The

high hydrogen ion content (19.2 me/100g) in the zeolite could also contribute to the high

production of methane (flammable biogas) as reported by (Kotsopoulos et al., 2008; Uri, 1992).

The particle size of the zeolite which have a high percentage of clay (35%) (Table 3) could also

complement the production of methane by serving as an immobilized matrix for enzyme

activity and in retention of the moisture content of the slurry (Milian et al., 2001; Kotsopoulos

et al., 2008). The addition of additives such as boric acid, NiSO4, CoSO4 and Zn did not show

any increase in the biogas yield when compared with the negative control (Bioreactor 2) and

positive control (Bioreactor 1treated). This observation is contrary to the findings of Geetha et

al (1990); Seenayya et al (1992), and Jarvis et al (1997). The additive only reduces the time lag

(the onset of flammable biogas) when compared with negative control (with the time lag of 24)

(Table 8). Winfrey and Zeiku (1977) reported that sulphate inhibits methanogenesis at 0.2mM

concentration. However, these could be attributed to the reduction in biogas yield observed in

Bioreactors 4 (NiSO4) and 5 (CoSO4).

Table 8 shows that the ambient temperature has a significant correlation with the daily biogas

yield and the slurry temperature of Bioreactors 1, 2, 3, 4, 5, 6 and 7 which is significant at

p≤0.05. The pH of the Bioreactor has been reported as a very important factor in biogas

production (Anonymous, 1989). The pH of the Bioreactor 1 showed a positive correlation with

Bioreactors 2, 3, 4, 5, 6 and 7 which is significant at p≤0.05.

The result of this study implies that pre-treated saw dust (Bioreactor1) is a better substrate for

the optimization of the onset of biogas flammability and biogas production from combination

of saw dust and cow dung. Addition of zeolite (Bioreactor 7) to the combination of saw dust

and cow dung also optimized both the time lag and the biogas yield when compared with the

negative control (Bioreactor 2).

The quality of the biogas produced (table 10) was optimized in Bioreactor 5 (CoSO4) with 72%

CH4 (methane) and 27% CO2 (carbon dioxide) when compared with the negative and positive

controls (Bioreactor 2 and 1, respectively). These could be attributed to the cobalt dependent

80

metalo-enzymes involved in biogas production and the synergy that existed between the

additives and the wastes. Bioreactors 3, 4 and 7, with 70% CH4, showed a significant increase

in the quality of biogas produced when compared with the control (Table 10).

4.1 CONCLUSION

The result of this investigation has shown that the time lag (onset of flammable biogas

production), biogas yield and the quality of biogas produced from blending saw dust and cow

dung could be optimized significantly by pretreatment of the saw dust with 1.45N of NH4OH

solution, addition of zeolite and additives such as cobalt sulphate. The overall result shows that

pretreated saw dust (bioreactor 1 (1:1)) optimized the biogas yield and the onset of flammable

biogas production (time lag) of saw dust/cow dung blend, followed by the addition of zeolite

prior to digestion of the blend. Addition of NiSO4, CoSO4, Zn and boric acid to the saw

dust/cow dung blend reduced the time lag of flammable biogas produced, but did not optimize

the biogas yield. The addition of CoSO4, also enhanced the quality of biogas produced. Hence

saw dust could be utilized effectively by converting it into the production of biogas and this

will help address the challenge of its economic disposal. Moreover, the optimization of biogas

production is an important process which encourages large-scale production of biogas for both

domestic and industrial uses.

4.2 RECOMMENDATION

This study has shown that saw dust and cow dung can be used to produce biogas which could

be used to some extent to address the energy challenge (renewable energy) and environmental

problems. However, there is need for further studies such as mathematical modeling of the

studied system, determination of other agents (such as additive, maintaining the mesophilic

temperature and pH range) for anaerobic digestion.

81

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solid waste co-digestion model. Waste research. 43: 2717-2727.

94

APENDIX ONE

Regression

Descriptive Statistics

Mean Std. Deviation N

DAY 14.50 8.226 28

BIOR 1 (1:1)TREATED 1.953571 1.4114563 28

BIOR 2 (1:1) UNTREATED 1.064286 1.0559231 28

BIOR 3(1:1) +BORIC ACID .371429 .7412987 28

BIOR 4(1:1) +NIKEL

SULPHATE

.432143 .7013498 28

BIOR 5(1:1)+COBALT

SULPHATE

.846429 .7290187 28

BIOR 6(1:1)+ZINC .489286 .5698208 28

BIOR 7(1:1)ZEOLITE 1.075000 1.2940390 28

Correlations

DAY

BIOR 1

(1:1)TREATED

BIOR 2 (1:1)

