Browsing by Author "Gunsch, Claudia K"

A multilevel high-throughput sequencing approach for identifying microbial community interactions and informing precision microbiome engineering

The 2010 Deepwater Horizon disaster remains one of the largest oil spills in history. This event caused significant damage to coastal ecosystems, the full extent of which has yet to be fully determined. Crude oil contains both toxic substances that are detrimental to microbes and compounds that may be used as food and energy resources by some microbial species. As a result, oil spills have the potential to cause significant shifts in microbial communities. In this study, we assessed the impact of oil contamination on the function of endophytic microbial communities associated with saltmarsh cordgrass (Spartina alterniflora). Soil samples were collected from two locations in coastal Louisiana, USA: one severely affected by contamination from the Deepwater Horizon oil spill and one relatively unaffected location. Spartina alterniflora seedlings were grown in both soil samples under greenhouse conditions, and GeoChip 5.0 was used to evaluate the endophytic microbial metatranscriptome shifts in response to host oil exposure. Microbial functional shifts were detected in functional categories related to metal homeostasis, organic remediation, and phosphorus utilization. These findings show that host oil exposure elicits multiple changes in metabolic response from their endophytic microbial communities, producing effects that may have the potential to impact host plant fitness.

The growth and proliferation of invasive bacteria in engineered systems is an ongoing problem. While there are a variety of physical and chemical processes to remove and inactivate bacterial pathogens, there are many situations in which these tools are no longer effective or appropriate for the treatment of a microbial target. For example, certain strains of bacteria are becoming resistant to commonly used disinfectants, such as chlorine and UV. Additionally, the overuse of antibiotics has contributed to the spread of antibiotic resistance, and there is concern that wastewater treatment processes are contributing to the spread of antibiotic resistant bacteria.

Due to the continually evolving nature of bacteria, it is difficult to develop methods for universal bacterial control in a wide range of engineered systems, as many of our treatment processes are static in nature. Still, invasive bacteria are present in many natural and engineered systems, where the application of broad acting disinfectants is impractical, because their use may inhibit the original desired bioprocesses. Therefore, to better control the growth of treatment resistant bacteria and to address limitations with the current disinfection processes, novel tools that are both specific and adaptable need to be developed and characterized.

In this dissertation, two possible biological disinfection processes were investigated for use in controlling invasive bacteria in engineered systems. First, antisense gene silencing, which is the specific use of oligonucleotides to silence gene expression, was investigated. This work was followed by the investigation of bacteriophages (phages), which are viruses that are specific to bacteria, in engineered systems.


For the antisense gene silencing work, a computational approach was used to quantify the number of off-targets and to determine the effects of off-targets in prokaryotic organisms. For the organisms of Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv the mean number of off-targets was found to be 15.0 + 13.2 and 38.2 + 61.4, respectively, which results in a reduction of greater than 90% of the effective oligonucleotide concentration. It was also demonstrated that there was a high variability in the number of off-targets over the length of a gene, but that on average, there was no general gene location that could be targeted to reduce off-targets. Therefore, this analysis needs to be performed for each gene in question. It was also demonstrated that the thermodynamic binding energy between the oligonucleotide and the mRNA accounted for 83% of the variation in the silencing efficiency, compared to the number of off-targets, which explained 43% of the variance of the silencing efficiency. This suggests that optimizing thermodynamic parameters must be prioritized over minimizing the number of off-targets. In conclusion for the antisense work, these results suggest that off-target hybrids can account for a greater than 90% reduction in the concentration of the silencing oligonucleotides, and that the effective concentration can be increased through the rational design of silencing targets by minimizing off-target hybrids.

Regarding the work with phages, the disinfection rates of bacteria in the presence of phages was determined. The disinfection rates of E. coli K12 MG1655 in the presence of coliphage Ec2 ranged up to 2 h-1, and were dependent on both the initial phage and bacterial concentrations. Increasing initial phage concentrations resulted in increasing disinfection rates, and generally, increasing initial bacterial concentrations resulted in increasing disinfection rates. However, disinfection rates were found to plateau at higher bacterial and phage concentrations. A multiple linear regression model was used to predict the disinfection rates as a function of the initial phage and bacterial concentrations, and this model was able to explain 93% of the variance in the disinfection rates. The disinfection rates were also modeled with a particle aggregation model. The results from these model simulations suggested that at lower phage and bacterial concentrations there are not enough collisions to support active disinfection rates, which therefore, limits the conditions and systems where phage based bacterial disinfection is possible. Additionally, the particle aggregation model over predicted the disinfection rates at higher phage and bacterial concentrations of 108 PFU/mL and 108 CFU/mL, suggesting other interactions were occurring at these higher concentrations. Overall, this work highlights the need for the development of alternative models to more accurately describe the dynamics of this system at a variety of phage and bacterial concentrations. Finally, the minimum required hydraulic residence time was calculated for a continuous stirred-tank reactor and a plug flow reactor (PFR) as a function of both the initial phage and bacterial concentrations, which suggested that phage treatment in a PFR is theoretically possible.

In addition to determining disinfection rates, the long-term bacterial growth inhibition potential was determined for a variety of phages with both Gram-negative and Gram-positive bacteria. It was determined, that on average, phages can be used to inhibit bacterial growth for up to 24 h, and that this effect was concentration dependent for various phages at specific time points. Additionally, it was found that a phage cocktail was no more effective at inhibiting bacterial growth over the long-term than the best performing phage in isolation.

Finally, for an industrial application, the use of phages to inhibit invasive Lactobacilli in ethanol fermentations was investigated. It was demonstrated that phage 8014-B2 can achieve a greater than 3-log inactivation of Lactobacillus plantarum during a 48 h fermentation. Additionally, it was shown that phages can be used to protect final product yields and maintain yeast viability. Through modeling the fermentation system with differential equations it was determined that there was a 10 h window in the beginning of the fermentation run, where the addition of phages can be used to protect final product yields, and after 20 h no additional benefit of the phage addition was observed.

In conclusion, this dissertation improved the current methods for designing antisense gene silencing targets for prokaryotic organisms, and characterized phages from an engineering perspective. First, the current design strategy for antisense targets in prokaryotic organisms was improved through the development of an algorithm that minimized the number of off-targets. For the phage work, a framework was developed to predict the disinfection rates in terms of the initial phage and bacterial concentrations. In addition, the long-term bacterial growth inhibition potential of multiple phages was determined for several bacteria. In regard to the phage application, phages were shown to protect both final product yields and yeast concentrations during fermentation. Taken together, this work suggests that the rational design of phage treatment is possible and further work is needed to expand on this foundation.

Silver nanoparticles (AgNPs) are increasingly being integrated into a wide range of consumer products, such as air filters, washing machines, and textiles, due to their antimicrobial properties [1]. However, despite the beneficial applications of AgNPs into consumer products, it is likely that their use will facilitate the release of AgNPs into wastewater treatment plants, thereby possibly negatively impacting key microorganisms involved in nutrient removal. For this reason, it is important to characterize the effects of AgNPs in natural and engineered systems and to measure the antimicrobial effect of AgNPs on wastewater microorganisms. Polyvinyl alcohol coated AgNPs have already been linked to decreased nitrifying activity [2] and it is important to determine if AgNPs coated with other materials follow similar trends. Furthermore, it is likely that, with repeated exposure to AgNPs microbial communities could evolve and develop resistance to silver. Thus, a long-term effect of silver nanoparticle exposure could be a reduction of the efficacy of such products in a similar fashion to the development of microbial antibiotic resistance [3]. Therefore, it is critical that the impacts of these materials be ascertained in wastewater treatment systems to prevent long-term negative effects.

The objectives of this dissertation were to: 1) characterize the effect of several different AgNPs on the ammonia oxidizing bacterium (AOB) Nitrosomonas europaea and investigate possible mechanisms for toxicity, 2) test the effects of consumer product AgNPs on a wide range of heterotrophic bacteria, 3) evaluate the effects of AgNPs on bench scale wastewater sequencing batch reactors, and lastly 4) assess the impacts on microbial communities that are applied with AgNP spiked wastewater biosolids.

