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Investigation of the Underlylng Microbial Actmties BIOFILTRATION OF DIMETHYL DISULPHIDE AND AMMONIA: INVESTIGATION OF THE UNDERLYLNG MICROBIAL ACTMTIES A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by MICHAEL JOHN GIBSON In partial fulfilment of requirements for the degree of Doctor of Philosophy December, 200 1 O Michael John Gibson, 200 1 Natianal Liirary Bibliiuenationale 1+1 ,cmada du Canada uisibions and Acquisitions et 3-8' wraphic Services services bibliographiques The author has granteci a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Li'brary of Canada to BiblothèQue nafiode du Canada de reproduce, loan, distn'bute or selî reproduire7prêter, distribuer ou copies of this thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfichdfilm, de reproduction sur papier ou sur format électronique. The author retains ommhip of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts f?om it Ni la thèse ni des extraits substantiels rnay be printed or otherwise de ceiie-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT BIOFILTRATION OF DRWCTHYL DISULPHIDE AND AMMONIA: INVESTIGATION OF THE UNDERLYING MICROBIAL ACTMTIES Michael John Gibson Advisor: University of Guelph, 200 1 Dr. L. Otten Biofiltration relies on microbial degradation of the contaminants to maintain the absorptive capacity of the filter media. Biofilters are extensively used to treat gas streams with multiple contaminants with different chernical properties. This study investigates the co-removal of dimethyl disulphide (DMDS) and amrnonia fiom a gas stream using compost based biofilters. Pure cultures of Thiobacillus thioparus (ATTC 8 1 58), Nitrosornonas europaea (ATTC 25978) and Niîrobacter winogrudskyi (ATTC 2539 1) were used to inoculate the media. T~hiopurusis capable of oxiditing DMDS to sulphate, while N europaea and N. winogradskyi peerform the two stage autotrophic oxidation of ammonia to nitrate, nitrification. A quantitative polymerase chah reaction (PCR)method was developed to allow tracking of T. thiopams and N. europaea in the filter media. The PCR method developed used most probably nurnber (MPN) statistics to make population estimates fiom a senes of positivehegative tests conducted on senes dilutions of DNA extracted from the filter media. The MPN-PCR method had a lower detection limit of 100 cells per gram of dry media and an upper detection lirnit of 109 cells per gram of dry media, with a 95% confidence factor of 4.67. T.tiiioparus populations observed in the media samples ranged fiom 7.9 x 102 to 1.3 x 10' cells per gram of dry media. N europuea populations in the media samples ranged fiom 1.6 x 10) to 6.3 x 104cells per gram of dry media. In situ octivity rates for T. rhiopams change significantly, 95% confidence level, fiom 2.6 x 10-'* gram sulphur per ce11 during growth phase to 4.4 x 10'" grams sulphur per ce11 during stationary phase. T. thioparus was inhibited by ammonia levels as low as 50 ppmv in the gas phase, but if DMDS removal was established pnor to arnmonia addition chemical removal of the ammonia prevented this inhibitory effect. Autotrophic nitrifiers persisted in the biofilters treating ammonia only and arnmonia in combination with DMDS, while populations Ui the biofilters treating DMDS only dropped below detection. While they persisted there is evidence that they may not be responsible for the observed amrnonia removal. In the combined treatments the sulphate produced fiom the DMDS oxidation was suffcient to account for al1 of the observed ammonia removal. Other evidence, including preliminary denaturing gradient gel electmphoresis (DGGE) analysis, suggests that heterotrophic nitrifiers may be making a significant contribution to the biofiltration of ammonia. Acknowledgements There are many people who have supported me during this undertaking. First, I would like to thank Dr. Otten for giving me the opportunity to conduct this work, his faith in my abilities remained even when my own faltered. Dr. Trevors accepted me, an engineer no less, into his laboratory allowing me the opportunity to experience the thrill, and the fiutration, that is microbiology lab work. I would aiso like to thank Dr. Lee for his assistance in helping me achieve my objectives with the PCR andysis. #en 1 look back on this time, some of my fondest mernories will be of the tirne spent in the labs of Drs. Trevors and Lee with the group of students and post-doctoral fellows they had assembled there. 1 would particularly like to thank pst-doctoral fellows Kam Leung and Mike Cassidy for their guidance. 1 would also like to acknowledge the support of my wife and parents as well as my extended family. When involved in something as consuming as a Ph.D. it is wonderful to have reminders that you are something more. 1 dedicate this to the memory of my dear friends Lana McLaren and Richard Reynolds. Table of Contents Table of Contents List of Tables List of Figures Chapter 1. Introduction 1.1 Biofiltration 1.2 Odour Generation During Composting 1.3 Biofiltration of DMDS 1.4 Biofiltration of Ammonia 1.5 Indentification of Microorganisms 1.6 Polymerase-Chain-Reaction 1.7 Quantitative PCR 1.8 MPN-PCR 1.9 DNA Extraction from Soils for PCR Chapter 2. 