TABLE OF CONTENTS
Page
Title Page………………………………………………………………………………………………………..i
Certification………………………………………………………………………………………..ii
Dedication……………………………………………………………………………………..….iii
Acknowledgement………………………………………………………………………………………….iv
Table of content……………………………………………………………………………………………. v
List of Tables……………………………………………………….…………………………….. vi
Abstract……………………………………………………………………………………………..xii
CHAPTER ONE
1.0 Introduction……………………………………………………………………………………1
1.1 Aim of study…………………………………………………………………………………..6
1.2 Objectives of the study………………………………………………………………………………………………6
CHAPTER TWO
2.0 Literature review………………………………………………………………………………7
2.1 Origin of nut………………………..…………………………………………………………7
2.2 Storage and selection of TetracarpidiumConophorum …………………………………………8
2.3 fungus damage and control of TetracarpidiumConophorumshell……………………………9
2.4 medicinal value of TetracarpidiumConophorum ………………………………….……………….9
2.5 Nutritional value of TetracarpidiumConophorum………………………………………….10
2.6 Industrial value of TetracarpidiumConophorum…………………………………………………11
CHAPTER THREE
3.0 Materials and method ……………….……………………………………………………… 12
3.1 Study Area……………….……………………….……………….……………..…………. 12
3.2 Material…………………………….……………………………………………..…………. 12
3.3 Medial used…………………………………………………….….……………………………. 12
3.3.1 Chemical reagent used……………………………………………………………………. 12
3.4 Collection of sample………………………………………………………..……………… 13
3.4.1Walnut shell sample.………………………………………………………………………. 13
3.4.2 Chicken Dropping Sample………………………………………………………………… 13
3.5Media preparation and sterilization………….………………..……………………………. 13
3.6 Microbiological Analyses of Samples…………………………………………………….13
6
3.6.1 African walnut Shell…………………………………………………………………….13
3.6.2 Chicken Droppings………………………………………………………………………14
3.7 Microbial count……………………………………………………..……………………. 16
3.7.1 Isolation of pure isolates……………………..………………………………….…………. 17
3.7.2 Preservation of isolated microorganism………………………………………………… 17
3.8. Biochemical Characterization of Bacterial Isolates……………………………………… 18
3.8.1 The Gram Reaction……………………………………………………………… …….18
3.8.1.1 Gram Staining…………………………………………………………………………..19
3.8.1.2 3% Potassium Hydroxide Grams reaction…………………………………………..19
3.8.2 Catalase test……………………………………………….…………………………….20
3.8.3 Sugar fermentation test (sucrose, mannitol, lactose and fructose)….…..……… ……20
3.8.4 Sugar test fermentation…….……………….…………….……………..……………….20
3.8.4 Characterization of Fungi Isolates…………………..…………………………… …….21
2.9 Biodegradation………………………………………………………………………………22
3.9.1 Biodegradation of Grind walnut Shell and Chicken Droppings………………………..22
CHAPTER FOUR
4.0 Result and Discussion…………………………………..………………….………………….23
4.1 Result …………………………………………………….………………………………….23
4.1.1 Total viable microbial count on the samples………………………………………………23
4.1.2 Total microbial Viable Count from Natural Degrading Boiled African Walnut Shell……26
4.1.3 Microbial Viable Count from Natural Degrading Raw African Walnut Shell…………30
4.1.4 Total viable microbial count of degrading from African Walnut Shell Inoculated
with Chicken Droppings at ratio 1:1………………………………………………….34
4.1.5Total viable microbial count of Degrading from African Walnut Shell Inoculated