First, Nitrosomonas europaea was was selected because wastewater nitrifying microorganisms carry out the first step in nitrification and are known to be sensitive to a wide range of toxicants [4].The antimicrobial effects of AgNPs on the AOB N. europaea were measured by comparing nitrite production rates in a dose response assay and analyzing cell viability using the LIVE/DEAD® fluorescent staining assay. AgNP toxicity to N. europaea appeared to be largely nanoparticle coating dependent. While PVP coated AgNPs have shown reductions up to 15% in nitrite production at 20 ppm, other AgNPs such as gum arabic (GA) coated showed the same level of inhibition at concentrations of 2 ppm. The first mechanism of inhibition appears to be a post-transcriptional interference of AMO/HAO by either dissolved Ag or ROS, in treatments where membranes are not completely disrupted but nitrite production decreased (2 ppm GA AgNP and 2 ppm PVP AgNP treatments). The disruption of nitrification is dependent on AgNP characteristics, such as zeta potential and coating, which will dictate how fast the AgNP will release Ag+ and ROS production Finally, total membrane loss and release of internal cellular matter occur.

In order to test the effects of AgNP products available to consumers, simple bacterial toxicity tests were carried out on well-studied heterotrophic bacteria. A model gram-positive and gram-negative bacterium (B. subtilis and E. coli, respectively) was selected to assess any differences in sensitivity that may occur with the exposure to AgNPs. A third model gram-negative bacterium (P. aeruginosa) was chosen for its biofilm forming capabilities. In addition to testing pure nanoparticles, three silver supplements meant for ingestion, were randomly chosen to test with these three bacteria. Growth curve assays and LIVE/DEAD staining indicate that the consumer product AgNPs had the most significant inhibition on growth rates, but not membrane integrity. Overall, P. aeruginosa was most negatively affected by all AgNPs with nearly 100% growth inhibition for all 2 ppm AgNP treatments. TEM imaging also confirmed cell wall separation in P. aeruginosa and internal density differences for E. coli. The effects on B. subtilis, a gram-positive bacterium, were not as severe but toxicity was observed for several AgNPs at concentrations greater than 2 ppm. Citrate AgNPs appeared to have the most impact on membrane integrity, while other mechanisms such as internal thiol binding might have been at work for other AgNPs.

The effects of varying concentrations of pure AgNPs on complex microbial wastewater reactors are currently being tested. Eight bench-scale sequencing batch reactors were set up to follow the typical "fill, react, settle, decant, idle" method with an 8 hour hydraulic retention time and constant aeration. Reactors were fed synthetic wastewater and treatment efficiency is measured by monitoring effluent concentrations of COD, NH4+, and NO3-. The reactors were seeded with 500 mL of activated sludge from a local wastewater treatment plant. After reaching steady state, the reactors were spiked with 0.2 ppm gum arabic and citrate coated AgNPs. Treatment efficiency was monitored and results showed significant spikes and ammonia and COD immediately following the first spike, but the microbial community appeared to adapt for future AgNP spikes. Microbial community analysis (terminal restriction fragment length polymorphism) showed confirmed this hypothesis.

Overall, this dissertation asserts that by examining AgNP coating type, Ag+ dissolution rates and Stern layer surface charge, it may be possible to predict which AgNPs may be more detrimental wastewater treatment, but not all AgNPs will have the same effect. The results obtained herein must be expanded to other types of AgNPs and microorganisms of ecological importance.

The antimicrobial properties of silver have been taken advantage of by societies for thousands of years. Its use has come back in favor in the form of silver nanoparticles, which are highly efficacious antimicrobial agents. Silver nanoparticles are incorporated into a myriad of products specifically designed for clinical use, but also for general use by consumers. Silver nanoparticles can be found in textiles such as clothing and stuffed toys, and in home appliances including washing machines and curling irons. A large number of products specifically marketed for use by children are also available to consumers, including pacifiers, sippy cups, and even breast milk storage bags. The hazards and toxicities associated with silver nanoparticles are not well understood, however modes of toxicity have been reported for ionic silver. It is assumed that the main mechanism of toxicity of silver nanoparticles relates to the release of ionic silver, however studies have indicated an additional nano-effect, likely due to nanoparticle size, differential coatings, and means of sustained dosing of ionic silver. However we are sure that these silver nanoparticles will accumulate in the waste stream, likely arriving during different stages of a product’s lifespan. A main sink of these nanoparticles travelling through both natural and engineered environments is wastewater treatment plants. As a society we rely on the biological removal of nutrients, which takes place primarily in the activated sludge of secondary treatment. Studies have already indicated possible, temporary decreases in removal efficiencies as well as changes in microbial communities, including losses of diversity, following exposure to silver nanoparticles. Therefore, it is of paramount importance to examine the effects of both silver nanoparticles and ionic silver on the community and function of wastewater bacteria.

Sequencing batch reactors were operated to mimic wastewater treatment. They were fed synthetic wastewater and after reaching acclimation, were dosed over time with varying concentrations of both ionic and nanosilver. Cell samples were collected periodically to assess the presence and identity of cultivable silver resistant bacteria and to map the microbial community changes taking place under different treatments using Next Generation Sequencing. Isolates were tested for the presence of known silver resistance (sil) genes as were activated sludge samples from a collection of domestic wastewater treatment plants, by designing TaqMan probe assays and performing quantitative PCR. The silver resistant isolates were also used to test the growth implications, as well as sil gene expression changes, following treatment with ionic silver and a variety of silver nanoparticles with various coatings, all at multiple concentrations. This was accomplished by performing multiple batch experiments and then using the TaqMan assays and reverse transcription-quantitative PCR.

Overall, microbial community changes were observed in the sequencing batch reactors, and there were differences noted based on treatment, including ionic silver versus nanosilver and between the two silver nanoparticle coatings. Most notably, the possibility of nitrification in wastewater treatment being particularly susceptible was strongly indicated. Individual wastewater bacteria isolates all contained sil genes, as did the majority of the wastewater treatment plant activated sludge, however the levels of actual sil gene expression were inconsistent. This particular finding supports a current body of work indicating that there are alternate modes of bacterial silver resistance in play that we are just becoming aware of.

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.

Water and sanitation are two of the world's most urgent current challenges (Elimelech, 2006). With a population racing towards seven billion people, over one sixth of the human population does not have access to adequate water and sanitation. Drinking water is inaccessible for approximately 783 million people living in the developing world (WHO, 2014). This is especially critical for people at risk of exposure to deadly pathogens such as Vibrio cholerae, Shigella, and Salmonella, such as those living in Haiti as Vibrio cholerae is now ubiquitous (Enserink, 2010). On the sanitation side, more than 2.5 billion people in the world still lack access to adequate resources (WHO, 2014). Almost half of these people have access to no sanitation facilities at all and practice open defecation (WHO, 2014). Thousands of small children still die every day from preventable diseases caused by inadequate sanitation (WHO, 2014). As global climate change is expected to exacerbate these issues, there is an urgent need for the development of sustainable treatment technologies to ensure a better tomorrow for our world (Ford, 1999). Safe water and sanitation technologies, while often disjointed, should be considered together as pathogens transmitted via drinking water are predominantly of fecal origin (Ashbolt, 2004; Montgomery, 2007).

In this dissertation project, I explore the use of both drinking water and wastewater treatment technologies which are cost effective and rely on locally available materials in low-income countries. For the drinking water treatment side, I focus on the use of biosand filters in Haiti with a specific interest in understanding their ability to remove the pathogen Vibrio cholerae, the causative agent for cholera. The wastewater treatment technology consists of biofilters packed with cocopeat, a waste product generated during coconut husk processing, and I investigate their use for the treatment of septic tank effluent in Vietnam. Both of these projects combine lab and field work. The specific objectives of this dissertation project are to 1) compare the removal efficiency of V. cholerae to indicator bacteria in field biosand filters and determine the parameters controlling removal; 2) investigate the correlation between removal efficiency of pathogens in field biosand filters having operated for varying lengths of time to schmutzdecke bacterial composition and influent water characteristics; 3) determine the effect of number of charges, total organic carbon loading, and schmutzdecke composition on V. cholerae removal efficacy; 4) isolate the effect of biological removal mechanisms and physical/chemical removal mechanisms on V. cholerae removal efficiency and determine the correlation to TOC concentration in water; 5) evaluate cocopeat as a packing medium for biofilters in terms of nitrogen, phosphorus and biological oxygen demand removal from simulated wastewater as compared to other traditional packing media; and 6) conduct an assessment of cocopeat-packed, vertical flow constructed wetlands treating septic tank effluent in the Mekong Delta of Vietnam.

