Dissertation
A taxonomic, functional, and expression level analysis of organohalide respiring bacteria and auxiliary microbial communities native to PCB contaminated sediments
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2021
DOI: 10.17077/etd.006041
Abstract
Although industrial production of Polychlorinated biphenyls (PCBs) ceased decades ago, these recalcitrant and toxic compounds still contaminate US Superfund sites and represent a serious risk to human and environmental health. One promising strategy to remediate PCB-contaminated sediments utilizes organohalide-respiring bacteria (OHRB) that dechlorinate PCBs. To transform PCBs, OHRB possess genes, known as rdhA genes, that encode for the enzymes which catalyze the dechlorination reaction. These enzymes are known as reductive dehalogenases, and remain unexplored in PCB contaminated sediments despite their utility in bioremediation projects that seek to harness the power of microbially catalyzed transformations to detoxify contaminated sediments. Here, I aimed to identify the PCB dechlorinators, reductive dehalogenase genes, and key microbial community members within sediment mixed microbial communities under environmentally relevant conditions. The first sets of experiments sought to explore the abundance of OHRB, phylogenetic lineage of naturally occurring rdhA genes, and extent of natural attenuation that occurred in PCB contaminated sediments collected from a field site in Altavista, VA. Total PCB concentrations in sediments ranged from 6.34 to 12,700 mg/kg and PCB congener profiles in sediment sample were similar to the PCB mixture Aroclor 1248 among the 27 sediment samples collected. Additionally, congener profiles at several locations showed enrichment of lower chlorinated and ortho-chlorinated congeners enriched relative to the likely contaminating mixture, Aroclor 1248. The sediment microbial community structure varied among samples but was dominated by Proteobacteria and Firmicutes. The relative abundance of putative dechlorinating Chloroflexi (which includes known PCB dechlorinators within the genera Dehalococcoides) comprised 0.01–0.19% of the microbial community in the sediment samples, with Dehalococcoides spp. representing 0.6–14.8% of this group. For this work, I conducted a PCR survey for rdhA genes of all known phylogenetic lineages and found 11 sequences related to rdhA genes in a PCB-respiring Dehalococcoides mccartyi strain CG5 and a PCB-dechlorinating Dehalococcoides mccartyi strain CBDB1.
After the first study revealed the occurrence of rdhA genes in PCB contaminated sediments, I developed a microcosm study aimed to determine if the microbial community native to the field site contaminated sediments could dechlorinate PCBs and then identified the PCB dechlorinators. Additionally, because functional genes that act as biomarkers for PCB dechlorination remain elusive, I aimed to isolate partial rdhA sequences and determine their phylogenetic lineage. I developed anaerobic sediment microcosms and found that the genus Dehalococcoides dominated the community of potential PCB degraders. Over the 430-day microcosm incubation, Dehalococcoides 16S rRNA sequences increased two orders of magnitude to 107 copies/g of sediment, and at the same time, PCB118 decreased by as much as 70%. In addition, the OHRB community dechlorinated a range of penta- and tetra-chlorinated PCB congeners including PCBs 66, 70 + 74 + 76, 95, 90 + 101, and PCB110 without exogenous electron donor. I quantified candidate rdhA genes over a 430-day incubation period and found rd14, a reductive dehalogenase gene first identified in the genome of Dehalococcoides mccartyi strain CG5, was enriched to 107 copies/g of sediment. At the same time, I found that a previously suggested PCB dechlorination biomarker gene, pcbA5, was enriched to only 105 copies/g of sediment. A survey for additional rdhA genes revealed sequences similar to strain CG5’s rd4 and rd8. In addition to demonstrating the PCB dechlorination potential of native microbial communities in contaminated freshwater sediments, the results suggested candidate functional genes with previously unexplored potential could serve as biomarkers of PCB dechlorination processes.
Once I identified Dehalococcoides that harbor the rdhA gene rd14 as the genera likely responsible for PCB dechlorination in sediment microcosms, I aimed to characterize the metagenome of the microbial community native to the field site and further explore the phylogenetic lineage of naturally occurring rdhA genes. Additionally, because OHRB require nutrients and cofactors from auxiliary members of the microbiome to survive, and bioremediation utilizes the complexities of microbial communities to drive PCB degradation, I aimed to compare the metagenome and metranscriptomes of microbial communities that supported OHRB in PCB contaminated sediments and to gain insight into possible community interactions. To accomplish those aims, I sequenced the metagenomes of two relatively unenriched microbial communities that originated from PCB contaminated sediments that differed in both PCB concentration and profile. A combined read and assembly-based approach to metagenome analysis revealed similarities in community structure and generated 63 high quality draft genomes that represent the families Methanomicrobiales, Desulfobacteraceae, Synergistaceae, Xanthomonadaceae, Planctomycetaceae, Nitrospiraceae, Porphyromonadaceae, and Nocardiaceae. Additionally, I assembled and annotated a Dehalococcoides draft genome that contains 24 rdhA, seven of which diverge from previously identified ortholog groups, and confirmed the presence of a previously proposed biomarker gene known as rd14. Further, I found that the metagenome assembled genome of Dehalococcoides did not harbor rdhA sequences related to genes pcbA1, pcbA4, and pcbA5.
