Utilizing “Omics” Based Approaches to Investigate Targeted Microbial Processes By Vanessa Lynn Brisson A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Engineering – Civil and Environmental Engineering in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Lisa Alvarez-Cohen, Chair Professor Kara Nelson Professor Fiona Doyle Spring 2015 Utilizing “Omics” Based Approaches to Investigate Targeted Microbial Processes Copyright © 2015 By Vanessa Lynn Brisson Abstract Utilizing “Omics” Based Approaches to Investigate Targeted Microbial Processes by Vanessa Lynn Brisson Doctor of Philosophy in Engineering – Civil and Environmental Engineering University of California, Berkeley Professor Lisa Alvarez-Cohen, Chair Metabolomic, genomic, and metagenomic analyses were used to provide insight into two different environmentally relevant microbial processes: bioleaching of rare earth elements from monazite sand and reductive dechlorination of chlorinated ethenes. Although rare earth elements are important for a variety of technologies, current extraction techniques are severely environmentally damaging. The research presented here demonstrates that some microorganisms are capable of biological leaching of rare earth elements from monazite, opening the possibility of a novel, environmentally sustainable bioleaching extraction process. Metabolomic analysis of a monazite bioleaching microorganism was used to further our understanding of the bioleaching process. Chlorinated ethenes are common groundwater contaminants with human health risks. Dehalococcoides mccartyi bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, D. mccartyi dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. Genomic and metagenomic analyses were used to advance our understanding of D. mccartyi in a mixed microbial community and in isolation. Successful isolation and characterization of monazite bioleaching microorganisms provided a proof of concept for monazite bioleaching as an environmentally friendly alternative to conventional extraction of rare earth elements from monazite, a rare earth phosphate mineral. Three fungal strains were found to be capable of bioleaching monazite, utilizing the mineral as a phosphate source and releasing rare earth cations into solution. These organisms include one known phosphate solubilizing fungus, Aspergillus niger ATCC 1015, as well as two newly isolated fungi: an Aspergillus terreus strain ML3-1 and a Paecilomyces spp. strain WE3-F. The rare earth elements were released in proportions similar to those present in the monazite, which was dominated by cerium, lanthanum, neodymium, and praseodymium. Although monazite also contains the radioactive element thorium, bioleaching by these fungi preferentially solubilized rare earth elements over thorium, leaving the thorium in the solid residual. Adjustments in growth medium composition improved bioleaching performance measured as rare earth release. Cell-free spent medium generated during growth of A. terreus strain ML3-1 and Paecilomyces spp. strain WE3-F in the presence of monazite retained robust bioleaching capacity, indicating that compounds exogenously released by these organisms contribute substantially to leaching activity. Organic acids released by the organisms were identified and quantified. Abiotic leaching with laboratory prepared solutions of the identified organic acids was not as effective as bioleaching or leaching with cell-free spent medium at releasing rare earths from monazite, 1 indicating that compounds other than the identified organic acids contribute to leaching performance. Metabolomic analysis of a monazite bioleaching microorganism was performed in order to better understand the bioleaching process. Overall metabolite profiling, in combination with biomass accumulation data, identified a lag in growth phase when this organism was grown under phosphate limitation stress. Analysis of the relationships between metabolite concentrations, rare earth element solubilization levels, and bioleaching growth conditions identified several metabolites potentially associated with bioleaching. Further investigation using laboratory prepared solutions of 17 of these metabolites indicated significant leaching contributions from citric and citramalic acids only. These contributions were relatively small compared to bioleaching effectiveness of microbial supernatant, suggesting that other still unknown factors contribute to bioleaching activity. Further investigations of bioleaching supernatant using gel permeation chromatography indicated that the compounds involved in leaching form only weakly held complexes, like those of citric acid, with the solubilized rare earth elements, rather than forming more strongly held complexes. The phylogenetic composition and gene content of a functionally stable trichloroethene degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2,500 bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides, Desulfovibrio, Methanobacterium, Methanospirillum, as well as a Spirochaete, a Synergistete, and an unknown Deltaproteobacterium. D. mccartyi contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2,500 bp. Examination of metagenome sequences affiliated with D. mccartyirevealed 406 genes not found in previously sequenced D. mccartyi genomes, including nine cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a D. mccartyi strain has been found to possess genes for synthesizing this cofactor critical to reductive dechlorination. Besides D. mccartyi, several other members of this community appear to have genes for complete or near-complete cobalamin biosynthesis pathways. Seventeen genes for putative reductive dehalogenases were identified, including 11 novel ones, all associated with D. mccartyi. Genes for hydrogenase components (271 in total) were widespread, highlighting the importance of hydrogen metabolism in this community. PhyloChip microarray analysis confirmed the stability of this microbial community over time. Bioinformatic analyses using genomic and metagenomic data were used to further advance investigations of organisms from the genus Dehalococcoides. In the first of these analyses, metagenomic sequencing data from three dechlorinating microbial communities was used to evaluate the specificity of a genus wide microarray targeting Dehalococcoides genes from four sequenced Dehalococcoides genomes. Based on this analysis, the microarray was found to detect sequences with a minimum estimated sequence identity of 90 to 95%, remaining highly specific for the target sequences while allowing for small sequence variation. However, the microarray did not detect all genes with > 95% sequence identity, and failed to detect some genes with apparently 100% sequence identity. In the second analysis, a comparative genomics analysis was used to evaluate the prevalence of the recently reported incomplete Wood- Ljungdahl pathway of Dehalococcoides. This analysis revealed that the genetic pattern of genes 2 associated with this incomplete pathway is unique to the Dehalococcoides genus among sequenced bacterial and archaeal genomes. 3 Dedication To Gabriel, Abigail, and Madeline i Table of Contents Abstract............................................................................................................................................1 Dedication.........................................................................................................................................i Table of Contents.............................................................................................................................ii List of Figures.................................................................................................................................vi List of Tables................................................................................................................................viii Acknowledgements.........................................................................................................................ix Chapter 1: Introduction and Background...................................................................................1 1.1 “Omics” techniques...................................................................................................................2 1.2 Targeted microbial processes.....................................................................................................3 1.2.1 Bioleaching of rare earth elements from monazite...........................................................3 1.2.2 Microbial reductive dehalogenation of chlorinated ethenes.............................................7 1.3 Dissertation overview................................................................................................................9 Chapter 2: Bioleaching of Rare Earth Elements from Monazite Sand..................................11 2.1 Introduction..............................................................................................................................12