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京都大学 / 新制・課程博士 / 博士(理学) / 甲第23741号 / 理博第4831号 / 新制||理||1691(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 酒井 章子, 教授 髙林 純示, 教授 松下 智直 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

This study consists of three research phases. First, we developed corrective action strategies to mitigate the impact of calcium hypochlorite and cadmium pulse shocks for the Plum Island Wastewater Treatment Plant (WWTP) in Charleston, SC. The corrective action strategies were developed in consultation with industrial consultants and operational personnel from the utility. These strategies were tested using a laboratory scale system, which was constructed and operated similar to the parent facility. Two corrective actions were tested for calcium hypochlorite, while only one strategy was tested for the cadmium at the laboratory scale. This study shows that no corrective action strategies are required for an acute hypochlorite stress. This is due to the fact that hypochlorite is highly reactive and dissipates rapidly on contact with the wastewater matrix, thus causing only low level process deterioration. In fact, implementation of corrective action strategies results in greater process deterioration as compared to the non-intervention approach. The corrective action tested for cadmium stress showed potential for reducing the peak impact of the toxin and allowed for faster process recovery as compared to the unstressed control. For the second phase, the corrective actions were tested at a pilot scale facility operated at the Plum Island wastewater treatment plant. We tested two different corrective action strategies for cadmium, while only one strategy was tested for hypochlorite during the pilot scale study. Similar to the laboratory scale experiments, we conclude that no mitigative approaches are necessary for an acute hypochlorite stress. Additionally, the implementation of mitigative approaches for the pilot scale cadmium stress events resulted in greater process deterioration as compared to the non-intervention approach. In contrast to the laboratory scale experiments, theoretical effluent blending calculations showed that corrective actions may not reduce the impact of the cadmium stress. This was attributed to the lower intensity of process deterioration caused by the simulated cadmium stress. The pilot scale study shows that prior to implementing a corrective action strategy, the operator should determine the probable extent of process deterioration due to the detected chemical contaminant before deciding if a corrective action is needed. The pilot scale study also evaluated the effectiveness of current sensor technologies towards the upstream detection of influent anomalies and reliable monitoring of process performance during an upset event. Multivariate analysis on the rate of change of influent sensor signals was reliably able to detect the presence of both toxins tested during this study. For the third phase of this research, we investigated the impact of cadmium stress on the structure and function of bioreactor microbial communities. We observed significant increases in post-stress heterotrophic and autotrophic bacterial respiration rates for the bioreactors subjected to cadmium stress. The higher respiration rates were due to an increase in bacterial abundance in the cadmium stressed reactors. We were also able to show that the increase in bacterial abundance was not due to changes in community structure or due to cadmium induced deflocculation. In fact, this study demonstrates that transient cadmium stress reduces predator abundance within the activated sludge community and this reduction in predator grazing was responsible for the increase in bacterial abundance. This research highlights the importance of higher life forms, specifically eukaryotic microorganisms, in regulating bacterial community dynamics in systems undergoing chemical perturbations. / Ph. D.

Numerous studies have looked at biotic succession at the aboveground level; however, there are no studies describing fungal community change associated with long-term ecosystem development. To understand ecosystem development, the organisms responsible for shaping and driving these systems and their relationships with the vegetation and soil factors, it is critical to provide insight into aboveground and belowground linkages to ultimately include this new information into ecosystem theory. I hypothesized that fungal communities would change with pedogenesis, that these changes would correlate with vegetation community change, and that they should show change of composition and diversity as the seasons change. Chapter 1 discusses the main topics related to this dissertation. Chapter 2 includes a publication draft that describes a study of sand-dune soil samples from northern Michigan that were analyzed to pinpoint the structural change in the fungal community during the development of the ecosystem. The samples were analyzed by pyrosequencing the soil DNA, targeting the internal transcribed spacer region. Chapter 3 contains a coauthored published paper that describes plant invasion of fields in Virginia to determine how they impact soil bacterial and fungal communities. The bacterial and fungal communities that were invaded by 3 different plant species exhibited similar changes, regardless of plant species, suggesting that some functional traits of invasives may have similar impacts on belowground communities. Chapter 4 remarks the conclusions of this research. / Master of Science / Ecosystems, including the soils underneath, are the environments that surround us perform a large number of critical human-relevant functions (playing roles in production of food, filtration of water for drinking, sequestration of carbon and nitrogen to build soil organic matter, and buffer against flooding). Yet, how these systems naturally develop over time are still in need of detailed study. One particular area of interest and need is the study of belowground fungal communities. It is not commonly known, but plants and ecosystems are highly dependent on the underground web of fungal hyphae that transform nutrients and provide water to plants. A first step in gaining this understanding utilized a natural ecosystem development gradient known as a chronosequence. It was expected that fungal communities would change as soil and ecosystem development progressed and that they would mimic changes in soil and vegetative properties. Discerning if these linkages occur is the first step to assessing how they work together to create ecosystems and their valuable environmental services. Chapter 1 provides a discussion of the main topics in this dissertation. Chapter 2 is at the heart of the dissertation via a study of fungal communities in a developmental soil ecosystem in northern Michigan in addition, in Chapter 3, I include a coauthored published paper that describes plant invasion of fields in Virginia. Chapter 4 remarks on the major conclusions of this Master thesis, supporting the role that vegetation and fungal community change in soil are associated with one another.