In the first part of this dissertation, biosand filters in the Artibonite Valley of Haiti, the epicenter of the cholera epidemic, were tested for total coliform and V. cholerae removal efficiencies. In addition, schmutzdecke samples were collected in order to measure the amount of EPS in the biofilm, as well as characterize the microbial community. Total coliform and V. cholerae concentration were measured using novel membrane filtration technique methods. It was found that total coliform concentration does not indicate V. cholerae concentration in water, and total coliform removal efficiency does not indicate V. cholerae removal efficiency within biosand filters. Additionally, parameters controlling biosand filter performance include: schmutzdecke composition, time in operation, and idle time.

In the second part of this dissertation, V. cholerae challenge tests were performed on laboratory-operated biosand filters receiving high, medium or low TOC influents in order to determine the effect of number of charges, total organic carbon loading, and schmutzdecke composition on V. cholerae removal efficacy, as well as to isolate the effect of biological removal mechanisms and physical/chemical removal mechanisms on V. cholerae removal efficiency and determine the correlation to TOC concentration in water. To this end, three biosand filters were operated in the lab. Each received lake water or diluted lake water with high, medium or low concentrations of TOC. After being charged once per day for 6 days, the filters were charged with four consecutive charges of pure cultures of V. cholerae suspended in PBS buffer, at concentrations of 102, 103, 105, and 107 cfu/mL. This challenge was repeated each time the filters received an additional 6 charges, up to 66 total charges. This was done to determine how number of charges, TOC loading, and schmutzdecke composition affects removal efficiency. Schmutzdecke was analyzed for amount of EPS and microbial community. It was found that parameters controlling biosand filter performance include: TOC loading, schmutzdecke composition, time in operation, and physical/chemical attachment. Additionally, it was shown that physical/chemical attachment is critical during startup, especially at low TOC concentrations. At steady state, physical/chemical attachment is more important than schmutzdecke effects in filters receiving low TOC, and schmutzdecke effect is more important than physical/chemical attachment in filters receiving high TOC.

For the third section of this dissertation, columns packed with cocopeat, celite, or sphagnum peat were charged with simulated wastewater and removal efficiencies of nitrogen, phosphorus, and biological oxygen demand were measured. Additionally, different redox zones were tested to determine if cocopeat could successfully accomplish nitrification and denitrification. It was found that cocopeat is comparable to traditional packing media and can successfully accomplish nitrification and denitrification in the treatment of synthetic wastewater.

In the final section of this dissertation, constructed wetlands were built and packed with cocopeat to determine if cocopeat is a suitable packing media in constructed wetlands treating wastewater in Vietnam. Removal efficiencies of nitrogen, phosphorus, and biological demand were measured. Microbial community samples were collected periodically in order to analyze community shifts between wetlands and over time. This work concluded that cocopeat can be used successfully as a packing media in constructed wetlands treating wastewater for the removal of nitrogen, phosphorus, and total coliform.

Overall, this dissertation work contributes to the body of knowledge on point-of-use water and wastewater technologies. The biosand filter was studied in both lab and field conditions and it was found that total coliform is not a reliable indicator for V. cholerae, and that there are several factors controlling biosand filter performance, including idle time, TOC, filter time in operation, physical/chemical attachment, and schmutzdecke composition. Cocopeat was studied for its ability to promote nitrification and denitrification in lab-scale vertical flow columns treating synthetic wastewater. It was shown that cocopeat achieved similar levels of nitrification and denitrification as traditional packing media. Finally, cocopeat packed vertical flow constructed wetlands were operated in Vietnam for the treatment of septic tank effluent. This setup proved effective for the removal of nitrogen, phosphorus, and total coliform in the treatment of wastewater.

In this work, we offer readers the ability to numerically simulate flow through a sudden expansion themselves. We choose to study the sudden expansion due to its prevalence in engineered and natural water distribution networks (i.e., pipes and rivers, respectively). The simulation is written in the Wolfram Language, also known as Mathematica. The symbolic nature of this programming language enables readers to implement physical theory directly, resulting in a highly readable numerical flow solver; a stark contrast with commonplace commercial flow solvers, which operate like “black box” technologies, and low-level programming languages, which require an advanced level of syntax knowledge and programming proficiency. Upon completion of this laboratory exercise, users should be able to: (i) describe the main principles underpinning the numerical simulation of non-linear models, (ii) apply numerical models to investigate the accuracy of simplified analytical models, (iii) demonstrate a beginner-level understanding of Mathematica and, more broadly, symbolic coding environments, (ii) and most generally, (iv) understand the proper context for physical and numerical experimentation. The novelty of this work is attributed to the fact that no such simulation tool is detailed and provided in the literature for readers to utilize and alter at their discretion.

This work was developed and undertaken in collaboration with my co-authors, Dr. Anil Ganti (A.G.), and Dr. Zbigniew Kabala (Z.J.K), my master’s advisor. Author contributions are as follows: conceptualization, Z.J.K.; methodology, A.H.F, A.G. and Z.J.K.; software, A.H.F and A.G.; validation, A.H.F, A.G. and Z.J.K.; formal analysis, A.H.F; investigation, A.H.F, A.G. and Z.J.K.; resources, Z.J.K; data curation, A.H.F, A.G. and Z.J.K.; writing—original draft preparation, A.H.F and Z.J.K.; writing—review and editing, A.H.F, A.G. and Z.J.K.; visualization, A.H.F.; supervision, Z.J.K.; project administration, A.H.F and Z.J.K.

Partial funding for this project has been received from Duke University Undergraduate Program Enhancement Fund (UPEF) grant 399-000226.

More than 50% of wastewater biosolids are applied to agricultural fields as fertilizer in the U.S. This technique has been used for decades as a widely accepted beneficial reclamation method for biosolids, which meet the established regulatory levels for nutrients, metals, and pathogens. A major drawback to land application is the potential environmental release of non-regulated organic contaminants, which accumulate in biosolids during the wastewater treatment process. Recent studies have been performed to identify and quantify the presence of emerging contaminants in biosolids, and others have investigated the effects of compounds already identified as `priority pollutants' and whose use is waning. However, there is limited research on the effect of emerging organic contaminants on soil microbial ecology and nutrient cycling. Because many of the compounds found in biosolids are specifically designed to elicit biological modifications (e.g., antimicrobials), there is a risk that these compounds will disrupt microbial soil functions, decrease soil productivity, and ultimately affect the long term viability of these ecosystems, resulting in unforeseen economic and social costs. Therefore, there is a clear need to characterize the effects of novel contaminants on soil health.

This dissertation was divided into three distinct parts examining the impacts of emerging organic contaminants on soil microbial ecology with increasing complexity to better reflect environmental conditions. To assess the ecological impacts, the functional endpoint of denitrification was selected because it provides a vital indication of soil health. Denitrifying bacteria play a critical role in this process, and thus, were used as indicator organisms for determining contaminant ecotoxicological potential. Furthermore, antimicrobial agents (a.k.a., bactericides or biocides) were selected as model contaminants because they are designed specifically to deactivate microorganisms, are heavily used in the U.S with over $1 billion in yearly sales, and have been measured in biosolids.

Overall, the objectives of this dissertation were to: 1) develop a rapid, high-throughput functional assay that measured denitrification inhibition for screening potential ecological impacts of biosolids-derived antimicrobial agents, 2) determine the potential effects of common and emerging biosolids-derived antimicrobial agents on denitrification by a model soil denitrifier, Paracoccus denitrificans PD1222, 3) examine the impacts of the most commonly used antimicrobial, triclosan (TCS), on wastewater treatment efficiency in bench scale sequencing batch reactors (SBRs) coupled with anaerobic digesters, 4) examine the impacts of biosolids aged and spiked with TCS on denitrification under simulated agricultural soil conditions, and 5) evaluate potential impacts of TCS in `traditional' biosolids on denitrification in agricultural soil under field conditions.

