Browsing by Subject "Bioremediation"

This dissertation presents a framework to improve bioremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs). PAHs are of great concern because they are recalcitrant and toxic. PAHs enter the environment from a variety of sources such as incomplete combustion and coal tar distillation. The PAHs focused on in this dissertation have polluted soils as a result of creosote-based wood treatment operations that took place at Holcomb Creosote and Atlantic Wood Industries, Inc. (AWI) both of which are now classified as Superfund sites. There are numerous sites analogous to these two Superfund sites throughout the world which have been polluted through similar wood-treatment operations, as creosote was once industry’s foremost wood preservative.

There is room for existing PAH treatment options, which are mainly physical and chemical in nature, to be expanded to include more sustainable options. Commonly used technologies include excavation, in situ stabilization, and soil washing. Historically, bioremediation strategies relying on bacteria to transform pollutants have been challenged by the tight sorption of heavy- and middle-weight PAHs to soils, as this restricts aqueous phase transport required for bacterial degradation. Multiple studies have demonstrated fungi to be capable of degrading these inaccessible pollutants and other mixtures of hydrophobic pollutants (mycoremediation). Yet, when fungi have been introduced to polluted soils (mycoaugmentation), they have not been able to outcompete the native microbiota long enough to degrade the contaminants of concern over the long term. It is possible that a thorough characterization of the indigenous fungi at a given site may provide some insights into the development of targeted in situ mycoremediation strategies.

Although incorporating site microbes has been generally acknowledged as important for some time, the techniques enabling thorough assessment of microbial ecosystems are relatively new. Consequently, little is known about PAH-associated microbiomes in general, and even less is known about PAH-associated fungal communities. The work presented in this dissertation aims to address this knowledge gap by leveraging recent advances in high-throughput sequencing technology to design and validate targeted mycoremediation strategies. To this end, the overarching goal of this dissertation was to develop and test a framework for incorporating native fungi into a bioremediation strategy to expand such sustainable remediation options to sites where they have not been relevant in the past.

In the first aim of this dissertation research, advances in high-throughput sequencing were used to identify potential biostimulation targets in soils moderately polluted with PAHs. The next generation sequencing (NGS) platform, Illumina, was utilized to sequence the large sub-unit (LSU) gene commonly used as a marker gene in fungal community studies. Relationships were examined between concentrations of over 31 different polycyclic aromatic hydrocarbons and the pollutant-associated communities to test whether there were any fungi capable of tolerating high levels of these toxic contaminants. In this aim, fungal genera were identified that contained species closely related to known PAHs and petroleum hydrocarbon degraders. In all, this work identified 32 targets for biostimulation, based on Spearman rank correlations between prevalence and mid- and high-molecular weight PAHs. Ascomycetes were found to have higher levels of diversity than any other phylum in this subset of biostimulation targets. These data suggest that ascomycete fungi are more likely to be present in heavily polluted soils than basidiomycete fungi (which had previously been subjects of much interest). Overall, this work illustrates that polluted soils harbor fungal biostimulation targets, specifically within Ascomycota.

The second aim of this thesis research was to use the precision bioremediation assessment in highly polluted soils and then to evaluate a range of amendments with the goal of identifying strategies to stimulate the fungal communities that dominate these PAH-associated fungal communities. Here we applied the approach we fine-tuned in the first aim to the AWI soils, as these soils have some of the highest documented PAH-concentrations. Again, Ascomycota were found to be more prevalent in these soils, so an isolate obtained from AWI was used to compare alternative stimulation techniques between three substrates they are known to grow on: chitin, cellulose, and wood. We used anthracene degradation as a proxy for PAH degradation, which we monitored in sacrificial simplified bioreactors responding to the three amendments. T. harzianum is also known to have enzymes which degrade PAHs, but it is unknown which ecological role uses those enzymes, and thus which ecological role we should promote. T. harzianum was grown in the presence of chitin, cellulose, and wood as substrates in liquid culture with anthracene. Chitin was found to stimulate the highest anthracene removal, with a 0.1% (w/v) amendment resulting in ~93% degradation. While ~13% less than chitin, 1% (w/v) cellulose was also found to stimulate ~46% more anthracene degradation than wood, which had no improvement over the abiotic losses (~33% on average). This is notable because the “go to” method for stimulating fungi in the past has been wood supplements. This work provided insight into alternative stimulation strategies to target specific ecological roles that may better degrade PAHs in situ.

