Evaluating the impact of ecological growth strategies of bacteria on plasmid transfer and function for bioremediation of polycyclic aromatic hydrocarbons
Abstract
Remediation of contaminated soils and sediments traditionally involves physical methods that are environmentally intrusive and expensive. Bioremediation is a more environmentally friendly, cost-effective treatment strategy that utilizes naturally occurring indigenous and/or exogenous microorganisms in combination with physico-chemical stimulation to promote degradation of contaminants. However, the predominant challenge of bioremediation that involves adding exogenous microorganisms is the lack of long-term survival of augmented organisms due to their lack of adaptation to environmental conditions often found at contaminated sites. Genetic bioaugmentation aims to overcome this challenge by introducing relevant genes from exogenous bacteria into indigenous microbes adapted to the polluted environment utilizing conjugation, or plasmid transfer, to increase overall contaminant biodegradation. Genetic bioaugmentation relies on many processes yet to be fully understood including how growth strategies of donors and recipients affect plasmid transfer and the functionality of their degradative properties. Additionally, methods for tracking plasmid transfer in situ are lacking, which contributes to the lack of knowledge surrounding properties affecting genetic bioaugmentation as a sustainable remediation approach. The overarching goals of this dissertation were to first develop methods for in situ quantification of conjugation of plasmids capable of degrading polycyclic aromatic hydrocarbons (PAHs), which could then be used to investigate the effect of bacterial growth strategies on conjugation and PAH degradation in both simple, synthetic communities and complex, natural sediment communities. Two plasmids, pNL1 and NAH7, were used to track conjugation and PAH degradation due to their ability to degrade naphthalene and other PAHs, their similarity in transfer mechanisms, and the fact that they are well-characterized plasmids. The first objective of this dissertation was to develop methods for detecting conjugation of PAH-degrading plasmids that could be applied in situ without the need for culturing. To achieve this, plasmids and donor chromosomes were genetically modified to develop fluorescence-based methods for conjugation detection. This method was then used to investigate the effect of growth strategies, GC content, and phylogenetic relatedness on conjugation in simple communities of one donor and one recipient. We observed significant correlations between all three parameters and, specifically, more conjugation into slow growing K-strategists compared to their fast-growing r-strategist counterparts. All three parameters affected conjugation of the PAH-degradative plasmids. The work stemming from this first objective led to the development of a novel method for monitoring conjugation, and, more broadly, those results provided insights into biological factors that affect conjugation and therefore, successful genetic bioaugmentation. The second objective was to assess the impact of growth strategy on conjugation and naphthalene degradation in a simple bacterial community. Conjugation of two PAH-degrading plasmids into recipients with mixed growth strategies and subsequential degradation of naphthalene in respective communities was assessed using methods developed in the first objective. There were clear conjugation preferences into slow-growing K-strategists for the pNL1 plasmid but no clear preferences for the NAH7 plasmid. However, communities of K-strategists consistently had more naphthalene degradation than their r-strategist counterparts. These findings provide insight into K-strategists being ideal targets for genetic bioaugmentation in simple communities, but further needed to be translated to complex communities. The final objective was to determine if the findings in simple communities translated over to complex, natural sediment communities, thus providing insights into the field implementation of genetic bioaugmentation. Plasmid donors were added to bioreactors with PAH-contaminated sediment containing a natural microbial community. The bioreactors were biostimulated differentially to promote growth of microbes with varying growth strategies and both conjugation and PAH degradation were quantified. Overall, the growth strategies of transconjugants were most closely associated with effects on conjugation and persistence of transconjugants. The highest amount of conjugation occurred during the latter experimental stages when transconjugants were more representative of slow-growing K-strategists. Although the impact of growth strategy on PAH biodegradation was unclear within the experimental time frame, differential nutrient biostimulation was found to be a successful approach for promoting differences in transconjugant growth strategies, providing some insights for developing a genetic bioaugmentation framework. Overall, the work in this dissertation suggests that bacterial growth strategy can be stimulated by differential nutrient biostimulation, and that slow growing K-strategists are optimal targets for genetic bioaugmentation. This dissertation provides the beginning of a framework for identifying favorable conditions needed to promote successful implementation of genetic bioaugmentation targeted to the existing recipient community present at a PAH contaminated site. This fills a critical research gap for the translation of genetic bioaugmentation into field application of precision microbiome engineering.
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Varner, Paige Marie (2022). Evaluating the impact of ecological growth strategies of bacteria on plasmid transfer and function for bioremediation of polycyclic aromatic hydrocarbons. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/26820.
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