The first phase of research pertaining to Objectives 1 and 2 examined the baseline interactions between biosolids-derived antimicrobial agents and soil microbial ecology. However, to isolate the effect of an individual contaminant from the myriad of contaminants found in biosolids, there was a need for developing a rapid, high-throughput method to evaluate general ecotoxicity. In the first part of this dissertation, we developed a novel assay that measured denitrification inhibition in a model soil denitrifier, Paracoccus denitrificans Pd1222. Two common (TCS and triclocarban) and four emerging (2,4,5 trichlorophenol, 2-benzyl-4-chlorophenol, 2-chloro-4-phenylphenol, and bis(5-chloro-2-hydroxyphenyl)methane) antimicrobial agents found in biosolids were analyzed as model contaminants. Overall, the assay was reproducible and measured impacts on denitrification over three orders of magnitude exposure. The lowest observable adverse effect concentrations (LOAECs) were 1.04 μM for TCS, 3.17 μM for triclocarban, 0.372 μM for bis-(5-chloro-2-hydroxyphenyl)methane, 4.89 μM for 2-chloro-4-phenyl phenol, 45.7 μM for 2-benzyl-4-chorophenol, and 50.6 μM for 2,4,5-trichlorophenol. Compared with gene expression and cell viability based methods, the denitrification assay was more sensitive and resulted in lower LOAECs. Of the six compounds examined, four resulted in LOAECs that were below or within an order of magnitude of concentrations that were measured in the environment, indicating potential ecological impacts.

In the second part of the dissertation, the impacts of emerging contaminants were examined first under laboratory conditions mimicking wastewater treatment processes (Objective 3) and then agricultural fields (Objective 4). For this phase, TCS, which is the most widely used antimicrobial agent and identified in the first phase for potential ecological impacts, was used as the model contaminant. To mimic wastewater treatment processes, bench scale SBRs coupled with anaerobic digesters were set up and operated. The SBRS and digesters were seeded with activated and anaerobically digested sludge from the North Durham Water Reclamation Facility (NDWRF, Durham, NC). Reactors were fed synthetic wastewater with or without 0.73 &muM of TCS. Samples were taken periodically to monitor chemical oxygen demand (COD), ammonium (NH₄+), nitrate (NO₃-), nitrite (NO_{2-), total suspended solids (TSS), volatile suspended solids (VSS), dissolved oxygen (DO), and phosphate (PO43-) and pH. In addition, biomass samples were collected for DNA extraction and microbial community analysis using terminal restriction fragment length polymorphism (T-RFLP) of 16S SSU rDNA. Methane production was also monitored for the anaerobic digesters. In addition, the final digested biosolids that were generated from the SBRs fed with and without TCS were analyzed for TCS concentration, TSS, VSS, TKN, phosphorus (as P2O5), potassium (as K2O), and pH. Overall, biological processes associated with nitrogen removal (nitrification and denitrification), were impacted by TCS entering the SBRs regardless of the starting microbial community. Both of the SBRs that were not receiving TCS reached steady-state at greater than 92% NH4+, removal within the first week of operation, whereas the SBRs receiving TCS took 42 and 63 days to reach steady-state removal at that level. However, while NH4+ removal was temporarily inhibited, elevated levels of NO3- and NO2- in the effluent of the TCS fed SBRs, suggested longer-term impacts on nitrite oxidizing bacteria (NOB) and denitrifiers. After Day 58, the NO3- effluent concentration for the SBRs receiving TCS was 3.9 ± 0.16 mg/L, which was 2.4 times greater than the NO3- effluent of the SBRs not receiving TCS (1.7 ± 0.08 mg/L). Similarly, after Day 58, the NO2- effluent of the SBRs receiving TCS reached a steady-state concentration of 8.7 ± 0.75 mg/L. The mean NO2- concentration in the controls after Day 58 was 7.7 times lower at 1.1 ± 0.78 mg/L, but was still trending towards 0 when the reactors were stopped. No inhibition was observed for COD and PO43- removal. In addition, non-metric multidimensional scaling (NMS) ordination analysis showed that the microbial communities between SBRS fed with and without TCS were similar on Day 0, but increased in difference to Day 41, around when the major changes in nitrification were observed. After a slight increase in similarity between the control and TCS SBR microbial communities on Day 41, the communities increased in difference to Day 63.}

To mimic agricultural field conditions, containers of soil were amended with the biosolids generated from the SBRs. The containers were maintained in a growth-chamber to simulate field lighting and watering conditions. Three biosolids treatments were examined: 1) biosolids generated from the SBRs not fed TCS, but that still had low backgrounds of TCS (a.k.a., Control Biosolids); 2) biosolids generated from the SBRs fed with TCS (a.k.a., Aged TCS Biosolids); and 3) biosolids that were generated by the SBRs not fed TCS, but spiked with TCS 24 h before application (a.k.a., Spiked TCS Biosolids). Alfalfa was planted in half of the containers receiving the Control and Aged TCS Biosolids to assess differences due to vegetation. To assess the overall ecotoxicity of biosolids aged and spiked with TCS, the function, abundance, and diversity of the soil denitrifying communities were examined. The impacts on total bacteria abundance and diversity were also examined for comparison. Specifically, the denitrifying enzyme activity (DEA) assay was used to measure functional impacts, quantitative polymerase chain reaction (qPCR) was used to measure impacts on abundance, and T-RFLP was used to measure impacts on diversity. Correlations between these methods were also examined for possible interactions between denitrifier function and community structure and to provide insight into targets of inhibition. Lastly, a denitrification inhibition score was developed to quantify global impacts of TCS on denitrification. The containers with plants that received biosolids aged with and spiked with TCS showed potential long-term inhibition based on measurement of soil denitrification at 26.9 ± 4.6 μg/kg and 68.6 ± 26.9 μg/kg of TCS, respectively. Denitrifier abundance and diversity, however, were more sensitive to TCS in biosolids and inhibition was observed throughout the experiment, with maximum inhibition on Days 7 and 28. Inhibition of denitrifier abundance and diversity was observed at TCS concentrations as low as 17.9 ± 1.93 μg/L, which was about 10 to 3000 times lower than concentrations reported by other studies that showed impacts on other functional endpoints (i.e., respiration, phosphatase activity, NO₃- and NO₂- production, and Cy17 stress biomarker abundance), even after taking pH into account. Five significant correlations were developed, three of which related qPCR and the DEA assay, or abundance and activity. However, the analyses that were correlated did not yield the same results as far as significant inhibition in the presence of TCS. Thus, while the results suggested some relatedness between activity, abundance, and diversity, the results generally support the use of multiple methods to determine the ecotoxicity of biosolids-derived organic contaminants. As a result, a denitrification inhibition score was developed that took into account all three methods to determine the overall ecotoxicity of TCS in biosolids. Overall, the denitrification inhibition score showed that denitrification was inhibited by both biosolids that were aged and spiked with TCS over the extent of the 84 day experiment, but maximum inhibition occurred after a week to about a month. While the denitrification inhibition score indicated that the TCS in the biosolids aged with TCS was less bioavailable than in the spiked biosolids, the impacts of the aged and spiked biosolids could have also been due to differences in TCS concentrations.