For the third and final aim of this dissertation research, the two most promising amendments were added with and without Trichoderma harzianum spores to test several mycoremediation treatment strategies in soil bioreactors and compare them with a (no carbon added) nutrient stimulation treatment. Pollutants were added as aged Atlantic Wood Industries soil delivering aged pollutants. Triplicate reactors from each treatment were sequenced at time zero, after two weeks, and after one month. At each sampling time, RNA was extracted, converted to cDNA, and submitted to Illumina MiSeq library preparation targeting the LSU region for fungal community analysis in addition to the V4 region of the 16S rDNA for bacterial community analysis. Statistical analyses using DESeq2 identified responders among the groups of reactors subjected to the different biostimulation treatments. Taxa from both the fungal and the bacterial communities responded differentially to the amendments. Fungi were found to comprise the majority of the significant responders. This work also found that mycoaugmented strains were not successful in establishing themselves as prominent members of the active community. This represents one of the earliest studies to directly measure mycoaugmentation failure. These data propose a hypothesis about functional redundancy inhibiting establishment of augmented fungi as already established fungi outcompete them for freshly added nutrients. Over 90% degradation was observed over the course of one month regardless of treatment-interestingly, the highest degradation was found in the nutrient amendment (no carbon added) treatment. These results show similar degradation across the soil bioreactors, yet different microbial growth, which supports the hypothesis that there is community-level functional redundancy and multiple metabolic food webs that result in the observed pollutant degradation.

Overall, this dissertation work demonstrates how significant advances in sequencing technology can be implemented in design and monitoring stages of bioremediation. This work also suggests that significant advances could be possible through the application of targeted metatranscriptomic analysis. Through incorporating such insights as described in this dissertation, this research brings the field of bioremediation one step closer to successfully engineering microbiomes to degrade contaminants of concern.

Bioremediation is a treatment strategy that involves the removal of chemical pollutants using biological agents. When compared to physico-chemical treatment approaches, bioremediation causes less site disturbance and offers a range of other economic and ecological benefits. Yet, this treatment approach is not routinely selected mainly because of the unpredictability of the biological agents in the heterogeneous environments encountered at sites. Generally, three main approaches are utilized to improve bioremediation efficacy. These approaches consist of: 1) allowing native bacteria to degrade the pollutant (bioattenuation); 2) stimulating indigenous bacteria to improve their natural degradative capacity (biostimulation); or 3) supplying exogenous microorganisms that are known to degrade a specific contaminant if no known degraders are present at the site (bioaugmentation). Genetic bioaugmentation is another bioremediation treatment strategy, which relies on the well-studied mechanism of horizontal gene transfer (HGT) in which plasmids from exogenous donors are transferred to indigenous recipients. This strategy circumvents the need for the exogenous strain to compete with indigenous microorganisms under site conditions, a challenge that is difficult to overcome. HGT is a widespread natural phenomenon that readily occurs especially under harsh environmental conditions such as that in heavily contaminated environments.

Although all of these approaches show promise, in general, little is known about how organisms assemble in contaminated environments, limiting the implementation of bioremediation treatment strategies under field conditions. The work presented in this dissertation aims to address this challenge by characterizing and engineering prokaryotic microbiomes in soils contaminated with polycyclic aromatic hydrocarbons (PAHs). The overarching goals of this dissertation were to first utilize next-generation sequencing (NGS) to characterize pollutant-exposed environmental microbiomes and then use these data to develop a generalizable framework, which combines metagenomic and physico-chemical data to inform bioremediation treatment strategy selection. Then, in order to effectively validate this framework, a method for monitoring catabolic plasmid conjugation was developed. This dissertation was broadly broken down into four objectives as described below.

