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Downloads/Bin/Fastq Quality Filter -Q20 -P90 -Q33 Biological Reduction of Selenium Oxyanions in the Presence of Nitrate anions using Anaerobic Microbes by Gaurav Subedi B.Sc., Jacobs University Bremen, 2010 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemical and Biological Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2016 © Gaurav Subedi, 2016 Abstract Biological selenium reduction has emerged as a viable solution for the removal of toxic selenium from the environment. However, the presence of nitrate hinders selenium reduction by acting as a competitive electron acceptor. The present thesis investigated the use of local mine-impacted sediment as an inoculum for selenium reduction and studied the affect of nitrate on the removal of selenium. Sediment samples, impacted by mining activities, were collected from two vastly different sites of the Elk River Valley. These sediments namely; Goddard Marsh and Mature Tailing Coal, were enriched for selenium reducing bacterial consortium under high selenium and varying nitrate concentrations to put additional selection pressure. Ultimately, two cultures from Goddard Marsh enriched under low and high nitrate condition as well as one culture from Mature Tailing Coal enriched under moderate nitrate condition were used to access the affect of nitrate on selenium reduction using central composite design matrix. The extent of Se reduction was highest in the Goddard Marsh enrichment with no nitrate while enrichment with moderate and high nitrate reduced selenium poorly. ANOVA results from the CCD experiment in Goddard Marsh enrichment with no nitrate indicated no affect of nitrate in Se reduction. Two primer sets targeting the selenate redutase (serA) from Thauera selenatis and nitrite reductase (nirK) from denitrifying population were used to quantify the population of selenium reducing and denitrifying population in the CCD experiment. Q-PCR assay successfully quantified serA genes in the cultures and correlated well with the initial Se concentration. Furthermore, the selenium reducing ability of enrichment cultures were compared with the bio-stimulated native sediments. Native sediments efficiently removed selenium from the culture medium while enrichment cultures preferentially removed nitrate over selenium. Metagenomic sequencing revealed the presence of many putative selenium reducers in the native sediments while Pseudomonas were more prevalent in the enrichment cultures. Denitrification, sulfate reduction and selenium assimilation genes were abundant in most sequences indicating its role in the reduction of selenium and nitrate. Thus, our study shows that efficient reduction of selenium in the presence of nitrate is possible with biological system. ii Preface This dissertation is an original, independent work of the author, G. Subedi. Materials (Soil Samples) for experiment was kindly sent to us by Dr. Alison Morrison from Teck Coal, except Mount Polley Soil samples which was collected by Dr. Susan Baldwin, Jon Taylor and Myself. The design of experiment as well as experimentation was done by myself while Dr. Susan Baldwin was involved in supervision, and guidance. She has contributed to analysis and interpretation of data as well as largely contributed to editing and revision of chapters in the dissertation. Most of the measurements (including selenium and nitrate) were performed in Civil Engineering Lab located at the University of British Columbia while some measurements of (nitrate and nitrite) were also done in the Baldwin Lab at Chemical and Biological Engineering. Primers (serA, srdBCA) used in Chapter 3 for Q-PCR experiment was designed by Jon Taylor and myself. Sample preparation for Illumina Mi-seq sequencing was done by Jon Taylor and was sent to Genome Quebec (http://gqinnovationcenter.com/index.aspx) for sequencing. Illumina sequences were analyzed in Metapathways software provided freely by the Hallam Lab. iii Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Tables ..................................................................................................................................x List of Figures .............................................................................................................................. xii List of Symbols and Abbreviations ............................................................................................xv List of Units ............................................................................................................................... xvii Acknowledgements .................................................................................................................. xviii Dedication ................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1 Chapter 2: Background and literature review ............................................................................5 2.1 Background of Selenium................................................................................................. 5 2.2 Selenium occurrence in the environment ........................................................................ 6 2.3 Biological assimilation and essentiality of Selenium .................................................... 8 2.4 Bioaccumulation and ecotoxicological impact of Selenium ........................................... 9 2.4.1 Places where Se occurrence has been or still is a problem ......................................... 9 2.5 Water quality guidelines in BC and USEPA ................................................................ 11 2.6 Biological cycling of Selenium ..................................................................................... 11 2.6.1 Selenium oxidation ................................................................................................... 14 2.6.2 Selenium reduction ................................................................................................... 16 2.6.3 Non-dissimilatory Selenium reduction ..................................................................... 21 iv 2.6.4 Se(IV) reduction........................................................................................................ 23 2.7 Mechanisms of Selenium Oxyanion reduction ............................................................. 24 2.7.1 Selenium transport/uptake ........................................................................................ 24 2.7.2 Dissimilatory reduction and selenate reductase ........................................................ 25 2.7.3 Selenite reduction...................................................................................................... 27 2.7.4 Assimilation .............................................................................................................. 27 2.7.5 Methylation ............................................................................................................... 28 2.7.6 Formation of metal selenides .................................................................................... 28 2.8 Treatment technologies available for removing Se from mining water ....................... 28 2.8.1 Physical treatment ..................................................................................................... 30 2.8.1.1 Reverse osmosis and Nanofiltration ................................................................. 30 2.8.1.2 Ion Exchange .................................................................................................... 31 2.8.2 Chemical treatment ................................................................................................... 32 2.8.2.1 Reduction with Zero-valent Iron ....................................................................... 32 2.8.3 Biological treatment .................................................................................................. 32 2.8.3.1 Wetlands and Passive Biochemical Reactors ................................................... 33 2.8.3.2 Active Bioreactors ............................................................................................ 33 2.8.3.2.1 Upflow Anaerobic Sludge Bed (UASB) Bioreactors .................................. 34 2.8.3.2.2 BseR/ABMet technology ............................................................................ 34 2.8.3.2.3 Bioreactors with specific selenate- or selenite-reducing microbes ............. 35 2.9 Summary, motivation and hypotheses .......................................................................... 35 2.10 Hypothesis and motivation for the work ....................................................................... 36 Chapter 3: Materials and method ..............................................................................................38 v 3.1 Background of the sites from where the samples were collected ................................
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