Objective 5 consisted of a long-term soil sampling campaign on four agricultural fields receiving Class B municipal biosolids. Soil samples were taken before and after biosolids application and were analyzed to elucidate potential impacts of TCS in the biosolids on denitrification. Again, to assess the overall impacts of TCS on the soil denitrifying community, the DEA assay, qPCR, and T-RFLP were used to measure impacts on function, abundance, and diversity, respectively. Similar to Objective 4, the analysis included an examination of potential correlations between denitrifying community structure and function, and quantification of global impacts using the denitrification inhibition score. As expected, the results in this pilot-study reflected the complexity of the system that was analyzed and many more samples, which account for variables including, but not limited to soil characteristics, biosolids characteristics, biosolids application rates, and chemical composition and quantities, would be needed to show any statistically significant differences. Nevertheless, several key results were obtained. Again potential long-term inhibition of denitrification was observed using the DEA assay, however the effects of exhaustion of resources, such as NO₃-, or significant changes in the local environment were suspected, but could not be verified. Inhibition was also observed for denitrifier abundance, but little to no inhibition was observed when examining the relative number of denitrifying species. Thus, while the abundance of denitrifiers was reduced, and denitrification was eventually depressed, the number of species in the soil remained constant. When looking at the denitrification inhibition score, which took all three measurements into account, increased inhibition over time was observed with the exception of the measurements on Days 30 and 103, which indicated overall, but weak inhibition of denitrification by the application of biosolids. NMS ordinations showed no correlation between the shift in denitrifying microbial community and TCS. Because of the complexity of the soil and biosolids and because of the myriad of contaminants likely in the biosolids, the results may not be significant and a more in-depth study was recommended.

Overall, the results presented in this dissertation provide a systematic evaluation of the effects of biosolids-derived TCS on agricultural soil microbial ecology. First, it was demonstrated that statistically significant inhibition of denitrification could be used as a potential indicator of biosolids-derived emerging organic contaminant ecotoxicity. The denitrification assay that was developed was then used to analyze ecotoxicological potential of six emerging biosolids-derived antimicrobial agents, and found inhibition of denitrification at environmentally relevant concentrations. The most widely used antimicrobial agent, TCS, was further shown to inhibit wastewater treatment processes, as well as, denitrification in simulated agricultural conditions after being aged with and spiked into biosolids. In addition, evidence showing potential inhibition of denitrification by TCS in `traditional' biosolids under field conditions was also obtained. Based on these results, this dissertation asserts that biosolids-derived emerging organic contaminants pose a potential risk to agricultural soil microbial ecology and overall soil health. Future studies, however, are needed to examine the impacts of other contaminants that might be flagged with the assay developed in this dissertation under more complex conditions mimicking the environment. Furthermore, other research is needed to examine the role microbial communities play in the bioavailability of emerging contaminants, especially TCS, and a more extensive, in-depth study is needed to characterize the individual impacts of emerging contaminants on soil microbial communities under field conditions.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that accumulate in soils and sediment due to their physicochemical properties. In these environmental matrices, PAHs are predominantly transformed and degraded by the native fungal and bacterial communities. However, microbial degradation of PAHs is a slow process that requires engineered approaches to improve degradation rates to meet remediation criteria.

Engineered bioremediation approaches consist of altering the microbial community by either increasing cell concentrations of specific, targeted organisms or by introducing catabolic genes that confer for a phenotype that can degrade the target contaminant. This approach is called bioaugmentation and is generally applied using the former strategy. Biostimulation is another method, which includes the addition of nutrients that may be limited to microorganisms and can help grow the indigenous microbial community and accelerate contaminant degradation. However, biostimulation is not a targeted approach and may stimulate the entire microbial community, not just organisms capable of degrading the target contaminant.

Bioaugmentation of sediments is challenging due to constraints surrounding the longevity, stability, and delivery of microorganisms. To address the limitations of this remediation approach, the work within this dissertation outlines methods for developing a consortium of PAH-degrading bacteria coordinated within a stable community, as well as a technology for delivering this consortium to creosote contaminated sediments.

The first objective was to identify and isolate PAH-degrading bacteria from creosote contaminated sediment. Sediment was collected from sites along the Elizabeth River, VA and a 16S rRNA amplicon library of sequences was analyzed to generally evaluate the influence of chemical contamination on the bacterial community structure. To detect PAH-degrading organisms within sediment communities, DNA-SIP using uniformly labeled stable isotopes of phenanthrene and fluoranthene were prepared in incubations with Republic Creosoting site sediment. Clones derived from this experiment revealed one prominent degrader of phenanthrene and two prominent fluoranthene degrading bacteria. In an attempt to isolate these and other PAH-degrading organisms for laboratory evaluation, culture-based methods were employed and resulted in the successful isolation of 6 unique bacteria, including one strain which was detected in the DNA-SIP experiments. Overall, it was determined that PAH-degrading bacteria exist in Republic Creosoting site sediments, although not in significant relative abundance compared to other bacteria. This finding suggests that these contaminated sediments could be a good candidate for a bioaugmentation approach.

Most of the research on bioremediation has focused on organisms in isolation and existing in a free-floating, or planktonic, cellular state. The second objective of this dissertation was to confirm the PAH-degrading capabilities of isolated bacteria and to coordinate these organisms into a biofilm structure, which provides protection and additional community benefits to participating microorganisms. To this end, we employed a high-throughput, reproducible assay to confirm whether or not isolated bacteria are capable of coordinating within a biofilm. We also used culture-based methods and performed incubations with multiple types of PAHs to determine if the isolated organisms can interact with PAHs of various size and ring number. Finally, we used a metabolic assay for the novel application of assessing the respiration capacity of the isolated PAH-degrading bacteria in the biofilm conformation, to determine if these organisms are metabolically active when they are situated within a biofilm. We found that all of the organisms isolated were capable of forming a biofilm that was metabolically active. Many of these organisms demonstrated the ability to degrade phenanthrene and fluoranthene, but only a few showed the potential for degrading pyrene. These results confirmed that the isolated organisms from Republic site sediment can degrade PAHs and form a biofilm structure, which will be beneficial for their application to sediments in a bioaugmentation strategy.

The final aim of this work was to evaluate the use of an activated-carbon amendment based technology for the delivery of a bacterial consortium to PAH-contaminated sediment. While validated for use as a remediation technology and delivery strategy for organisms capable of degrading polychlorinated biphenyls (PCBs), this approach has not yet been tested for use with sediments contaminated with PAHs.

Over the past few hundred years humans have been evolving into a primarily urban species. This evolution has coincided with an increase in allergy that may be caused by an underdeveloped immune system. Increasingly, research suggests this underdevelopment may be caused through loss of contact with beneficial, health promoting microorganisms known as the old friend’s hypothesis. This is of particular interest as humans continue to urbanize and begin spending more time indoors. there still exists several questions that need to be answered. Thus, it has become imperative to understand how exposure to diverse microbial populations changes the composition the lung and gut microbiomes and whether exposure through soils results in beneficial health outcomes. The first objective of this dissertation was to quantify the relative impacts of the different environmental media on the lung microbiome using a pig model. Using 16S rRNA gene amplicon sequencing, the microbiomes of the lungs, soil, water, feed, and air were determined at two different farms. Using the environmental data, a support vector machine model was created that could accurately classify the upper respiratory lung microbiome samples but not the alveolar samples. This suggests that while the upper respiratory system is regulated by the surrounding environment, this is a distinct community at the alveolar level that is regulated by selective pressures within the lung. The second objective utilized a mouse model exposed to three different soil types over 5 weeks to determine how the gut and lung microbiomes changed in response to the environmental alteration. This objective utilized 16S rRNA gene amplicon sequencing to determine the bacterial community within the cage, gut, and lung microbiomes. The differentially abundant amplicon sequence variants (ASVs) were identified to determine whether differentially abundant features in the soils were also found in the gut and lungs. This objective found that soils had a significant effect on both the gut and lung microbiomes that was driven largely by ASVs belonging to the family Lachnospiracae. The third objective explores whether exposure to the diverse microbial communities through soils resulted in a differential immune response when challenged with influenza A. To measure the differential immune response, RNA-seq was conducted with RNA extracted from whole lung tissue. From the differential expression data, the River soil exposed mice showed a significant increase in anti-inflammatory cytokines and lower overall neutrophil and macrophage cell counts. To determine what may be mediating this response, metagenome function was inferred from the 16S rRNA amplicon which showed several of the higher abundance Lachnospiracae ASVs were implicated in an upregulated propionate producing pathway, a small chain fatty acid associated with lower lung inflammation.

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.