UNTREATED

Pearson Correlation DAY 1.000 .726 .744

BIOR 1 (1:1)TREATED .726 1.000 .739

BIOR 2 (1:1) UNTREATED .744 .739 1.000

BIOR 3(1:1) +BORIC ACID .647 .572 .712

95

BIOR 4(1:1) +NIKEL

SULPHATE

.684 .549 .701

BIOR 5(1:1)+COBALT

SULPHATE

.643 .633 .765

BIOR 6(1:1)+ZINC .741 .632 .726

BIOR 7(1:1)ZEOLITE .605 .319 .583

Sig. (1-tailed) DAY . .000 .000

BIOR 1 (1:1)TREATED .000 . .000

BIOR 2 (1:1) UNTREATED .000 .000 .

BIOR 3(1:1) +BORIC ACID .000 .001 .000

BIOR 4(1:1) +NIKEL

SULPHATE

.000 .001 .000

BIOR 5(1:1)+COBALT

SULPHATE

.000 .000 .000

BIOR 6(1:1)+ZINC .000 .000 .000

BIOR 7(1:1)ZEOLITE .000 .049 .001

N DAY 28 28 28

BIOR 1 (1:1)TREATED 28 28 28

BIOR 2 (1:1) UNTREATED 28 28 28

BIOR 3(1:1) +BORIC ACID 28 28 28

BIOR 4(1:1) +NIKEL

SULPHATE

28 28 28

BIOR 5(1:1)+COBALT

SULPHATE

28 28 28

BIOR 6(1:1)+ZINC 28 28 28

96

BIOR 7(1:1)ZEOLITE 28 28 28

Correlations

BIOR 3(1:1)

+BORIC ACID

BIOR 4(1:1)

+NIKEL

SULPHATE

BIOR

5(1:1)+COBALT

SULPHATE

Pearson Correlation DAY .647 .684 .643

BIOR 1 (1:1)TREATED .572 .549 .633

BIOR 2 (1:1) UNTREATED .712 .701 .765

BIOR 3(1:1) +BORIC ACID 1.000 .585 .712

BIOR 4(1:1) +NIKEL

SULPHATE

.585 1.000 .647

BIOR 5(1:1)+COBALT

SULPHATE

.712 .647 1.000

BIOR 6(1:1)+ZINC .619 .899 .750

BIOR 7(1:1)ZEOLITE .417 .838 .542

Sig. (1-tailed) DAY .000 .000 .000

BIOR 1 (1:1)TREATED .001 .001 .000

BIOR 2 (1:1) UNTREATED .000 .000 .000

BIOR 3(1:1) +BORIC ACID . .001 .000

BIOR 4(1:1) +NIKEL

SULPHATE

.001 . .000

BIOR 5(1:1)+COBALT

SULPHATE

.000 .000 .

BIOR 6(1:1)+ZINC .000 .000 .000

97

BIOR 7(1:1)ZEOLITE .014 .000 .001

N DAY 28 28 28

BIOR 1 (1:1)TREATED 28 28 28

BIOR 2 (1:1) UNTREATED 28 28 28

BIOR 3(1:1) +BORIC ACID 28 28 28

BIOR 4(1:1) +NIKEL

SULPHATE

28 28 28

BIOR 5(1:1)+COBALT

SULPHATE

28 28 28

BIOR 6(1:1)+ZINC 28 28 28

BIOR 7(1:1)ZEOLITE 28 28 28

Correlations

BIOR

6(1:1)+ZINC

BIOR

7(1:1)ZEOLITE

Pearson Correlation DAY .741 .605

BIOR 1 (1:1)TREATED .632 .319

BIOR 2 (1:1) UNTREATED .726 .583

BIOR 3(1:1) +BORIC ACID .619 .417

BIOR 4(1:1) +NIKEL

SULPHATE

.899 .838

BIOR 5(1:1)+COBALT

SULPHATE

.750 .542

BIOR 6(1:1)+ZINC 1.000 .821

BIOR 7(1:1)ZEOLITE .821 1.000

98

Sig. (1-tailed) DAY .000 .000

BIOR 1 (1:1)TREATED .000 .049

BIOR 2 (1:1) UNTREATED .000 .001

BIOR 3(1:1) +BORIC ACID .000 .014

BIOR 4(1:1) +NIKEL

SULPHATE

.000 .000

BIOR 5(1:1)+COBALT

SULPHATE

.000 .001

BIOR 6(1:1)+ZINC . .000

BIOR 7(1:1)ZEOLITE .000 .

