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.
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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
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.
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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.
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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.
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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.
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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.
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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,
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% 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
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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:
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% 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
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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).
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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.
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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.
The quantity of cow dung used was kept constant while the quantity of water was varied as
above.
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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.
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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.
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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
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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.
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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.
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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.
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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
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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.
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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.
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)
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.
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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