Ballast water is a known vector for the global translocation of microorganisms. Research into the ballast microbiome recently accelerated following a ballast-associated outbreak of Vibrio cholerae in Peru during the 1990s that killed over 10,000 people. Over the last two decades there has been increasing regulation surrounding ballast water treatment with the aim of protecting human and environmental health, recently culminating in the approval of the International Maritime Organization Ballast Water Management Convention in September 2017. The Convention requires shipowners to install and use of ballast water treatment systems within an established timeline. However, many basic questions remain surrounding the composition of the ballast water microbiome. This dissertation strives to address several of these questions, which will allow shipowners, regulators, and enforcement agencies to make more informed decisions in an uncertain space.

The first aim of this dissertation is to characterize the bacterial microbiome of ballast water aboard vessels arriving at several ports, and identify characteristics to explain observed variations. Published research that utilizes high throughput sequencing (HTS) technology to explore microbial community dynamics is relatively rare. In this study, 16S rRNA gene sequencing and metabarcoding were used to perform the most comprehensive microbiological survey of ballast water arriving to hub ports to date. In total, 41 ballast, 20 harbor, and 6 open ocean water samples were characterized from four world ports (Shanghai, China; Singapore; Durban, South Africa; Los Angeles, California). In addition, total coliforms, Enterococcus, and E. coli were cultured to evaluate adherence to International Maritime Organization standards for ballast discharge. Five of the 41 vessels – all of which were loaded in China – did not comply with standards for at least one indicator organism. Dominant bacterial taxa of ballast water at the class level were Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia. Ballast water samples were composed of significantly lower proportions of Oxyphotobacteria than either ocean or harbor samples. Linear discriminant analysis (LDA) effect size (LEfSe) and machine learning were used to identify and test potential biomarkers for classifying sample types (ocean, harbor, ballast). Eight candidate biomarkers were used to achieve 81% (k nearest neighbors) to 88% (random forest) classification accuracy. Further research of these biomarkers could aid the development of techniques to rapidly assess ballast water origin.

The first portion of the second aim of this dissertation evaluates the prevalence of indicator organisms and antibiotic resistance genes (ARGs) in ballast water compared to harbor and ocean water. The Ballast Water Management Convention, which sets forth guidelines regarding indicator organisms in ballast water, entered into force in September 2017. Notably, antibiotic resistance is absent from the Convention. We collected a total of 74 ballast and harbor samples from Singapore; Shanghai, China; Durban, South Africa; and Los Angeles, California. Eight ocean samples were collected for comparison. This research examines the concentration of indicator organisms and prevalence of three antibiotic resistance genes (ARGs). The ARGs examined in this study range from ubiquitous (sul1 – sulfonamide) to common (tetM – tetracycline) to rare (vanA – vancomycin). In ballast samples, there were significantly higher concentrations of E. coli in Singapore and China when compared to South Africa (Singapore, p = 0.040) and California (Singapore, p < 0.001; China, p = 0.038). Harbor samples from China had significantly higher concentrations of E. coli than Singapore (p = 0.049) and California (p = 0.001). When compared to ocean samples, there were significantly higher concentrations of normalized tetM in ballast samples from California (p = 0.011) and Singapore (p = 0.019) and in harbor samples from California (p = 0.018), Singapore (p = 0.010), and South Africa (p = 0.008). These findings indicate that there are differential microbial loads in different ports. Furthermore, there appears to be elevated levels of certain ARGs in ballast and harbor water when compared to ocean water, which may indicate that ballast is either translocating higher concentrations of certain ARGs or that conditions in the ballast tanks are placing selective pressure in favor of some ARGs.

The second portion of the second aim of this dissertation evaluates the prevalence of fungal pathogens in ballast water compared to harbor and ocean water. Several recent studies have explored the ballast water microbiome, but few have examined the fungal mycobiome and, to our knowledge, no studies have examined fungal pathogens in ballast. The fungal mycobiome was characterized by collecting a total of 65 ballast, harbor, and ocean samples from four major ports and sequencing the fungal internal transcribed spacer (ITS) region. A literature review of the resulting taxa was performed to identify well-studied fungal pathogens. Hosts of the identified pathogens included corals, humans, animals, plants, and crops. Of ballast samples, 21.4% had at least one fungal taxon pathogenic to corals and 81.0% had at least one fungal taxon pathogenic to humans. The majority of the fungal community in 19% of ballast samples were pathogenic taxa. A significantly higher proportion of the fungal community was composed of pathogens in Shanghai compared to all other sample sites (p = 0.025). The identification of fungal pathogens in ballast, especially those affecting corals and humans, highlights the need to further research the ballast microbiome to protect human and environmental health from the threat of fungal pathogen introductions via ballast.

The first portion of the third aim of this dissertation examines correlations between the bacterial and fungal microbiome. This chapter utilizes high throughput sequencing (HTS) and machine learning to examine and integrate the 16S and 18S rRNA genes and fungal ITS region. These sequencing regions were examined using the SILVA v132 and UNITE reference databases. The highest correlation was found between the communities in Silva_16S and UNITE_ITS (0.74). There was a higher proportion of positive inter-kingdom correlations than positive intra-kingdom interactions (p = 0.032). Understanding the reasons for this difference will require additional research under more controlled conditions. Finally, a machine learning model was used to examine the accuracy of assignment when using each sequencing region and reference database. There was significantly higher accuracy when using SILVA v132 (0.814) when compared to UNITE (0.664) (p < 0.001). In the short term, future research with the goal of classifying ballast water samples based on location or ballast water residence time should be performed using the 16S rRNA gene and SILVA v132 reference database. Future research to curate other sequencing regions or the UNITE reference database in the aquatic ecosystem may improve the utility of these tools when attempting to classify ballast water.

The second portion of the third aim of this dissertation examines correlations between the bacterial microbiome and non-target chemical analysis. To our knowledge, no literature is available that examines the interaction between microbes and chemicals in ballast water. This study addresses this gap in the literature by examining correlations between bacterial taxa and non-target chemical compounds. All strong interdomain and intradomain Pearson correlations (i.e. |r| > 0.7) were positive (54 interactions); however, the majority of Pearson correlations at all levels were negative (25,497 of 33,920; 75.2%). The reasons for this pattern are unclear and further research to isolate specific bacterial taxa and non-target chemicals may provide useful insight. In addition, machine learning was performed using bacterial, chemical, and bacterial and chemical markers. The bacterial markers appeared to perform well at differentiating California, China, and South Africa; however, accuracy was poor when classifying Singapore samples. Chemical markers appeared to supplement this deficiency, and the lowest out-of-bag error was achieved using a combined bacterial-chemical marker set with 6 features (6.67%). Further research with a larger sample size is necessary to appropriately test the markers identified in this work; however, this research serves as a proof-of-concept for a combined bacterial-chemical machine learning classification approach to ballast and harbor water samples.

In order to meet disinfection guidelines, wastewater utilities must achieve a high level of treatment before discharging treated water for irrigation or industrial use. However, public pressure to reduce disinfection by-products and pharmaceutically-active compounds, recently-promulgated regulations on chlorine-resistant microorganisms such as Cryptosporidium parvum, and growth in population and water demand have driven an interest in alternatives to chlorination. The WateReuse Foundation has funded WRF 02-009 (Innovative Treatment Technologies for Reclaimed Water), which is a survey of current and emerging reuse water treatment technologies. The goal of the project is to evaluate treatment technologies can provide adequate recycled water effluent without the cost of reverse osmosis (RO) or the disinfection by-products (DBPs) formed during chlorination.

The inactivation of indigenous microorganisms (total and fecal coliform bacteria, and total aerobic spores) and spiked surrogate, respiratory, and enteric viruses (MS-2 bacteriophage, adenovirus type 4, reovirus type 3, and coxsackievirus type B5) and chemical degradation by wastewater treatment technologies was evaluated on the bench-scale. These include: low- and medium-pressure UV, LPUV/H₂O₂, ozonation, O₃/H₂O₂, peracetic acid (PAA), LPUV/PAA, chlorination, chloramination, and ultrafiltration. The applicability of the candidate disinfection methods, especially emerging and comparatively untested methods such as PAA and advanced oxidation processes (AOPs), was studied through comparison of their performance and the important water matrix parameters (e.g., alkalinity, BOD, TSS, etc.).

