Perchlorate and chlorate degradation by two organisms isolated from wastewater Microbial identification and kinetics Filipa Costa Pinto Prata Thesis submitted in fulfilment of the requirements for the degree of Master Science in CHEMISTRY President: Profª Doutora Matilde Marques, IST-UTL Promotors: Profª Doutora Cristina Costa, FCT-UNL Profª Doutora Cristina Viegas, IST-UTL Doutor Paulo Costa Lemos, FCT-UNL November 2007 ACKNOWLEDGMENTS I wish to express my sincere gratitude to Prof ª Doutora Maria Cristina Costa, for the opportunity to perform my dissertation, support and guidance. To Profª Doutora Cristina Viegas I would like to thank to accept to be my promotor. I would also especially like to thank Doutor Paulo Lemos and Profª Doutora Maria Ascensão Miranda Reis for their laboratorial guidance, suggestions and scientific advises which improved this work. I would like to thank the BioEng staff for their friendship during my laboratorial work and also my gratitude to Marta for her hold. Many thanks to my friends for their constant hold, encouragement and patience throughout the duration of this project. They always have a word of support and a smile to give me. Thank you Cristiana, Mariana and Bruna. Never enough thanks to one who doesn’t want to be named but he knows who he is and so do I. To my family I just want to thank for their care, support and ethical values that always motivated me to improve my knowledge and personality. ABSTRACT - - The biological removal of perchlorate (ClO 4 ) and chlorate (ClO 3 ) can be viewed as a very promising water treatment technology. The process is based on the ability of specific bacteria to use (per)chlorate as an electron acceptor in the absence of oxygen. The present research work was focused on the isolation and kinetic characterization of perchlorate reducing bacteria. The enrichment process started with a sludge sample taken from an anaerobic digester of a domestic wastewater treatment plant (Beirolas, Portugal). Two perchlorate-reducing bacteria (per1) and (per2) were isolated using different selection methods, platting and liquid transfer respectively. The purity of the isolates was confirmed by genetic characterization of 16S rDNA. The BLAST search showed that the microorganims shared a 99% sequence similarities to the 16S rDNA of Dechlorospirillum sp. DB (per1) and Dechlorosoma sp. PCC (per2). Batch tests were performed under anaerobic conditions with acetate as the electron donor and perchlorate and/or chlorate as electron acceptor. During perchlorate reduction by Dechlorospirillum sp. DB it was observed transient accumulation of chlorate. The isolates showed different behaviour concerning perchlorate and chlorate reduction. Chlorate was preferentially reduced when both electron acceptors were present, being perchlorate reduced after completely depletion of chlorate. The former performance was observed in both bacteria. Keywords : Bioremediation, Perchlorate and chlorate reduction, Isolation, Kinetic characterization, Dechlorospirillum sp. DB, Dechloromona sp. PCC. Perchlorate and chlorate degradation by two organisms isolated from wastewater ii TABLE OF CONTENT CHAPTER 1. LITERATURE STUDY………………………………………… 1 1.1. Introduction………………………………………………………………. 1 1.2. Perchlorate as a pollutant.....……………………………………………… 2 1.2.1. Properties...........…………………………………………………... 2 1.2.2. Perchlorate environmental occurence....…………………………… 4 1.2.3. Health effects..……………………………………………………... 7 1.2.4. Legislation........................................................................................... 7 . 1.3. Perchlorate treatment technologies……………...…………………………. 8 1.3.1. Physical processes..……………………..…...…………………….. 9 1.3.2. Chemical processes………..…………………………………….... 13 1.3.3. Biological processes................…..……………………………....... 14 1.4. The microbiology and biochemistry of perchlorate reduction………….. 16 1.4.1. Perchlorate reducing bacteria..……………………………………… 16 1.4.2. Electron donors used by PRB for growth…………...………........ 19 1.4.3. Nutritional requirements for PRB……..…………………………... 19 1.4.4. Biological perchlorate reduction…………………………………… 20 1.4.5. Enzymes responsible for (per)chlorate reduction………………….. 21 1.4.6. Factors that interfere with perchlorate enzyme induction………… 25 1.5. Outline of the thesis………………………………………………………... 26 27 CHAPTER 2. MATERIALS AND METHODS….……………………………… 2.1. Source of organisms……………………...………………………………. 27 2.2. Media............................................................................................................. 27 2.3. Bacterial isolation procedures and culturing conditions …………………. 28 Perchlorate and chlorate degradation by two organisms isolated from wastewater iii 2.4. Morphology………………………………………………………………… 29 2.5. 