Isolation Characterisation and Molecular Studies of some Pathogenic Bacteria in Composting Animal and Municipal Wastes in Zaria, Kaduna State, Nigeria.
Abstract
Composting of raw organic waste is an excellent example of high biological processes used for agricultural practices.
The open pile and windrow method were used in composting poultry, cow, and municipal solid waste for twelve (12) weeks.
Temperatures, pH and some selected heavy metals were determined during composting and in the finished products.
The presence of helminths was determined and the total plate count, total coliform counts, the antibiotic sensitivity patterns of Salmonella typhi, Vibrio cholerae and E.coli 0157:H7 and their molecular profiles were also studied.
All the heavy metals in the final compost was lower than the standard recommended by the USEPA, the temperature conditions did not meet the 55oC recommended for compost throughout the 12 weeks of composting .
All the helminths did not survive beyond two weeks of composting. The results also showed a steady decline in total plate and coliform counts, and showed no significant difference between both.
Composting techniques did not affect microbial growth but were significantly affected by the duration of composting. A total of 109 Vibrio, 152 E.coli, and 105 Salmonella isolates were tested using biochemical and serological tests.
Out of these number 97(88.9%) Vibrio isolates were confirmed to be Vibrio cholerae, 140 (92.1%) were E.coli 0157 and 98 (93.3%) were Salmonella typhi.
Table Of Contents
COVER PAGE…………. i
BLANK PAGE……….. ii
TITLE PAGE……….. iii
DECLARATION…….. iv
CERTIFICATION……… v
DEDICATION………… vi
TABLE OF CONTENTS………. xi
CHAPTER ONE
1.0 INTRODUCTION. 1
- Statement of the Problem……….. 3
- Justification…………….. 3
- Aim………………….. 4
- Specific Objectives………. 5
CHAPTER TWO
2.0 LITERATURE REVIEW………. 6
- Indicator Organisms, Pathogenic coli and Salmonella……. 9
- Protozoa and Helminthes………… 14
- Composting Systems………….. 15
- Occurrence of Pathogens in Human and Animal Waste and Compost………………… 16
- Survival of Pathogens in Human and Animal Waste and Compost…………………….. 19
- Other Bacteria…………….. 20
- Viruses…………… 20
- Agents of Transmissible Spongiform Encephalopathies……. 21
- Standards for Pathogens in Compost…….. 22
- Environmental Impacts………………… 23
- Ammonia………………… 24
- Greenhouse Gases…………. 25
- Leachate……………….. 26
- Effect of Composting on Chemical Parameters of the Finished Product……………… 27
2.12.2 pH……… 28
- Potentially Toxic Elements (PTEs)…….29
- Organic Contaminants………. 29
- Heavy Metals………….. 33
- Effect of Composting on the Biological Parameters of the Finished Product………… 35
CHAPTER THREE
- MATERIALS AND …. 36
- The Study Area………. 36
- Sampling Sites…….. 38
- Collection of Samples……. 38
- Composting Techniques……. 38
- Laboratory Samples………….. 39
- Determination of Physicochemical Parameters of the Waste Samples……… 39
- Temperature…………… 39
3.6.2 pH…………………….. 39
- Conductivity….. 40
- Determination of Heavy Metals in Waste…………… 40
- Compost Sample Digestion…… 40
- Microbiological Analyses of the Waste Samples………. 41
- Preparation of Media………. 41
- Determination of Total Bacterial Count……. 41
- Total Coliform Count………. 42
- Isolation of Escherichia coli from Samples…… 43
- Identification of coli O157:H7 Strains……… 42
- Agglutination test for coli 0.157:H7…………. 43
- Isolation of Vibrio cholerae………. 43
- Agglutination tests Vibrio cholerae……. 436
- Isolation of Salmonella spp………… 43
- Agglutination test for Salmonella typhi………… 44
- Detection of Helminths / eggs / larvae in Compost………. 44
- Molecular Characterization of Bacterial Isolates……….. 44
- Extraction of DNA……………… 44
- Restriction Endonuclease Digestion of Chromosomal DNA…..45
- Agarose gel of Chromosomal DNA, Restriction Endonuclease ………….. 46
- Staining and Photography of Agarose gel of Chromosomal DNA Restriction Endonuclease …. 47
- Polymerase Chain Reaction Detection of coli 0157: H7, Salmonella typhi, Vibrio cholerae 16 rDNA Gene Regions for Prokaryotes………. 47