Of the chemical disinfectants, molecular ozone and free chlorine were the most effective, with substantial coliform and virus kill at low doses. Combined chlorine in the form of monochloramine had a reduced disinfectant capacity than free chlorine, and peracetic acid (PAA) performed equally as well as free chlorine with respect to coliform bacteria in some instances but had little to no impact on spiked MS2 bacteriophage. None of the aforementioned disinfectants had an appreciable impact on indigenous aerobic spore-forming bacteria due to their physiology. UV and O₃ rapidly killed human enteric and respiratory viruses, but a consistent benefit by AOPs over their base technologies was not observed for any of their base technologies.

Low and medium-pressure UV inactivated free-floating indigenous coliform bacteria almost immediately, while slower inactivation rates at higher UV fluences illustrated the "tailing" behavior observed when bacteria are embedded in or shielded by particulate matter. Log-linear inactivation of spiked viruses and indigenous aerobic spores by UV was consistent across the utility waters. The UV-based advanced oxidation processes (UV/H₂O₂ and UV/PAA) destroyed spiked organic compounds at much higher rates than direct UV photolysis, while O₃, with or without H₂O₂ , oxidized spiked compounds and reduced estrogenicity (EEQ) at low doses. Recalcitrant chlorinated hydrocarbons such as TCEP were only moderately removed by the tested AOPs, but low doses of O₃ (3 ppm residual O₃) reduced estrogenic activity by 99%. Like other disinfection processes, AOP performance is dependant on pretreatment, especially concerning particulates.

Polycyclic aromatic hydrocarbons (PAHs) are a class of over 100 chemicals found at various EPA Superfund sites across the United States formed through the incomplete combustion of organic compounds. These environmental contaminants are of concern due to their carcinogenicity, mutagenicity, and teratogenicity. Bioremediation using microorganisms is an economically efficient and environmentally sustainable process for PAH transformation in the environment. However, bacterial bioremediation schemes are limited in their ability to transform high molecular weight (HMW) PAHs. Through exploiting non-specific extracellular fungal enzymes in a mixed fungal-bacterial consortia, HMW PAHs have the ability to become bioavailable to bacteria, and ultimately be transformed. This approach is not widely used in the field of bioremediation, as there is limited understanding around interkingdom relationships between bacteria and fungi. There is limited knowledge of the array of extracellular enzymes that may assist with the transformation of PAHs, and whether these enzymes can be biostimulated or antagonized to increase the removal of PAHs in the environment. Additionally, a knowledge gap exists with regards to the survivability of introduced fungal and bacterial isolates, and whether a mixed fungal-bacteria consortia will provide an advantage in soil communities. This dissertation work will focus on developing a joint fungal-bacterial consortia that will exploit the cross-kingdom interactions of fungi and bacteria to assist in the removal and transformation of PAHs. This will be examined through isolating and identifying fungal isolates from creosote contaminated soil and assessing them for their ability to be biostimulated to produce extracellular enzymes that can be analogized for PAH transformation. Once determined, fungal and bacterial isolates will be assessed together for their ability to produce a joint biofilm. Lastly, fungal, and bacterial isolates will be assessed for their transformation capability and survivability amongst the microflora community in PAH soils.

This dissertation's first research objective was to identify and characterize indigenous fungi for a biostimulation/bioaugmentation scheme targeting PAHs. The goal of this objective was to find promising indigenous fungal isolates for further investigation in the transformation of PAHs. First, creosote contaminated soil was diluted and plated onto an array of growth agars to provide a representative overview of the fungal soil community. Fungi were isolated, cultured, and screened for the enzymatic production of laccase (Lac), manganese peroxidase (MnP), and tannase, along with the ability to degrade cellulose and starch. Fungal isolates were then incubated alongside complex amendments and Lac production will be measured through absorbance. Fungal isolates and complex amendments were lastly incubated alongside PAHs to measure transformation. From this objective, it was found that wide variety of fungal isolates native to creosote contaminated sediment were found to be able to produce extracellular enzymes that may assist in the biotransformation of PAHs. It was also found that complex amendments may also be used to increase extracellular enzymatic production to assist with PAH transformation. The complex grasshopper amendment is shown to be the most promising complex amendment to use in a future scheme.

This dissertation's second objective was to observe and characterize intra- and interkingdom behaviors in a mixed fungal-bacterial consortium for the improved transformation of PAHs. This research goal aimed to investigate the interactions between fungi and bacteria that could aid in the transformation and degradation of PAHs, as well as understand the potential for antagonistic and mutualistic interactions within soil communities. In this objective, a series of fungal permutations were performed from a select group of previously isolated indigenous fungi. The fungal permutations were visually assessed for unique growth patterns and assessed for changes in enzymatic functions. Permutations were then examined in the presence of a complex amendment to compare enzymatic production of fungal consortia to monoculture isolates. A select group of fungal isolates were then incubated alongside PAH degrading bacteria and examined for biofilm formation using optical density. Finally, fungal-bacterial mixed consortia and select model PAHs were incubated with the model complex amendment and measured for transformation capability. It was found that fungi will develop different morphological responses in the presence of other isolates. From this objective it was found that a mixed consortia between Novosphingobium pentaromativorans and filamentous fungi such as Penicilium.513 and Trichoderma.508 were deemed to be more advantageous for bacterial cell adhesion than yeast and yeast-like fungi such as Scheffersomyces.502 and Aureobasidium.509. When placed against model PAHs: FLA, PHE, PYR, and BaP, it was found that nearly all the fungal isolates saw improvements in their ability to degrade when placed alongside the bacteria. Additionally, when co-cultures were provided with a grasshopper amendment, increases in PAH degradation were observed.

The final objective of this dissertation work was to assess the performance of a mixed fungal bacterial consortia in a PAH-spiked soil microflora community. The purpose of this research objective is to assess the viability of a mixed fungal bacterial consortia in a soil reactor that mimics field conditions. Reactors were developed to assess community changes in PAH-spiked soil and non-spiked soil. Reactors were also inoculated with either monocultures or cocultures to explore how each variation affects the microbial community over time. Reactors were also biostimulated with a complex grasshopper amendment. Over a 60-day period, reactors were sacrificed at various points. RNA was extracted out of the soil at each time point and used to analyze the bacterial and fungal communities. Furthermore, soil samples were evaluated for PAH degradation at every time interval. No correlation between mixed fungal-bacterial consortia and PAH degradation was observed based on this objective. In comparison to their naturally attenuated counterparts, soil reactors that were subjected to biostimulation, bioaugmentation, and biostimulation-bioaugmentation analyses did not demonstrate appreciable variations in community structure.

This dissertation work will help engineers and researchers create efficient and sustainable PAH remediation strategies. This dissertation addresses long-standing knowledge gaps in mixed consortia fungal behavior and fungal-bacterial interactions. This work provides a framework for future investigations into cross-kingdom interaction, improved bioaugmentation methods, and optimized biostimulation for target organisms.

Antibiotic resistance rates have increased in both clinical and environmental bacteria over the past several decades. While the causes underlying these trends have been investigated in a clinical setting, little work has been done to estimate the contribution of antibiotic resistance genes (ARGs) derived from non-traditional sources like transgenic (GM) crops. The cultivation and consumption of transgenic crops continues to be a widely debated topic, as the potential ecological impacts are not fully understood. In particular, because ARGs have historically been used as selectable markers in the genetic engineering of transgenic crops, it is important to determine if the genetic constructs found in decomposing or consumed transgenic crops persist long enough in the environment and if they can be transferred horizontally to indigenous microorganisms.

