TABLE OF CONTENTS
TITLE . . . . . . . . . . I
APPROVAL . . . . . . . . . II
DEDICATION . . . . . . . . . III
ACKNOWLEDGEMENT . . . . . . . . IV
TABLE OF CONTENT . . . . . . . . V
LIST OF TABLES . . . . . . . . . VIII
LIST OF FIGURES . . . . . . . . . IX
REPORT OVERVIEW (ABSTRACT) . . . . . . X
CHAPTER ONE
- INTRODUCTION . . . . . . . . 1
- AIMS AND OBJECTIVES . . . . . . . 2
- SCOPE OF STUDY . . . . . . . . 3
1.4 SIGNIFICANCE OF STUDY . . . . . . . 3
CHAPTER TWO
LITERATURE REVIEW . . . . . . . . 4
2.1 MICROBIOLOGY AND ECOLOGY OF NITRIFICATION . . 4
2.2 TYPES OF NITRIFYING BACTERIA . . . . . 5
2.3 NITRIFYING POTENTIAL . . . . . . . 6
2.4 POND . . . . . . . . . . 6
2.5 FRESHWATER . . . . . . . . . 8
2.6 CHEMISTRY OF NITRIFICATION . . . . . . 9
2.7 NITRIFICATION IN THE MARINE ENVIRONMENT . . . 10
2.7.1 NITROGEN CYCLE . . . . . . . . 11
2.7.2 MARINE NITROGEN CYCLE . . . . . . 11
2.7.3 ENVIRONMENTAL IMPACTS OF MARINE NITROGEN CYCLE . 12
2.8 PLASMID EXTRACTION . . . . . . . 13
2.9.0 PLASMID EXTRACTION BY GENEJET™ PLASMID MINIPREP KIT. 13
2.9.1 DESCRIPTION . . . . . . . . 13
2.9.2 PRINCIPLE . . . . . . . . . 14
2.10 NANODROP SPECTROPHOTOMETER . . . . . 14
CHAPTER THREE
3.0 MATERIALS AND METHODS . . . . . . 16
3.2 RESEARCH DESIGN . . . . . . . . 16
3.3 DATA COLLECTION . . . . . . . . 16
3.4 MATERIALS AND REAGENTS . . . . . . 16
3.5 TOOLS FOR DATA COLLECTION . . . . . . 18
3.6 SAMPLE AND SAMPLING TECHNIQUES . . . . . 18
3.7 PROCEDURES . . . . . . . . . 18
3.8 STOCK CULTURE . . . . . . . . 19
3.9 GRAM STAINING . . . . . . . . 19
3.10 NITRIFICATION POTENTIALS . . . . . . 20
3.11 PLASMID EXTRACTION PROCEDUE . . . . . 20
3.12 NANODROP SPECTROPHOTOMETER . . . . . 22
3.13 STATISTICAL ANALYSIS OF NITRIFYING POTENTIAL
CHAPTER FOUR
4.0 RESULTS. . . . . . . . . . 23
CHAPTER FIVE
5.1 DISCUSSION . . . . . . . . . 30
5.2 VARIATION IN ABUNDANCE OF NITRIFIERS . . . . 30
5.3GRAM STAINING . . . . . . . . 31
5.2 AGGREGATION . . . . . . . . 31
5.3 NITRIFYING POTENTIAL . . . . . . . 31
5.4 PLASMID DNA QUANTIFICATION . . . . . . 31
CONCLUSION . . . . . . . . . 32
REFERENCES . . . . . . . . . 33
LIST OF TABLES
Table 1: NITRIFYING BACTERIA THAT OXIDIZE AMMONIA . . 6
Table 2: Nitrifying bacteria that oxidize nitrite . . . . 7
Table 3: Result from gram staining and microscopic view . . . . 23
Table 4. Nitrifying Potential of the bacteria . . . . . . 23Table 5. The Nano-drop spectrophotometer . . . . . . 29
LIST OF FIGURES
Figure 1: A CHAT REPRESENTING THE NITRIFICATION ACTIVITIES . 29
Figure 2: Chart of the plasmid DNA (Concentrations at A260 and A280) . . 30
ABSTRACT
Nitrification is the biological oxidation of ammonia into nitrite, followed by the oxidation of nitrite into nitrate by small groups of autotrophic bacteria and Achaea. NH3 removal is beneficial to the plant system as build up is dangerous. Presence and activities of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in freshwater fishpond of a circulating aquaponics system was examined in respect to random points in the aquaponics system (the fish tank, the bio-filter, and the line supplying water to the plants). The samples FT4, FT6, BF5, BF6, BF1, LN1, LN2, and LN3 were all rod-like in shape, when viewed with 40 objective light compound microscope.While samples BF7 and FT7 were both circular in shape. samples FT4, FT6, FT7, BF5, BF6, BF1, LN1, LN2 and LN3 retained their primary dye ( blueblack colouration). Sample BF7 retained its secondary colour (pink colouration). , samples FT4, FT5, LN2, LN3, LN1 were all in chains. Samples FT6, BF6, and BF1 were spaced. Samples BF7 FT7 were clustered together. From the statistical analysis, it was deduced that nitrifying bacteria can be isolated from any point in the aquaponics unit. Most nitrifying bacteria were discovered to have good yield of plasmid DNA. The potential
nitrification activities and oxidation rates were shown to be linear and activity of ammonia-oxidizingand nitriteoxidizing bacteria was highest in samples from the bio-filter.