16S ribosomal DNA extraction and sequencing…………………………… 29 2.5.1. Extraction and confirmation 16S ribosomal DNA…………………… 29 2.5.2. PCR amplification and purification………………………………… 30 2.6. Phylogenetic analysis……..………………………………………………... 31 2.7. Batch growth kinetics……………………………………………………… 31 2.8. Analytical techniques……………………………………………………… 32 2.9. Calculations……………………………………………………………….. 33 2.9.1. Specific growth rate………………………………….….................. 33 2.9.2. Specific uptake rate…………..………………………………………. 33 2.9.3. Substrate uptake yield………..…………………………………….. 33 2.9.4. Chloride formation yield…………………………………………… 33 2.9.5. Biomass yield………………………………………………………. 33 CHAPTER 3. RESULTS AND DISCUSSION …………………………..……… 34 3.1. Results………………………………………..……………………….……. 34 3.1.1. Morphological and genetic characterization of the isolates….............. 34 3.1.2. Growth kinetics………………..…………………………………… 35 3.2. Discussion…………………………….………………………………….. 43 CHAPTER 4. CONCLUSIONS AND FURTHER RESEARCH....………….… 49 CHAPTER 5. BIBLIOGRAPHY……………..………..………………………... 51 Perchlorate and chlorate degradation by two organisms isolated from wastewater iv LIST OF TABLES Table 1.1: Physical and chemical properties of selected perchlorate compounds...... 4 Table 1.2: Perchlorate respiring bacterial isolates...................................................... 17 Table 2.1: Media and reagents used for enrichment and isolation .…… ……….…... 27 Table 3.1:Specific growth rates of described perchlorate and chlorate reducing bacteria…………………………………………………………………… 44 Table 3.2: Biomass yields in the presence of different electron acceptors determined in this study and reported by others........................................ 46 Table 3.3: Resume of all kinetics parameters (n, number of data points considered for parameter calculations)…… …............................................................ 48 LIST OF FIGURES Figure 1.1: Energy profile for the rate-limiting step in perchlorate reduction, abstraction of the first oxygen atom. ………………….…………….…. 3 Figure 1.2: Perchlorate manufacturers and users in US, April 2003 ………………. 5 Figure 1.3: Concentrations levels of perchlorate found in wine samples from various Continents…………………………………………………….. 6 Figure 1.4: Mechanism of anion exchange – chloride for perchlorate.…………….. 10 Figure 1.5: Reverse osmosis (RO). The influent water is forced through a membrane that is impermeable to dissolved salts.……………………... 11 Figure 1.6: Electrodialysis. Water flows through alternate semipermeable membranes while under the influence of an electric field .…………….. 12 Figure 1.7: Simple electrolytic cell of the reduction of perchlorate.……………….. 13 Figure 1.8: Schematic diagram of ion transport and bioreduction in the ion exchange membrane bioreactor …………………………………..… 15 Figure 1.9: Phylogenetic distribution of (per)chlorate and chlorate reducing microorganisms based on total 16S rDNA ………………………….. 18 Perchlorate and chlorate degradation by two organisms isolated from wastewater v Figure 1.10: Schematic of perchlorate-reducing pathway, based on accepted roles of (per)chlorate reductase and chlorite dismutase enzymes………….. 20 Figure 1.11: Model of the pathway involved in the respiratory reduction of (per)chlorate by (per)chlorate reducing bacteria ……………………… 21 Figure 1.12: Model of the pathway involved in the reduction of chlorite by perchlorate reducing bacteria………………………………………… 23 Figure 2.1: Schematic representation of the reactor used for batch tests………….. 28 Figure 2.2: Schematic representation of the reactor used for batch tests………….... 31 Figure 3.1: Optical microscopy observation of the enriched cultures; A: (per1) and B: (per2) (100x)……………...…………………… ……………..…….. 34 Figure 3.2: Acetate and perchlorate uptake and transient accumulation of chlorate - as function of time during the reduction of 10mM of ClO 4 by Dechlorospirillum sp. DB. Note the different concentration scale for - ClO 3 . Dry Weight (DW) as function of time is also represented…....... 36 Figure 3.3: Acetate and chlorate uptake as function of time during the reduction of - 10mM of ClO 3 by Dechlorospirillum sp. DB. Dry weight (DW) and chloride formation as a function of time are also represented…………. 37 Figure 3.4: Acetate, perchlorate and chlorate uptake as function of time during the - - reduction of 5mM of ClO 4 + 5mM of ClO 3 by Dechlorospirillum sp. DB. Dry weight (DW) and chloride formation as a function of time are also represented……………………………………………………… 38 Figure 3.5: Perchlorate and chlorate uptake as function of time during the - - reduction of 5mM of ClO 4 + 5mM of ClO 3 by Dechlorospirillum sp. DB.………...…………………………………………………………… 39 Figure 3.6: Acetate and perchlorate uptake as function of time during the reduction of 10mM of ClO 4- by Dechlorosoma sp. PCC. Dry weight (DW) as function of time is also represented..……………………………