EFFECTIVENESS OF WINDROW COMPOSTING METHODOLOGY IN KILLING A THERMO-TOLERANT SPECIES OF SALMONELLA DURING MORTALITY COMPOSTING

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EFFECTIVENESS OF WINDROW COMPOSTING METHODOLOGY IN KILLING A THERMO-TOLERANT SPECIES OF SALMONELLA DURING MORTALITY COMPOSTING, Is A Well-Researched Topic, It Is To Be Used As A Guide Or Framework For Your Research.

ABSTRACT

In a large agricultural operation, such as the one at Cal Poly San Luis Obispo, disposal of deceased animals is an immense issue. The cost of transporting and rendering every dead animal is inhibitory to the general function of the agricultural operations and their thin budget. Therefore, we propose that composting mortalities could be an economical alternative. Composting is a recognized method for taking animal waste
products along with carbon waste and turning it into a pathogen-free, nutrient-rich topsoil. Carcass composting is in fact performed in other countries and states to varying degrees of success. However, the California EPA limits carcass composing to only private land. Therefore, the purpose of this work was to determine the efficacy of killing
pathogens by composting using bench top composting models. Ultimately, our goal is to provide “proof of concept” data in order to gain permission for a full-scale carcass compost pile to be set up at Cal Poly San Luis Obispo.

Using thermo tolerant Salmonella senftenberg as an indicator organism, we performed bench top trials of traditional and carcass compost in the lab. Samples were inoculated with S. senftenberg and kept at 55°C for 15 days in accordance with the California EPA and Test Method for the Examination of Composting and Compost (TMECC). Samples were then plated and processed for multiple tube analysis and most probable number. Samples were also partitioned for a viability qPCR with propidiummonoazide (PMA) to compare to the classic techniques. Using these methods we were then able to track and produce thermal death time data for S. senftenberg in both traditional and carcass compost. By comparing the types of compost, we were able to determine that the composting method presented by the California EPA and the TMECC
produces safe, pathogen free compost, even when inoculated carcasses were introduced. However, even with removal of dead cells by PMA, qPCR did not outperform the classical microbiological methods for as tracking pathogen killing.

TABLE OF CONTENTS

LIST OF TABLES………………………………………………………………………………………………. ix
LIST OF FIGURES ……………………………………………………………………………………………. xii
CHAPTER
1. INTRODUCTION ……………………………………………………………………………………………. 1
1.1 Composting………………………………………………………………………………………………… 1
1.2 Cal Poly San Luis Obispo Composting Unit …………………………………………………… 6
1.3 Mortality/Carcass Composting ……………………………………………………………………… 7
1.4 Composting Regulations…………………………………………………………………………….. 12
1.5 Salmonella senftenberg………………………………………………………………………………. 15
1.6 EPA Required Methods for Detecting Salmonella in Compost. ………………………. 18
1.7 Viability PCR with Propidium Monoazide Dye …………………………………………….. 21
1.8 Experimental Overview……………………………………………………………………………… 23
2. MATERIALS AND METHODS………………………………………………………………………. 24
2.1 Strains and Media ……………………………………………………………………………………… 24
2.2 Growth Curve Analysis of S. senftenberg …………………………………………………….. 24
2.3 Calculation of Cells in Culture Using Growth Curve……………………………………… 25
2.4 Thermal Death Time (TDT) Analysis of S. senftenberg in Water…………………….. 25
2.5 TDT Analysis of S. senftenberg Suspended in Compost…………………………………. 26
2.6 DNA Extraction from S. senftenberg……………………………………………………………. 26
2.7 PCR Detection of S. senftenberg …………………………………………………………………. 27
2.8 Preparation of Compost Samples…………………………………………………………………. 29

2.9 Preparation of Inoculated Compost Samples for DNA Extraction……………………. 30
2.10 Multiple-Tube Enrichment of S. senftenberg from Compost …………………………. 32
2.11 Analysis of Loss During Separation by Centrifugation and DNA Extraction…… 33
2.12 Preparation of Compost Samples with Inoculated Chicken …………………………… 33
2.13 DNA Extraction from Composting Chicken ……………………………………………….. 34
2.14 Propidium Monoazide Staining and DNA Extraction …………………………………… 34
2.15 Production of qPCR Standard Curve and Analysis of Extraction Rates ………….. 35
2.16 Live/Dead qPCR Using PMA Dye …………………………………………………………….. 36
2.17 qPCR Data Analysis ………………………………………………………………………………… 38
2.18 Statistical Analysis…………………………………………………………………………………… 38
3. RESULTS ……………………………………………………………………………………………………… 39
3.1 Growth Rate of S. senftenberg…………………………………………………………………….. 39
3.2 Thermal Death Time (TDT) for S. senftenberg in Water ………………………………… 40
3.3 Analysis of qPCR Primers for S. senftenberg Detection …………………………………. 41
3.4 TDT of S. senftenberg Contained in Tubes Inserted into Compost…………………… 42
3.5 Analysis of Loss During Separation by Centrifugation and DNA Extraction……. 43
3.6 TDT of S. senftenberg in Compost ………………………………………………………………. 45
3.7 TDT of S. senftenberg Inoculated into Chicken Carcass Compost …………………… 51
3.8 Statistical Analysis…………………………………………………………………………………….. 56
4. DISCUSSION………………………………………………………………………………………………… 59
BIBLIOGRAPHY………………………………………………………………………………………………. 65
APPENDICES
A. Compost Nutrient and Microbial Analysis …………………………………………………….. 73

B. Raw Data with Associated Error…………………………………………………………………… 74
C. FDA MPN Table ………………………………………………………………………………………… 85
D. Microbial Competition Pilot Study……………………………………………………………….. 86

Additional information

Author

Spencer Gabriel Myers

No of Chapters

4

No of Pages

99

Reference

YES

Format

PDF

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