with Chicken Droppings at ratio 2:1………………………………………………….38
4.1.6 Morphological and microscopic characteristics of isolate colonies from the samples….42
4.1.7 Morphological and Microscopic characteristics of microbial isolate colonies from
the Degrading African Walnut Shell……………………………………………………46
7
4.1.8. Morphological and Microscopic characteristics of microbial isolate colonies from
the Degrading African Walnut Shell……………………………………………………50
4.1.9 Morphological and Microscopic characteristics of microbial isolate colonies from
the Degrading African Walnut Shell…………………………………………………….54
4.1.10 Biochemical Characteristics of the isolate………………………………………………58
4.1.11 Biochemical Characteristics of the isolate………………………………………………62.
4.1.12 Total Tritratable Acid of Degrading African Walnut Shell………………………………66
4.1.13 PH reading of Degrading African walnut shell………………………………………….70
4.2 Discussion ………………………………………………………………………………… 73
CHAPTER FIVE
5.0 Conclusion and Recommendation…………………………………………………… ………76
REFERENCES………………………………………………………………………………….77
8
LIST OF TABLES
Tables Page
1 Total viable microbial count isolated from Boiled African walnut shell………….……23
2 Total viable microbial count isolated from Raw African walnut shell ………………..24
3 Total viable microbial count isolated from chicken Droppings………………………..25
4 Total Bacteria viable count from natural degrading boiled African walnut shell…..….27
5 Total yeast viable count of isolates from natural degrading boiled African
walnut shell 28
6 Total mold viable count isolates from natural degrading Boiled African
walnut shell…………………………………………………………………………………………………….29
7 Total Bacteria viable count isolate from natural degrading Raw African
walnut shell …………………………………………..………………………………..31
8 Total Yeast viable count of isolate from natural degrading Raw African
walnut shell ……………………………………………………..……………………..32
9 Total mold viable count isolate from natural degrading Raw African
walnut shell ……………………………………………………………………………33
10 Total Bacterial viable count of isolates from Degrading African
Walnut Shells Inoculated with Chicken Dropping at ratio 1:1…..…………………35
11 Total Yeast viable count of isolates from Degrading African Walnut
Shells Inoculated with Chicken Dropping at ratio 1:1..……………………….……36
12 Total Mold viable count of isolates from Degrading African Walnut Shells
Inoculated with Chicken Dropping at ratio 1:1 ……………………………………….37
13 Total Bacterial viable count of isolates from Degrading African
Walnut Shells Inoculated with Chicken Dropping at ratio 2:1………………………39
14 Total Yeast viable count of isolates from Degrading African Walnut Shells
Inoculated with Chicken Dropping at ratio 2:1 ……………………………………..40
15 Total Mold viable count of isolates from Degrading African Walnut Shells
Inoculated with Chicken Dropping at ratio 2:1 …………………………………..…..41
16 Morphological and microscopic characteristics of bacteria isolate
colonies from African Walnut Shell Boiled Bacteria…………………………….…43
17 Morphological and microscopic characteristics of bacteria isolate
colonies from African Walnut Shell Raw Bacteria…………………………………..44
9
18 Morphological and microscopic characteristics of bacteria isolate
colonies from Chicken Droppings……………………………………………………45
19 Morphological and Microscopic characteristics of bacteria isolate colonies
from Natural Degrading Boiled African Walnut Shell……………………………….47
20 Morphological and Microscopic characteristics of fungi isolate colonies from
Natural Degrading Boiled African Walnut Shell………………….……………48
21 Morphological and Microscopic characteristics of bacteria isolate colonies
from Natural Degrading Raw African Walnut Shell…………………..……………..49
22 Morphological and Microscopic characteristics of fungi isolate colonies
from Natural Degrading Raw African Walnut Shell……..………………………….51
23 Morphological and Microscopic characteristics of Fungi isolates from
Chicken Droppings……………………………………………………………………52
24 Morphological and Microscopic characteristics of Bacteria isolate colonies from
Degrading African Walnut Shells Inoculated with Chicken Dropping at ratio 1:1……53
25 Morphological and Microscopic characteristics of fungi isolate colonies from
Degrading African Walnut Shell Inoculate with chicken droppings at ratio 1:1…..…55
26 Morphological and Microscopic characteristics of Bacteria isolate colonies from
Degrading African Walnut Shells Inoculated with Chicken Dropping at ratio 2:1…..56
27 Morphological and Microscopic characteristics of fungi isolate colonies
from Degrading Raw African Walnut Shell Inoculate with chicken droppings
at ratio 2:1……………………………………………………………….…….…….57
28 Biochemical test on Bacterial isolates from Natural Degrading Boiled African
Walnut Shell…………………………………………………………………………..59
29 Biochemical test on Bacterial isolates from Natural Degrading Raw African
Walnut Shell…………………………………………………………………………..60
30 Biochemical test on bacterial isolates from Chicken Droppings………………………61
31 Biochemical test of Bacterial isolates from Degrading Raw African
Walnut Shell Inoculate with chicken Droppings at ratio 1:1……………………..…63
32 Biochemical test of Bacterial isolates from Degrading Raw African
Walnut Shell Inoculate with chicken droppings at ratio 2:1…………………………64
33 Total Titratable Acid (TTA) of Natural Degrading Boiled African
Walnut Shell…………………………………………………………………………..66
34 Total Titratable Acid (TTA) of Natural Degrading Raw African Walnut Shell……..67
10
35 Total Titratable Acid (TTA) of Degrading African Walnut Shell Inoculated
with Chicken Dropping at Ratio 1:1…………………………………………………68
36 Total Titratable Acid (TTA) of Degrading African Walnut Shell Inoculated
with Chicken Dropping at Ratio 2:1………………………………………………….69
37 PH of Natural Degrading Raw African Walnut Shell………………………………..71
38 PH of Natural Degrading Boiled African Walnut Shell………………………….….72
39 PH. of Degrading African Walnut Shell Inoculated with Chicken Dropping
at Ratio 1:1……………………………………………………………….……………73
40 PH. of Degrading African Walnut Shell Inoculated with Chicken Dropping
at Ratio 2:1…………………………………………………………………………….74
11
Abstract
Microbial analyses were carried out on the shell of the edible nuts of African walnut
(Tetracarpidium conophorum). This study was aimed at determining the microbial load of boiled
and raw African walnut shell and chicken dropping which can be used as inoculums in
biodegradation process in order to solve the problem of agro waste pollution in the environment.