A differential expression analysis of the metatranscriptomes revealed that expression of 6220 genes was significantly different between the two microcosm conditions. I found that transcripts involved in production of cobamide, a cofactor essential factor Dehalococcoides survival, belonged to members of the phylum Euryarchaeota, Firmicutes, Chloroflexi, and Proteobacteria. Further, expression of genes involved in cobamide production were significantly more abundant in the microcosms with greater PCB contamination, which suggests the importance of cobamide production as a critical interaction to facilitate Dehalococcoides growth in PCB contaminated sediments. Finally, to foster reproducibility efforts I utilized version control, package management, and literate programming in the read and assembly based mixed ‘omics analysis. Along the same lines, I plan to deposit all data generated in the ‘omics study into public repositories and publicly release scripts, code, and guidance documents that will allow others to reproduce the ‘omics data analysis workflow.
In conclusion, the work presented here provides valuable insights to the field of OHRB catalyzed PCB bioremediation. Specifically, I provided the first analysis that identified naturally occurring rdhA genes in PCB contaminated sediments, and ultimately linked those genes to Dehalococcoides spp. that harbor seven rdhA genes that belong to novel ortholog groups. Further, I demonstrated that the PCB specific biomarker genes identified in pure cultures, pcbA1, pcbA4, and pcbA5, are less abundant in environmental samples than rd14, which may serve as a more predictive biomarker gene to indicate PCB dechlorination potential. I also identified members of the Euryarchaeota, Firmicutes, Chloroflexi, and Proteobacteria phylums as potential mediators of Dehalococcoides growth in PCB contaminated sediments through interactions that provide Dehalococcoides with cobamide cofactors. Altogether, these results provide a most thorough analysis of microbial communities in PCB contaminated sediments to date.
Details
- Title: Subtitle
- A taxonomic, functional, and expression level analysis of organohalide respiring bacteria and auxiliary microbial communities native to PCB contaminated sediments
- Creators
- Jessica M Ewald
- Contributors
- Timothy Mattes (Advisor)Keri Hornbuckle (Committee Member)Jerald Schnoor (Advisor)Andres Martinez (Committee Member)Gregory LeFevre (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Civil and Environmental Engineering
- Date degree season
- Summer 2021
- DOI
- 10.17077/etd.006041
- Publisher
- University of Iowa
- Number of pages
- xxii, 238 pages
- Copyright
- Copyright 2021 Jessica M Ewald
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references.
- Public Abstract (ETD)
Polychlorinated biphenyls (PCBs) are man-made chemicals banned by the EPA in 1979 for their toxicity, persistence in the environment, and tendency to accumulate in animal tissue. Initially produced for industrial applications, PCBs are highly stable and resist degradation. Improper disposal or accidental release of these compounds into surface waters occurred frequently before the ban. As a result, PCBs persist in aquatic sediments and remain contaminants of concern at 30% of sites designated by the US government for hazardous waste clean up. To avoid human PCB exposure and restore environmental health at these sites, we must develop a cost effective strategy to break down PCBs. Common strategies used presently to detoxify PCB contaminated sites include dredging. Dredging physically removes the contaminated sediments, and as a result it disrupts the sites ecological systems, increases PCB concentrations in surrounding air and water, and cost millions of dollars to implement. For these reasons, a need exists for alternative strategies to clean up PCBs in sediments.
One promising alternative to dredging utilizes a particular group of anaerobic bacteria that remove chlorine atoms from the PCB molecule, and therefore render the molecule more amenable to degradation by other bacterial groups. These unique anaerobes are known as organohalide respiring bacteria (OHRB). OHRB have been successfully used to clean up a variety of man-made chemicals with minimal ecological disruption. Typically, the ability of OHRB to transform a given contaminant is assessed by measuring known gene sequences. However, the OHRB genes required to transform PCBs remain elusive, and therefore limit our ability to use them as a tool to clean up PCB contaminated sediments. Here, I aimed to identify those valuable gene sequences at a PCB contaminated site through the application of several microbiological tools like quantitative polymerase chain reaction and high throughput sequencing. I combined that analysis with analytical methods to measure PCBs in the sediments. Additionally, I grew OHRB collected from a PCB contaminated site in a laboratory and studied patterns in their gene sequences. The same technology that sequenced the human genome allow us to sequence bacterial DNA and identify genes required to transform PCBs.
In addition to identifying the genes that OHRB possess to grown in PCB contaminated sediments, this research assessed the identify and function of microbes that may support OHRB growth. Bacteria are like people; in that they rely on members of their community for resources and support. If the auxiliary community members, i.e. non-OHRB, present at a contaminated site cannot support OHRB growth, then PCB transformation will not proceed. To assess interactions between community members that encourage OHRB to transform PCBs, I analyzed high through put sequencing data that reflected the entire microbial communities’ identity and function. In addition to the analyzing the non-OHRB community, I assembled fragments of DNA sequences into longer contiguous sequences that represent a draft genome of a previously unknown OHRB that belongs to the bacterial group Dehalococcoides mccartyi. From those contiguous sequences, I applied techniques that predict the function of genes and determined that 24 genes relevant to bioremediation and learned that six of the sequences belong to previously unclassified groups. These gene sequences, when measured with microbiological tools, provide tools to predict if a microbial community will effectively transform PCBs in a contaminated environment.
Ultimately, my research focused on identifying the genes and communities of microorganisms that OHRB rely on to transform hazardous man-made PCBs. With the knowledge generated by my research, these specialized OHRB can be more effectively utilized to clean up PCB contaminated sediments and reduce the risk that these compounds pose to humans and environmental health.
- Academic Unit
- Civil and Environmental Engineering
- Record Identifier
- 9984124360502771
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