N DAY 28 28

BIOR 1 (1:1)TREATED 28 28

BIOR 2 (1:1) UNTREATED 28 28

BIOR 3(1:1) +BORIC ACID 28 28

BIOR 4(1:1) +NIKEL

SULPHATE

28 28

BIOR 5(1:1)+COBALT

SULPHATE

28 28

BIOR 6(1:1)+ZINC 28 28

BIOR 7(1:1)ZEOLITE 28 28

ANOVAb

Model Sum of Squares Df Mean Square F Sig.

1 Regression 1310.812 7 187.259 7.255 .000a

99

Residual 516.188 20 25.809

Total 1827.000 27

a. Predictors: (Constant), BIOR 7(1:1)ZEOLITE, BIOR 1 (1:1)TREATED, BIOR 3(1:1) +BORIC ACID,

BIOR 5(1:1)+COBALT SULPHATE, BIOR 2 (1:1) UNTREATED, BIOR 4(1:1) +NIKEL SULPHATE,

BIOR 6(1:1)+ZINC

b. Dependent Variable: DAY

Coefficientsa

Model

Unstandardized Coefficients

Standardized

Coefficients

t Sig. B Std. Error Beta

1 (Constant) 5.793 2.078 2.788 .011

BIOR 1 (1:1)TREATED 2.575 1.225 .442 2.102 .048

BIOR 2 (1:1) UNTREATED .935 1.936 .120 .483 .634

BIOR 3(1:1) +BORIC ACID 2.443 2.079 .220 1.175 .254

BIOR 4(1:1) +NIKEL

SULPHATE

-1.378 3.613 -.118 -.382 .707

BIOR 5(1:1)+COBALT

SULPHATE

-1.093 2.505 -.097 -.436 .667

BIOR 6(1:1)+ZINC 1.980 5.226 .137 .379 .709

BIOR 7(1:1)ZEOLITE 2.164 1.719 .340 1.259 .222

a. Dependent Variable: DAY

100

APENDIX TWO

Regression 2

Descriptive Statistics

Mean Std. Deviation N

AMBIENT 25.257 2.6132 28

BIOR 1(1:1)TREATED 29.534 2.9953 28

BIOR 2 (1:1) UNTREATED 131.368 537.7091 28

BIOR 3(1:1) +BORIC ACID 29.536 3.0126 28

BIOR 4(1:1) +NIKEL

SULPHATE

29.893 3.0514 28

BIOR 5(1:1)+COBALT

SULPHATE

30.054 2.9511 28

BIOR 6(1:1)+ZINC 29.832 3.0155 28

BIOR 7(1:1)ZEOLITE 29.077 2.8691 28

Correlations

AMBIENT

BIOR

1(1:1)TREATED

BIOR 2 (1:1)

UNTREATED

Pearson Correlation AMBIENT 1.000 .730 -.034

BIOR 1(1:1)TREATED .730 1.000 -.076

BIOR 2 (1:1) UNTREATED -.034 -.076 1.000

BIOR 3(1:1) +BORIC ACID .783 .958 -.046

BIOR 4(1:1) +NIKEL

SULPHATE

.765 .956 -.068

101

BIOR 5(1:1)+COBALT

SULPHATE

.742 .968 -.098

BIOR 6(1:1)+ZINC .715 .960 -.049

BIOR 7(1:1)ZEOLITE .799 .964 -.137

Sig. (1-tailed) AMBIENT . .000 .431

BIOR 1(1:1)TREATED .000 . .351

BIOR 2 (1:1) UNTREATED .431 .351 .

BIOR 3(1:1) +BORIC ACID .000 .000 .408

BIOR 4(1:1) +NIKEL

SULPHATE

.000 .000 .365

BIOR 5(1:1)+COBALT

SULPHATE

.000 .000 .310

BIOR 6(1:1)+ZINC .000 .000 .402

BIOR 7(1:1)ZEOLITE .000 .000 .244

N AMBIENT 28 28 28

BIOR 1(1:1)TREATED 28 28 28

BIOR 2 (1:1) UNTREATED 28 28 28

BIOR 3(1:1) +BORIC ACID 28 28 28

BIOR 4(1:1) +NIKEL

SULPHATE

28 28 28

BIOR 5(1:1)+COBALT

SULPHATE

28 28 28

BIOR 6(1:1)+ZINC 28 28 28

BIOR 7(1:1)ZEOLITE 28 28 28

102

Correlations

BIOR 3(1:1)

+BORIC ACID

BIOR 4(1:1)