- Enterobacterial Repetitive Intergenic Consensus PCR……….48
- Preparation of Inocula…………49
- Determination of Zone of Inhibition……….. 49
3.10 Statistical Analysis……………..50
CHAPTER FOUR
4.0 RESULTS…….. 51
Comparison of the Mean and the Standard Deviation for the Physicochemical Parameters of the compost… 52
Comparison of the Mean and the Standard Deviation for the Physicochemical Parameters of the Compost by 12 Weeks……… 53
The Mean Effect for Each Parameter on the Compost……………………………………… 54
The Mean Values for all the Parameters on the Composting Techniques Used……… 56
The Mean Temperature (oC) Values for the Various Composting Techniques Used. 57
The Weekly Mean Temperature Values for the Various Wastes Composted………… 58
The Mean pH Values for the Composting Techniques Used……………………………… 60
The Weekly Mean pH Values for the Various Waste Composted……………………….. 61
The Mean Conductivity (µs/cm) Values for the Composting Techniques Used…….. 62
The Weekly Mean Conductivity (µs/cm) Values for the Various Waste Composted
Using the Two Composting Techniques…………………………………………………………. 63
The Level of Heavy Metals Present in the Various Waste Composted (ppm)……….. 64
4.2. Microbial Indicator Levels……65
Mean of the Total Plate and Counts of the Compost (log10 cfu-g) 66
Mean and Standard Deviation of Compost for each Week Counts (log10 cfu-g) 67
The Weekly Mean Total Plate Counts for the Various Waste Composted (log10 cfu-g) 69
The Total Plate Count for the Two Composting Techniques used for 12 Weeks (log10 cfu-g) 70
The Total Coliform Count for the Various Composting Techniques Used (log10 cfu-g) 71
The Weekly Mean Total Coliform Counts for the Various Waste Composted (log10 cfu- g) 73
Occurrence of Salmonella/Vibrio spp and Feacal Coliform for the Various samples…. 74
Characterization and Distribution of Isolates from the Different Compost 75
Frequency of Contamination of Municipal Solid Waste Samples with Helminthes 76
Frequency of Contamination of Poultry Waste Samples with Helminths 78
Frequency of Contamination of Cattle Waste Samples with Helminthes 79
Zones of Inhibition for Selected Antibacterial Testing on E. coli O157: H7. 81
Zones of Inhibition for Selected Antibacterial Testing on Salmonella typhi 82
Target Pathogens and their Sequence Profile…….. 87
CHAPTER FIVE
- DISCUSSION…….89
- Temperature Changes During Composting………. 892
- pH Changes During Composting……… 940
- Electrical Conductivity…….. 951
- Heavy Metals….. 961
- Microbial counts during composting….. 892
- Bacteria…. 904
- Helminths…..915
- Re-growth…… 916
- Antibiotic Sensitivity Tests…… 96
- Polymerase Chain Reaction………98
- CONCLUSIONS……… 99
- Recommendations…100
Introduction
Background Of Study
Composting is an excellent example of a highly complex biological process, involving many species of bacteria and fungi which convert a low-value material into a higher value product.
A wide range of biological wastes can be composted including materials generated by agriculture, food processing, wood processing, sewage treatment, industrial, municipal waste and animal wastes.
Composting is a process of controlled biological decomposition of biodegradable materials under managed conditions that are predominantly aerobic and allow the development of thermophilic temperatures as a result of heat produced biologically, in order to achieve compost that is stable and less harmful when used.
The agricultural or domestic use of compost may possibly increase the risk of disease transmission by a number of mechanisms. The most obvious is direct contact by humans handling the material or animals grazing pastures or crops on which the material has been used as a organic fertilizer.
This could be particularly important for organisms such as enterohaemorrhagic Escherichia coli O157:H7 which is thought to have a low infectious dose (possibly as few cells may initiate an infection in susceptible humans) (Tarr, 1995; Anon, 1997; Ritu, et al., 2011).
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