The nuts were sorted and washed with tap water to remove flesh residues and other contaminants
and divided into two lots. The first lot was boiled for 1.5 h. The second lot was used raw; the raw
nut and the boiled nut were then unshelled and the shells were dried at 60◦C for 5days in the oven.
The dried shells were grind and analyzed. Bacterial and fungal evaluations of the shell from nut
were done using the agar diffusion technique with serial dilutions of the grind shell and the same
process for chicken droppings. Forty Grams (40g) of the grind walnut shells were weighed and put
in a sterile cover bowl and 20ml of sterile distilled water was added to the samples under a sterile
condition and was gently mixed with sterile spatula. The grind walnut shell and chicken dropping
was done in Ratio 1:1 and 2:1. Microorganisms were isolated and identified. The microbial analysis
revealed that the boiled shell accommodate a large number of microorganism compared to that of
raw. The result show that the microorganisms present in the walnut shell were increasing as the day
is increasing and latter begins to decrease.
12
CHAPTER ONE
1.0 INTRODUCTION
African walnut Tetracarpidium conophorum (also called Plukenetia conophora) belong to the
family of Euphorbiaceae and is found in South east and South west Nigeria and Cameroon. Walnut
(Tetracarpidium conophorum) is an important crop that is cultivated throughout the world’s
temperate regions for its edible nuts (Srinivasan and Viraraghavan, 2008). T. conophorum is a
climbing shrub 10-20 ft. long, it is known in the Southern Nigeria asukpa (Igbo), Western Nigeria
as awusa or asala (Yoruba). It is known in the littoral and the Western Cameroon as kaso or ngak
[Tchiegang et al., 2007]. They are usually planted under an indigenous tree that can provide strong
support for the heavy weight of the climber when fully established on the crown of the tree, and in
cases where they cannot be harvested manually; they are left for full maturation after which the pod
falls off by itself and are picked, removed from the rotten pods, washed and sold in the market
(Hemery, 2006).
T. conophorum, like many plants in Africa and other parts of the world has been proven to have
decorative, nutritive, medicinal, agricultural and industrial values over the years. Conophor plants
are cultivated principally for the nuts which are usually cooked and consumed as snacks (Enujiugha
and Ayodele, 2003).
Tetracapridium conophurum contained in a pod which may house; one shelled nut (single), two
shelled nut (double) and three shelled nut (triple). The walnut shells could be black or brown from
the plant. The nut is whitish upon cracking from the shell. The nut has a thin layer in between
two halves (when a nut is divided into two equal parts) of nut. The seed (subglobose) is
about 2.5cm long and has wooly materials that attach the nut to the shell when cracked open. A
bitter taste is usually observed upon drinking water immediately after eating the nuts. This
could be attributed to the presence of chemical substances such as alkaloids [Ayodele, 2003]
13
Walnut shell is a waste generated in the walnut (tetracarpidium conophorum) harvest,
containing natural compounds with antioxidant properties. The walnut shell has antioxidant
compounds such as flavonoids which have been determined (Akbari et al., 2012). Production is
growing every year because it is popular as a highly nutritious food. However, this causes
problems, one being finding ways of using the walnut shells (WS) because they are waste. This
causes environmental pollution it will take a long time for them to complete their natural cycles,
and has a low utility value (Oliveira et al., 2008). It is therefore necessary to find new ways of using
walnut shell. Burning agricultural residues causes environmental problems such as air pollutiose,
soil erosion and decreasing soil biological activity [Copur et al., 2007]. Utilizing agricultural
residues not only prevents environmental concerns but also can mean farmers second income from
plantation [Ayrilmis et al., 2009].