+NIKEL

SULPHATE

BIOR

5(1:1)+COBALT

SULPHATE

Pearson Correlation AMBIENT .783 .765 .742

BIOR 1(1:1)TREATED .958 .956 .968

BIOR 2 (1:1) UNTREATED -.046 -.068 -.098

BIOR 3(1:1) +BORIC ACID 1.000 .980 .966

BIOR 4(1:1) +NIKEL

SULPHATE

.980 1.000 .972

BIOR 5(1:1)+COBALT

SULPHATE

.966 .972 1.000

BIOR 6(1:1)+ZINC .964 .973 .978

BIOR 7(1:1)ZEOLITE .967 .972 .977

Sig. (1-tailed) AMBIENT .000 .000 .000

BIOR 1(1:1)TREATED .000 .000 .000

BIOR 2 (1:1) UNTREATED .408 .365 .310

BIOR 3(1:1) +BORIC ACID . .000 .000

BIOR 4(1:1) +NIKEL

SULPHATE

.000 . .000

BIOR 5(1:1)+COBALT

SULPHATE

.000 .000 .

BIOR 6(1:1)+ZINC .000 .000 .000

BIOR 7(1:1)ZEOLITE .000 .000 .000

103

N AMBIENT 28 28 28

BIOR 1(1:1)TREATED 28 28 28

BIOR 2 (1:1) UNTREATED 28 28 28

BIOR 3(1:1) +BORIC ACID 28 28 28

BIOR 4(1:1) +NIKEL

SULPHATE

28 28 28

BIOR 5(1:1)+COBALT

SULPHATE

28 28 28

BIOR 6(1:1)+ZINC 28 28 28

BIOR 7(1:1)ZEOLITE 28 28 28

Correlations

BIOR

6(1:1)+ZINC

BIOR

7(1:1)ZEOLITE

Pearson Correlation AMBIENT .715 .799

BIOR 1(1:1)TREATED .960 .964

BIOR 2 (1:1) UNTREATED -.049 -.137

BIOR 3(1:1) +BORIC ACID .964 .967

BIOR 4(1:1) +NIKEL

SULPHATE

.973 .972

BIOR 5(1:1)+COBALT

SULPHATE

.978 .977

BIOR 6(1:1)+ZINC 1.000 .969

BIOR 7(1:1)ZEOLITE .969 1.000

Sig. (1-tailed) AMBIENT .000 .000

104

BIOR 1(1:1)TREATED .000 .000

BIOR 2 (1:1) UNTREATED .402 .244

BIOR 3(1:1) +BORIC ACID .000 .000

BIOR 4(1:1) +NIKEL

SULPHATE

.000 .000

BIOR 5(1:1)+COBALT

SULPHATE

.000 .000

BIOR 6(1:1)+ZINC . .000

BIOR 7(1:1)ZEOLITE .000 .

N AMBIENT 28 28

BIOR 1(1:1)TREATED 28 28

BIOR 2 (1:1) UNTREATED 28 28

BIOR 3(1:1) +BORIC ACID 28 28

BIOR 4(1:1) +NIKEL

SULPHATE

28 28

BIOR 5(1:1)+COBALT

SULPHATE

28 28

BIOR 6(1:1)+ZINC 28 28

BIOR 7(1:1)ZEOLITE 28 28

ANOVAb

Model Sum of Squares df Mean Square F Sig.

1 Regression 139.410 7 19.916 8.857 .000a

105

Residual 44.973 20 2.249

Total 184.384 27

a. Predictors: (Constant), BIOR 7(1:1)ZEOLITE, BIOR 2 (1:1) UNTREATED, BIOR 1(1:1)TREATED,

BIOR 3(1:1) +BORIC ACID, BIOR 6(1:1)+ZINC, BIOR 4(1:1) +NIKEL SULPHATE, BIOR

5(1:1)+COBALT SULPHATE

b. Dependent Variable: AMBIENT

Coefficientsa

Model

Unstandardized Coefficients

Standardized

Coefficients

t Sig. B Std. Error Beta

1 (Constant) 3.920 3.026 1.296 .210

BIOR 1(1:1)TREATED -.374 .427 -.429 -.876 .391

BIOR 2 (1:1) UNTREATED .001 .001 .152 1.237 .230

BIOR 3(1:1) +BORIC ACID .601 .531 .693 1.132 .271

BIOR 4(1:1) +NIKEL

SULPHATE

-.010 .583 -.012 -.018 .986

BIOR 5(1:1)+COBALT

SULPHATE

-.205 .617 -.232 -.332 .743

BIOR 6(1:1)+ZINC -1.041 .541 -1.201 -1.924 .069

BIOR 7(1:1)ZEOLITE 1.791 .596 1.966 3.004 .007

a. Dependent Variable: AMBIENT