2.1 Biofilter Study Objectives 2.2 Microbial Analysis Objectives Chapter 3. Methods 3.1 Laboratory Biofilter System 3.2 Airflow Measurements 3.3 Cas Analysis 3.4 Preparation of Cas Standards 3.5 Media Preparation 3.6 Cultures 3.7 Direct Counting of Liquid Cultures 3.8 Inoculum Preparation 3.9 Start-Up of Biofilter Experlments 3.10 Gas Sampling 3.1 f Sampling of Media 3.12 Moisture Determination by Microwave 3.13 Chernical Anaiysis of Media 3.14 DNA Extraction and Preparation 3.15 PCR Amplification PCR Product Purification Sequencing of PCR Products Chapter 4 Esperimental Design Effect of Inoculum Combined versus Individual Contaminant Treatment Long Term Combined Removal Nuhient Supplementation Effects on DMDS Removal Ceramic Media Chapter S. Results Development of PCR Protocol MPN-PCR Results Biofilter Results Chapter 6.0 Discussion PCR Overall f erformance of Biofilter Studies Physical Chemical Treatment vs Biological Treatment Biological Activity Coupling of DMDS and Ammonia Removals Ammonia Inhibition of DMDS Removal DMDS Inhibition of Ammonia Removal Evidence of Heterotrophic Nitrifiers Chapter 7.0 Preliminary DGGE Investigation Introduction to DGGE DGGE Methodology DGGE Results Discussion of DGGE Results Cbapter 8.0 Recommendations Chapter 9.0 Conclusions References Appendices Appendix A - PCR Images Appendix B - Media Analysis Results Appendix C - Primers and Sequencing Results Appendix D - Media Compositions List of Tables Chapter 1 Table 1. Operating costs for air treatrnent technologies Table 2. Removal rate constants for T. thioparus inoculated peat biofilter Table 3. Literature reported nitrification rates Chapter 3 Tabk 4. Identification of cultures used to inoculate biofilters Chapter 5 Table 4. Summary of population estimates for standards Table 5. Summary of maximum and average sulphur removal rates for al1 studies Table 6. Summary of maximum and average ammonia removal rates for al1 studies List of Figures Cbapter 1 Figure 1. Pathway for oxidation of DMDS by Thiobacillus thioparus. Figure 2. Microbial nitrogen transformations and oxidation States Chapter 3 Figure 3. Schematic of laboratory biofiltration systern Chapter 5 Figure 4. Electrophoresis gel image using psuedo-nested PCR method Figure 5. PCR results using specific primers for Neuropaea and T.thiopums Figure 6. Removal of DMDS and ammonia by biofilter with unsterilized-inoculated media, replicate A Figure 7. Removal of DMDS and ammonia by biofilter with unsterilized-inoculated media, replicate B Figure 8. Removal of DMDS and ammonia for biofilter where media was neither sterilized nor inoculated, replicate A Figure 9. Removal of DMDS and ammonia for biofilter where media was neither sterilized nor inoculated, replicate B Figure 10. DMDS and ammonia removal for biofilters with sterilized media Figure 1 1. DMDS and ammonia removal for biofilter with combined loading, replicate A Figure 12. DMDS and ammonia removal for biofilter with combined loading, replicate B Figure 13. Comparing DMDS removal between biofilters receiving both DMDS and arnmonia loading with those receiving DMDS loading only Figure 14. Comparing ammonia removal between biofilters receiving both DMDS and ammonia and those receiving ammonia loading only Figure 15. Ammonium, nitrate and nitrite andysis of media samples fiom biofilters treating both ammonia and DMDS Figure 16. Ammonium, nitrate and nitrite analysis of media samples fiom biofilters treating ammonia only Figure 17. Accumulation of inorganic and total nitrogen compared to cumulative nitrogen removal for biofilters receiving both DMDS and ammonia Figure 18. Accumiilation of inorganic and total nitrogen compared to cumulative nitrogen removal for biofilters receiving ammonia only Figure 19. Comparison of sulphur accumulated in filter media with cumulative removal of sulphur fiom air for biofilters receiving both DMDS and ammonia Figure 20. Comparison of sulphur accumulated in filter media with cumulative removal of sulphur from air for biofilters receiving DMDS only Figure 2 1. Population density of Tthioparus in filter media 95 Figure 22. Activity rates for T.thioparus in biofilters 95 Figure 23. Population density of Neuropaea in filter media 96 Figure 24. Activity rates for Neuropaea in biofilters 96 Figure 25. Amrnonia removal for biofilters loaded with media which had been stored Figure 26. DMDS removal for biofilters loaded with media 97 which had been stored Figure 27. idet concentrations of DMDS during nutrient 98 supplementation study Figure 28. DMDS removal during nutrient supplementation 98 StUdy Figure 29. Ammonia and DMDS removal for biofilter packed with ceramic media Figure 30. DMDS removal under cool lab conditions 99 Figure 3 1.
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