CHAPTER ONE
1.1 INTRODUCTION
Most of the nitrogen available to the biosphere exists as N2 in the atmosphere, and is not useful to most organisms until it is “fixed” either biologically or abiotically (by lightning or aurorae, or industrially). Once it is fixed into NH3, usually it is either assimilated and transformed into organic N or nitrified into NO3-. (NASA-Amens 1996). Nitrification is the process by which ammonia is converted to nitrites (NO2-) and then nitrate (NO3-). This process naturally occurs in the environment, where it is carried out by specialized bacteria (Remay, 2000). The bacteria that carry out nitrification are called “Nitrifying bacteria” (AOBs and NOBs).
In the environments with high inputs of a nutrient such as freshwater fish ponds, mineralization of organic substances as a result of over-feeding and excretion increases the ammonia concentration which is harmful to fish and shrimp (Goldman et al., 1985). Since microbial processes affect water quality parameters such as dissolved oxygen (DO), ammonium, nitrite, nitrate etc. (Moriarty, 1996), hence bacteria in ponds play important role in maintaining the water column chemistry (Vibha, 2011).
Aquaponics is the integration of a hydroponic plant production system with a recirculating aquaculture system. In a simple aquaponic system, nutrient-rich effluent from the fish tank flows through filters (for solids removal and biofiltration) and then into the plant production unit before returning to the fish tank (Christopher, 2015).
Ammonia becomes toxic to plants at certain concentration. This toxicity ranges from causing stunted growth in the plant to inhibiting germination of the seedling (Brain, 2014).
The present study was undertaken to determine the occurrence of nitrifying bacteria (Ammonia oxidizing bacteria [AOB] and Nitrite oxidizing bacteria [NOB]), in relation to the plants ability to utilize the ammonia produced from fresh-water fishpond in an aquaponic system in National Biotechnology Development Agency (NABDA) Abuja.
1.2 STATEMENT OF PROBLEM
In large urban areas, conventional agriculture is almost impossible, and this is as a result of lack of space for establishment of agricultural field. And it has consequently resulted to unsustainable supply of fresh, local, organic produce.
Aquaponics system which should have provided a reliable answer to the problem also has a little challenge on its own. Ammonia is produced by the fish’s respiratory system and is discharged through their gills. Buildup of ammonia in the fish tank eventually kill them (dead fishes will also produce ammonia). Also buildup of ammonia concentration in the system becomes toxic to the plant, such as causing stunted growth (Brain, 2014). Introduction of Nitrifying bacteria can help shorten the lag phase in starting up aquaponics system, which usually pose loss of resources and frustration on beginners.
1.2 AIMS AND OBJECTIVES
The general objective of the study is to determine if there are ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria NOB in Fresh water pond of Aquaponics system in NABDA Abuja. The specific objectives include;
- Isolation of bacteria from the fresh water fishpond of aquaponics system in NABDA.
- To identify ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) associated with aquaponics system .
- To identify specific locations of these bacteria in the culture system.
- To identify and characterize these bacteria.
1.3 SCOPE OF STUDIES
Under the auspices of this study, microbial work was carried out in the aspect of isolation of the organisms from the fishpond and gram-identification of the isolates.
Also a biochemical test will be carried out to evaluate the nitrifying potential of the isolates.
Molecular work will be also done by isolating plasmids from the bacteria.
Then a Nano technique will be used to quantify the concentration of the plasmids per isolate, using a Nano-drop spectrophotometer.
1.4 SIGNIFICANCE OF STUDY
This research work will provide information on the presence and types of bacteria or a bacteria culture in the Aquaponics or hydroponics system. Also the work will explore the plasmids available in the bacteria for future use in recombinant DNA technology for cloning and possibly in bio-remediation.
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