A shell of the African walnut nut is formed by three basic substances, namely cellulose (40.5%),
hemicellulose (23.8%) and lignin (20.3%) Various methods have been proposed for hydrolysis of
cellulose and hemicellulose in wood biomass as a means of obtaining carbohydrates, which are
good sources for bioethanol preparation. A large amount of walnut shell remains after harvesting.
Currently, walnut shell is mainly used as fuel for incineration applications. The shell can be used as
a carbonaceous sorbent to control of mercury from industrial liquid streams and activation of co2 for
waste water treatment (Zabihi et al., 2010).
Tetracapidium conophorum can be cooked, roasted or sun dried and the roasted seeds could be
ground like melon seeds and used as a thickener in soup preparation. The plant is known in Africa
especially in the Eastern and Western parts of Nigeria for its antibacterial efficacy (Okerulu and
Ani, 2001). Decoction of leaves and seeds serve as beverage which relieves abdominal pains and
fever (Malu et al., 2009). Dried walnuts can be ground and turned into flour which can be used as
composite flour during baking or in-place of milk in tea preparation (Stevens and Domelam, 2003).
14
T. conophorum, like many plants in Africa and other parts of the world has been proven to have
decorative, nutritive, medicinal, agricultural and industrial values over the years. A cardio
protective dietary fat profile is recommended for the treatment of type 2 diabetes (Gillen et al.,
2005). The leaves, bark, and fruit of T. conophorum are used medicinally, and their uses include
masticatory, giddiness, thrush, antihelminthic, toothache, syphilis, dysentery, and as an antidote to
snakebite (Odugbemi and Akinsulire, 2008). In the Southern Nigeria ethno-medicine, African
walnut is used as a male fertility agent and in the treatment of dysentery (Ajaiyeoba and Fadare,
2006)
Biodegradation is defined as the biologically catalyzed reduction in complexity of chemical
compounds (Alexander, 2004). Indeed, biodegradation is the process by which organic substances
are broken down into smaller compounds by living microbial organisms (Marinescu et al., 2009).
When biodegradation is complete, the process is called “mineralization”. However, in most cases
the term biodegradation is generally used to describe almost any biologically mediated change in a
substrate (Bennet et al., 2002). Microorganisms can degrade numerous of organic pollutants owing
to their metabolic machinery and to their capacity to adapt to inhospi environments. Thus,
microorganisms are major players in site remediation. Several microorganisms, including fungi,
bacteria and yeasts are involved in biodegradation process. Algae and protozoa reports are scanty
regarding their involvement in biodegradation (Das and Chandran, 2011). However, their efficiency
depends on many factors, including the chemical nature and the concentration of pollutants, their
availability to microorganisms, and the physicochemical characteristics of the environment (El
Fantroussi and Agathos, 2005)
Chicken litter is a waste by-product of poultry production and is comprised of feces, wasted feeds,
bedding materials, and feathers (Kim et al., 2012). Chicken litter is usually recycled as an organic
fertilizer or soil amendment for direct application to agricultural land (Enticknap et al., 2006).
However, chicken litter may contain loads of human pathogens, such as Salmonella spp., that have
15
great potential to directly or indirectly contaminate fresh produce and cause food-borne disease
outbreaks (Wilkinson et al., 2011). Raw chicken litter has been widely applied to arable land as
organic fertilizer or soil amendment to improve the soil fertility and structure. Chicken droppings
contain nitrogen and phosphorus (Adeleye, 1991), which are necessary for African walnut nut shell
degradation. In addition, chicken droppings harbor bacteria and fungi that can utilize African
walnut shell efficiently.
Previous studies have shown the feasibility of using agricultural waste to produce animal feeds and
as substrate for mushroom production. Various agricultural by-products such as walnut waste,
maize cobs, peanut shell, cassava waste, wheat bran, maize husk, coconut shell, bagasse, and
banana peel are also utilized in the removal of heavy metals and toxic materials from wastewater
(Thomas et al., 2008). Several works had been done on the walnut seed such as the determination of
oxalate, phylates and tannin (Enujiugha and Ayodele, 2003). The present study is to determine the
microbial load of boiled and raw African walnut shell and chicken dropping which can be used as
inoculum in biodegradation process in order to solve the problem of agro waste pollution in the
environment.
1.1 AIM OF THE STUDY
To assess the effectiveness of chicken droppings in enhance African walnut shell degradation
1.2 OBJECTIVES OF THE STUDY
1. To determine the microbial load of the walnut shell and chicken litter
2. To isolate the microorganism which responsible for the degradation of African walnut
shell
3. To determine the pH. and acid production of the samples at different intervals of 5days
4. To identify the microorganism present on the samples
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