The discovery of antibiotics has been considered as one of the most remarkable scientific accomplishments of the last century. However, the extensive usage of antibiotics has led to the rapid emergence of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB), which have been recognized as one of the biggest threats to human and environmental health. While ARGs and ARB are ubiquitous in diverse environments, aquatic environments play a particularly crucial role in their prevalence and dissemination. Furthermore, the microbial complexity and various pollutants persisting in aquatic environments significantly contribute to the evolution and spread of ARGs and ARB. However, the knowledge regarding the distribution pattern of ARGs on a large scale, as well as the interaction between microbial community, specific pollutants, and ARGs and ARB, is currently limited. In this study, I conducted systematic work at multiple scales, to fill crucial knowledge gaps that could support the future management of the spread of antimicrobial resistance. In particular, on the ecosystems level I depict the biogeographical patterns of ARGs in freshwater reservoirs, on the community level I explored the selection patterns of combinations of antibiotics on multidrug resistant strains in complex community context, and finally, on the population level I investigated the impact of cigarette smoke, and waste products on the dissemination of ARGs. In my first study, samples were collected from 24 freshwater reservoirs across southeast China and the biogeographical patterns of bacterial communities and ARG profile were characterized using 16S rRNA gene high-throughput sequencing and high-throughput-quantitative PCR. A distance-decay pattern for both, microbial communities and ARG profiles, were observed. However, larger differences between reservoir ARG profiles than microbial community compositions were detected. Further, I found that the biogeographical patterns of bacterial communities were simultaneously driven by stochastic and deterministic processes, while ARG profiles were not explained by stochastic processes, and the relationship between bacterial communities and ARG profiles was weak. In summary, this study indicated a decoupling of bacterial community composition and ARG profiles in inland waters under relatively low-human-impact at a large scale. In a second study, the selection dynamics for multidrug resistance between isogenic pairs of E. coli strains under exposure to multiple selective agents in the absence and presence of the microbial community were investigated using microcosm experiments. The presence of the community significantly decreased the selection for multidrug resistant strain under exposure to a single antibiotic. While pressure through the second antibiotic significantly decreased the activity and diversity of the community, its ability to reduce selection was consistently maintained at levels comparable to those recorded in the absence of the second antibiotic. This indicates that the observed effects of community context on selection dynamics are rather based on competitive or protective effects between the focal strains and a small proportion of bacteria within the community, than on general competition for nutrients. Last but not least, the effect of cigarette-derived pollutants on the proliferation of ARGs was explored using multifaced approaches. Cigarette smoke condensate in an artificial lung sputum medium significantly elevated the transfer rates of a multi-drug-resistance encoding plasmid between Pseudomonas strains. The overproduction of reactive oxygen species (ROS) as part of the bacterial stress response was directly connected to the increasing transfer rates. Similarly, cigarette ash leachate in an environmental medium significantly increased the plasmid transfer rates, and overproduction of ROS was equally detected. Furthermore, used cigarette filters with entrapped toxicants were submerged in a wastewater stream and colonized by distinct microbial communities compared to those colonizing unused control filters. The microbial communities colonizing used cigarette filters were significantly enriched in AMR, potential pathogenic bacteria and mobile genetic elements. Overall, the insights gained within this thesis into the spread of AMR at multiple scales constitute a valuable contribution to support future management and monitoring of ARGs in diverse ecosystems.