The first Objective of this dissertation addresses the question of persistence. Others have also estimated the DNA adsorption capacity of various clays, but have done so by manipulating the surface charge and size of particles tested which may overestimate sorption and underestimate the DNA available for horizontal transfer. In the present study, isotherms were generated using model Calf Thymus DNA and transgenic maize DNA without surface modification. Montmorillonite, kaolinite, and 3 soil mixtures with varying clay content were used in this study. The adsorption capacity of pure montmorillonite and kaolinite minerals was found to be one to two orders of magnitude less than previously estimated likely due to the distribution of clay particle sizes and heteroionic particle surface charge. However, it appears that a substantial amount of DNA is still able to adsorb onto these matrices (up to 200 mg DNA per gram of clay) suggesting the potential availability of free transgenic DNA in the environment may still be significant.

In addition to the soil environments described above, the persistence of these genes should be investigated in agricultural soils. Two important tools exist for understanding the potential contribution of transgenic crop-derived ARGs to environmental antibiotic resistance: (A) the microbiomes associated with transgenic maize and (B) decomposition behavior of transgenic crop biomass. The purpose of Objective Two is to characterize GM maize microbiomes and biomass decomposition to better how transgenic maize may be affecting antibiotic resistance rates among soil bacteria. To investigate this, bulk soil, rhizosphere soil, and internal endophyte prokaryotic microbiomes associated with conventional and transgenic maize were compared using lllumina MiSeq 16S amplicon sequencing. Internal endophytes were significantly influenced by time and location within the maize, but not by maize type. Nitrogen-cycling bacteria in the rhizosphere of transgenic pest-resistant (BT) maize experienced a decrease in diversity at day 56 of maize cultivation that was moderately correlated with the level of Cry1Ab protein exudates in soils surrounding transgenic BT maize. Secondly, bla ARG expression was tracked across 40 weeks of maize biomass decomposition in soils associated with conventional and transgenic BT maize. Bla expression significantly increased in soils associated with BT maize relative to conventional maize soils after 32 weeks or decomposition.

In addition to agricultural environments, wastewater treatment plants (WWTPs) may also contain genes derived from transgenic crops. Consumption of transgenic crops and foods containing them is common in the United States. Once ingested, DNA within transgenic crops can conceivably behave in three ways: 1) DNA will be degraded by acids or DNase I and II enzymes found within the digestive system, 2) DNA will be taken up by cells in the gut (host or bacterial), or 3) DNA will pass through the digestive system and excreted partially or wholly intact. In addition to degradation or uptake, transgenic DNA may be excreted as solid waste by the host. As free DNA contained within food matrices is protected from both enzymatic digestion and acid hydrolysis, the genes contained within foods containing GMOs may be allowed to pass through the host’s digestive tract intact and enter into WWTP environments. Objective 3 assessed this possibility by screening activated sludge and digester sludge obtained from France and the United States for the following four possible transgenes: p35 promoter gene, nos terminator gene, nptII ARG, and bla ARG. All wastewater samples were first screened using end-point PCR and all positive results were further analyzed with qRT-PCR to determine the relative concentration of each gene in wastewater. All domestic activated and digester sludge samples were found to contain at least one transgene, with the majority of samples containing all four. However, wastewater from France, where transgenic crops are neither cultivated nor consumed, contained significantly fewer copies of p35, nos, and nptII genes. Based on these findings, it is possible that some of these genes are derived from partially digested transgenic crop material present in human waste. This is supported in part by the significant differences in p35, nos, and nptII concentrations in French WWTP systems relative to domestic WWTP systems, as well as additional screening for Cauliflower Mosaic Virus (CaMV) ORF VII genes and larger fragments of possible transformation plasmids. Overall it is possible for genes contained within transgenic crops and processed foods containing their byproducts to survive the human digestive process and enter into WWTP environments, though the source of the detected genes could not be definitely confirmed.

The fate and behavior of these for genes in WWTPs remains largely uncharacterized. Further investigation of these trends in WWTP anaerobic digesters is of paramount importance, especially in the case of the bla and nptII ARGs, as a large fraction of WWTP-generated biosolids is used for land application. In addition, because of the relatively high background levels of antibiotic contaminants in wastewaters there is the further possibility that free ARGs may be horizontally transferred to WWTP bacteria, thus increasing antibiotic resistance in the environment. The ability of WWTP bacteria to take up these transgenes in this environment is a factor of (1) the observed rates of horizontal gene transfer (HGT) in anaerobic environments, (2) the abundance of genes of interest within WWTP anaerobic digesters, and (3) the presence of a selective pressure. Like most other topics related to HGT, observing and quantifying the rates of these event in anaerobic environments has proved to be a difficult task. To investigate the behavior of free ARG DNA in WWTP environments, anaerobic digesters were constructed using anaerobic feed sludge from the North Durham (NC) WWTP. Digesters belonged to one of the following treatments and were operated at 47oC in quadruplicate for a period of 60 days: 102 added bla and nptII transgene copies/mL, 104 added bla and nptII transgene copies/mL, or 106 added bla and nptII transgene copies/mL. Control digesters were also constructed. Across 60 days of operation, persistence of free DNA in anaerobic digester wastewater was significantly influenced by DNA fragment length. LUG genes significantly decreased after day 7, but nptII and bla ARGs persisted past 30 days of operation. Bla ARGs were only detected in iDNA within 106 transgene copies/mL treatments at day 60 of incubation. It is possible that this increase is due to bacterial uptake of free bla in eDNA as a result of an antibiotic-driven selective pressure, inducing the integration of bla into bacterial cells. NptII genes were below detection in iDNA at no time points after digester construction. Finally, the naturally occurring concentrations of bla and nptII ARGs in anaerobic digesters are consistent with the findings presented in Chapter 5. However, the existence of these ARGs as primarily eDNA is novel and presently unreported in the literature.

Large amounts of human pharmaceutical products are consumed worldwide. Many drugs and their metabolites, referred to as pharmaceutically active compounds (PhACs), are not fully metabolized prior to household discharge resulting in their common occurrence in wastewater treatment plants (WWTPs). In most instances, WWTPs present the first treatment opportunity for removing PhACs and preventing significant environmental exposure. Because most municipal WWTPs rely on the microbial component of the activated sludge process, there is a need to estimate the influence of PhACs in wastewater influent on the activated sludge microbial communities and the treatment performance of WWTPs. The objective of this dissertation was to determine the impact of selected PhACs (i.e., ketoprofen, naproxen, clofibric acid, carbamazepine and gemfibrozil) on activated sludge microorganisms and key individual microbial species in domestic wastewater treatment. Analyses were performed in batch reactors initially and then in laboratory-scale sequencing batch reactors (SBR) which mimic WWTP operations. Ammonia oxidizing bacteria (AOB) were selected as indicator organisms because of their importance in wastewater treatment and demonstrated sensitiveness to toxic compounds.

The batch experiments results suggested that microbial growth inhibition was correlated to organic loadings. In the presence of 0.2% (v/v) ethanol, significant inhibition, ranging from 34 to 43%, was observed for all PhACs other than clofibric acid.

Nitrification inhibition studies using Nitrosomonas europaea, a model AOB strain showed that ketoprofen, naproxen, carbamazepine and gemfibrozil inhibited nitrite production. The corresponding maximum nitrification inhibition rates were 25, 29, 22 and 26%, respectively. Inhibition was shown to increase with PhAC concentration for concentrations greater than 0.1 µM. Results from membrane integrity tests suggest that the inhibition may be due to the disturbance of the cell membrane by PhACs and such inhibition was shown to be irreversible.

Even though PhACs were shown to inhibit the nitrification rate in pure culture studies, the performance of SBRs exposed to individual PhACs was not adversely affected neither in terms of COD nor ammonia removal. Microbial fingerprinting for both total bacteria and AOB confirmed that no significant shifts occurred when microbial communities were exposed to PhACs. However, some PhACs introduced in binary mixture were found to both inhibit the nitrification of N. europaea as well as the performance of SBRs. The mixture composed of 0.5 μM ketoprofen and 0.5 μM naproxen showed significant inhibition (25%) on the nitrite production of N. europaea although neither 0.5 μM ketoprofen nor 0.5 μM naproxen had significant effect when presented alone. Similarly, both COD and ammonia removal were significantly impacted by binary mixtures of PhACs. These results suggest that mixture effects can play an important role in an overall treatment's nitrification potential and this phenomenon should be further investigated.