Organic micropollutant (OMP) biotransformation in aquatic systems and the role of dissolved organic matter (DOM) as auxiliary substrates

Anna-Ricarda Schittich

Research output: Book/ReportPh.D. thesis

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Freshwater contamination by organic micropollutants (OMPs) represents a major threat for ecosystems and human health. A major source of this pollution is located in peri-urban catchments, as agricultural runoff and wastewater effluents can lead to significant inputs of OMPs into freshwater systems. Therefore, improving our understanding of OMP behaviour in both natural and engineered systems, and investigating their intersections is essential in order to protect freshwater resources. Biodegradation represents a key process for the removal of OMPs and for limiting their concentrations in aquatic systems. In wastewater treatment plants (WWTPs), autotrophic co-metabolic OMP transformation by ammonia oxidizing microorganisms has been suggested as an important process for OMP transformation. However, the exact mechanisms and the classes of OMPs susceptible to co-metabolic OMP transformation are not yet fully understood. Moreover, co-metabolic OMP transformation commonly does not result in OMP mineralization and hence in full OMP removal. Another process that may result in OMP mineralization is heterotrophic metabolic (i.e., growth-linked) OMP biodegradation. This process might thus play an essential role for full OMP removal in both WWTPs and natural systems. Considering the typically low concentrations of micropollutants in aquatic systems (ng-μg L-1), heterotrophic OMP degraders, in addition to OMPs, may depend on natural occurring carbon sources present in the pool of dissolved organic matter (DOM). Such auxiliary carbon sources might be vital for the survival of heterotrophic OMP degraders. However, the classes of DOM constituents that are biodegradable and available for OMP degraders under different conditions, and their effects on OMP biodegradation remain poorly investigated.

The research performed in this PhD project aims at gaining a mechanistic understanding of co-metabolic and metabolic biodegradation of organic compounds (OMPs and DOM constituents) in aquatic systems. The main focus was on investigating links between the chemical structures of different organic compounds and their biodegradability. Furthermore, the role of different DOM constituents for heterotrophic OMP biodegradation was investigated in labscale experiments. Additionally, the quality and potential bioavailability of DOM from a peri-urban stream was characterized, in order to relate the results from lab-scale experiments to environmental conditions.

The first part of this PhD thesis evaluates the role of ammonia oxidizing microorganisms for co-metabolic OMP transformation in WWTPs based on a literature review and the theoretical prediction of biodegradation pathways. A specific focus was on linking structural moieties of OMPs to reactions supported by the enzyme ammonia monooxygenase (AMO). The analysis showed that a high percentage of the investigated OMPs contains structural moieties that would be susceptible to AMO mediated transformations. Furthermore, AMO catalyzed transformations could explain the formation of various biotransformation products previously observed in pure culture studies with ammonia oxidizers or in microbial wastewater communities. Hence, our study suggests that in systems which are dominated by ammonia oxidizers, AMO mediated OMP transformation might significantly contribute to the overall OMP transformation. However, it remains important to additionally investigate metabolic heterotrophic OMP transformation, since this process might, in contrast to co-metabolic transformations, result in complete OMP removal.

The main part of this thesis thus investigates metabolic heterotrophic OMP biodegradation. A specific focus was on elucidating the ability of a model OMP degrader (Novosphingobium sp. KN65.2) to biodegrade different DOM constituents and to elucidate its ability to compete for the uptake of these DOM constituents against a reference strain (Pseudomonas fluorescens sp. P17; generalist). Furthermore, the effect of four selected DOM constituents on OMP biodegradation was investigated. Theoretical pathway predictions were combined with laboratory experiments, fluorescence spectroscopy and parallel factor analysis (PARAFAC) to investigate growth-linked OMP-DOM biodegradation. This novel approach, combining different techniques, allowed us to gain a systematic understanding of OMP-DOM biodegradation mechanisms. All experiments were conducted with pure or binary cultures, chemically defined DOM constituents (aromatic amino acids and lignin derivatives), and the model OMP carbofuran (pesticide). Our results reveal a high specificity of the model OMP degrader for DOM constituents with parahydroxylated primary aromatic ring substituents and demonstrate that a range of naturally occurring substrates can support the strain’s growth, in addition to the OMP carbofuran. Competition experiments revealed a disadvantage of the OMP degrader to compete for the biodegradation of specific DOM constituents against the generalist. The disadvantage likely results from longer lag-phases observed for substrate degradation by the OMP degrader, indicating that experimental/environmental conditions which decrease these long lag-phases might significantly enhance the competitiveness of the model OMP degrader for the uptake of auxiliary substrates. Biodegradation kinetics of the OMP carbofuran were stable in the presence of all selected DOM constituents, indicating their suitability as auxiliary substrates for carbofuran biodegradation under the investigated conditions. However, the tested DOM constituents could be divided into two groups with distinct transformation mechanisms. This may, in the long-term, significantly influence their effect on OMP biodegradation. Our results thus highlight the importance of studying biodegradation mechanisms for an improved understanding of both the effect of DOM constituents on OMP biodegradation and the competiveness of OMP degraders for auxiliary substrate uptake.

Finally, DOM samples taken from a peri-urban Danish stream were qualitatively characterized using fluorescence spectroscopy and PARAFAC analysis, a powerful tool to trace dynamics of environmental DOM in aquatic systems. The analysis revealed a seven component PARAFAC model, including two protein-like, and five humic-like components. The two protein-like components showed a significantly higher relative contribution to the total fluorescence signal compared to the five humic-like components for most sampling stations and field campaigns. Since fluorescence in the protein-like region is commonly associated with more labile DOM, this might indicate a potential source of auxiliary substrates for OMP degraders. However, a slight but steady increase of these two components in total fluorescence along downstream sampling stations might instead indicate that these components (partly) represent refractory substances (i.e. fluorescent micropollutants). This is reasonable, considering that three WWTP outlets feed into the studied stream and highlights the need to further investigate OMPs at the intersection of natural and engineered systems.

In conclusion, this PhD thesis explored the co-metabolic and metabolic biodegradation of organic compounds (OMPs and DOM constituents) and contributed to an increased understanding of the relation between chemical structures and compound biodegradability by combining theoretical investigations and laboratory biodegradation experiments. In particular, experiments with pure cultures and chemically defined carbon sources demonstrated that subtle structural differences can significantly affect whether DOM constituents serve as growth substrate. These conceptualized studies should in the future be stepwise extended, for example, to additionally investigate the biodegradability of more complex DOM. This next step would provide detailed insights on biodegradation mechanisms together with an increased environmental representativeness.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherTechnical University of Denmark
Number of pages250
Publication statusPublished - 2022


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