The first objective was to investigate environmentally relevant adaptation events in sediment microbial communities and gut-associated microbial communities in Atlantic killifish exposed to PAHs. Sediment and gut samples were obtained from the Republic Creosote Co. site located along the Elizabeth River (ER) and the Kings Creek (KC) reference site and their microbiomes were comparatively analyzed using high-throughput sequencing. In addition, because it is known gut microbes regulate host metabolism, the gut-associated metabolome was also characterized. Overall, significant community shifts were identified between sites, suggesting an environmental microbiome evolved to withstand high levels of PAHs. Specifically, of the OTUs identified, 9 species were different between KC guts and KC sediment while 176 were different between Republic guts and Republic sediment. These data suggest that factors other than dietary influence affect the microbiota colonized in the Republic fish gut. With respect to the metabolome, the amino acid (AA) concentrations were found to be higher for 19 out of 21 AAs in the Kings Creek samples when compared to the Republic samples. This indicates both a potential consequence of the microbial shifts and impact on metabolism between in the PAH-exposed fish sub-populations. Overall, this work provides insight into chemical-associated microbial community and metabolomics shifts and some of the potential resulting impacts.

The second objective was to develop a framework for precision bioremediation in which optimal microbial taxa could be identified for biostimulation, bioaugmentation and genetic bioaugmentation at a given site. Here, we developed an approach that combined Illumina Miseq high throughput sequencing, chemical profiling and Spearman correlation analyses. This framework was developed using samples obtained from the Holcomb Creosote Co. Superfund site, which is contaminated with both PAHs and heavy metals. Using this framework, Geobacter was identified as a biostimulation target while Mycobacterium and Sphingomonas were identified as strong potential biostimulation and genetic bioaugmentation targets, respectively. Based on availability and sequencing data, a consortium of Mycobacterium fredericksbergense, Sphingomonas aromaticivorans F199 (containing the pNL1 catabolic plasmid) was selected as a possible bioaugmentation cocktail to treat the Holcomb Creosote Superfund soils for PAHs. Though it is possible to identify prospective bioremediation targets using this methodology, this approach remains limited mainly due to the inadequate amount of fully sequenced site-specific environmental strains and catabolic plasmids available in databases. Additional research is needed especially using shotgun metagenomic sequencing, rather than amplicon-based sequencing, to increase the availability of environmental microbiome data and thereby improve the identification of potential donor strains and catabolic plasmids.

The third objective consisted of developing a method to effectively monitor genetic bioaugmentation and validate the method in a series of lab scale precision bioremediation scenarios. Previous methods used to monitor for HGT events have relied on using either fluorescent labeling or culturing. However, these monitoring techniques have been shown to be ineffective in complex matrices and for large-scale field applications. Herein, qPCR probes were designed to effectively monitor plasmid conjugation in complex matrices on a large scale.

In the final inclusive objective, the microbial cocktail formed by the framework developed in Objective 2 and the monitoring technique developed in Objective 3 were validated in lab scale reactors using a realistic PAH-contaminated soil matrix obtained from the Holcomb Creosote Co. Superfund site. Overall, it was found that the targeted microbial consortium was able to improve bioremediation by constructing an engineered environmental microbiome capable of increasing the rate of PAH biodegradation with a minimal long-term impact on the communities. In particular, there were overall increases in the class Bacilli and decreases in Betaproteobacteria sustained over time. In addition, the efforts to monitor genetic bioaugmentation were successful and HGT through plasmid conjugation was quantified. Specifically, the probes were able to detect conjugation of the NAH7 plasmid immediately following genetic bioaugmentation. Not surprisingly, the Inc-P9 plasmid was not maintained in the community, as it did not provide a strong enough consistent benefit towards survival to justify the metabolic load. The probes were also successful in quantifying the pNL1 plasmid, though no sustained HGT events were detected.

Overall, this dissertation provides significant advancements to the field of precision bioremediation. In particular, this dissertation work begins to integrate metagenomic and chemical measurements using statistical methods to effectively identify bioremediation targets and providing tools to monitor bioremediation progress under field relevant conditions. It is anticipated that as environmental microbiome databases continue to be populated, the use of frameworks such as that outlined in this dissertation work will be instrumental for the identification of targeted bioremediation treatment strategies.