Master of Science
Gloor, Gregory B.
Naphthenic acids (NAs) are the main toxic component of oil refinery wastewater and require special processes to be removed. Harnessing bacterial biodegradation for NA removal has the potential to be effective, yet NA-degrading bacteria and pathways are poorly understood and uncharacterized. To improve our understanding of NA degradation, I characterize the metagenomes of novel NA-degrading bacterial communities seeded in NA-enriched granulated activated carbon (GAC) filters. I demonstrate methods that maximize the throughput of extraction, sequencing, and annotation of novel metagenomes - producing 72 MAGs and other 5432 circular contigs - 226 of which were putative phages. I also include state-of-the-art protein structure prediction and structure homology search tools, which greatly enrich annotations of novel sequences that are below the threshold for homology finding by sequence alone. Overall, these approaches unveiled a diverse and constantly changing consortium of novel bacteria and many potential NA-degrading genes.
Summary for Lay Audience
The amount of toxic wastewater accumulated by the oil industry has increased exponentially since it began its operations, and there is no cost-efficient, sustainable, yet effective way of reclaiming this wastewater. The major challenge is naphthenic acids (NAs), which are a natural byproduct of crude oil refining. NAs are complex, difficult to remove without special processes, and highly toxic. In wastewater treatment, however, there is potential for harnessing bacteria that can degrade NAs. These bacteria have been observed before, but there is no known bacteria or community that degrades the full range of NAs efficiently, and not many NA-degrading genes are known. The study of NA-degrading bacteria and how they remove NA will be foundational to creating future bioengineer wastewater treatment systems. In this thesis, I characterized NA-degrading microbial communities living in granulated activated carbon (GAC) filters from an oil refinery wastewater treatment collected over time. I first demonstrated how to collect DNA from these samples and sequence the DNA using Nanopore – a state-of-the-art DNA sequencing technology. This technology allowed me to develop methods to reconstruct entire bacterial genomes from GAC, and I observed a highly diverse bacterial community that is constantly changing over time and is mostly composed of bacteria never sequenced before. Also, I annotate these bacterial DNA sequences to unveil their biological capabilities and discover several genes likely related to NA degradation. Since these bacteria are novel, however, there were still a lot of uncharacterized DNA sequences that could be important. I tested a new annotation strategy on a small subset of viruses in the GAC community called bacteriophage, where I identified unknown sequences by predicting 3D models of the DNA products, the proteins, and compared them against other known 3D models of proteins. This approach drastically improved the ability to identify what the viruses are biologically capable of since protein structure comparisons are generally more reliable than sequence comparisons when inferring the functions of proteins. Though more work is needed to confirm NA-degrading bacteria and genes, this thesis sets a foundation for future analysis of NA-degrading bacteria and pathways.
Say, Henry H., "State-of-the-art Approaches for Sequencing, Assembling and Annotating Naphthenic Acid Degrading Bacterial Metagenomes" (2023). Electronic Thesis and Dissertation Repository. 9667.