Department Environmental Chemistry

Open master thesis topics 2019

Does enzyme evolution generated through changes of substrate specificity induce different O2 activation pathways

Preferred start date: Spring 2020

Short Description:

The faulty oxygenation of xenobiotic compounds leads to the formation of reactive oxygen species (ROS) after O2 activation. Those ROS are thought to promote mutations that ultimately lead to the adaptation of exposed microorganisms and their enzymatic machinery to new carbon sources.
In this master thesis we will test this hypothesis by assessing several biochemical indicators for O2 activation and substrate specificity using a microbial strain that has evolved through a substrate adaption processes.

To that end, we will work with Acidovorax sp. strain JS42 as well as mutant strains which were generated by switching from 2-nitrotoluene to 3-nitrotoluene as source of carbon and energy. This selection process has led to enzymes with enhanced affinity for the new substrate compared to the wild type enzyme. While mutations are documented by changes in amino acid residues, information on the efficiency of substrate oxygenation, O2 uncoupling, and the type of activated O2 species are lacking and will be provided in this work. 

Links: Project flyier (https://polybox.ethz.ch/index.php/s/vpQhPwf3FlulL5P); Group webpage (http://www.eawag.ch/en/department/uchem/organisation/gruppenseite-hofstetter/)

Supervision: Thomas Hofstetter (thomas.hofstetter@eawag.ch), Charlotte Bopp (charlotte.bopp@eawag.ch).

Oxygenation efficiency of nitroarene dioxygenases

Preferred start date: August 2019

Short Description:

Iron-containing oxygenases catalyse the initial step of the most important biodegradation reactions of aromatic pollutants but can also give rise to oxidative stress upon faulty oxidation reactions. Despite their importance for microbial contaminant metabolism, their catalytic mechanisms are poorly understood. With the study proposed in this master thesis, we aim at understanding basic aspects of the active site chemistry of Rieske non-heme ferrous iron dioxygenases that lead to both ``good'' results, that is pollutant degradation, and ``bad'' side effects through the generation of toxic reactive oxygen species. Using dioxygenases that are capable of oxygenating nitroaromatic compounds, we plan to establish quantitative relationships for the ``efficiency'' with which these enzymes incorporate molecular O2 into the aromatic contaminants vs. the amount of O2 that ends up in reactive oxygen species. We hypothesize that the degree of such O2 uncoupling is correlated with the expression of the kinetic isotope effect for the hydroxylation of the aromatic substrate. This hypothesis will be tested with measurements of mass- and electron balances as well as a characterization of enzyme kinetics. 

Links: Project flyier (https://polybox.ethz.ch/index.php/s/vpQhPwf3FlulL5P); Group webpage (http://www.eawag.ch/en/department/uchem/organisation/gruppenseite-hofstetter/)

Supervision: Thomas Hofstetter (thomas.hofstetter@eawag.ch), Charlotte Bopp (charlotte.bopp@eawag.ch).

Micropollutant distribution dynamics in a small creek:
From water to aquatic invertebrates

Starting Date: Autumn 2019/Spring 2020

Short Description:

The number of chemicals reaching surface waters from agricultural, industrial and personal use is steadily increasing. This can lead to effects on non-target organisms living in the aquatic environment. While for most of the compounds lab studies exist that analyse the uptake into and the effect onto aquatic organisms, it has been shown that there are strong deviation when compared to data collected in the field. For example is the internal concentration of the neonicotinoid pesticides in gammarid species around a 100fold higher than expected based on the measured water concentration and bioaccumulation factors determined in lab studies.

In this project, the temporal dynamics of the micropollution distribution into the most relevant compartments of a small creek (water, sediments, biofilms, detritus and Gammarus invertebrates) are analysed.  

Depending on the interests of the applicant and the starting data, the master thesis can focus on different stages of the analytical process.

Some possibilities:

- Target analysis of a few priority compound in one compartment, including sample work-up, LC-MSMS measurement and target screening.

- Target/Suspect screening for non-priority compounds in pre-measured sample of different compartments and analysis of the distribution dynamics.

-  Suspect/Non-target screening for possible transformation products in premeasured samples. (Only with later start of project)


keywords: LC-HRMS, pesticides, target/suspect screening, bioaccumulation, exposure dynamics

contacts: juliane.hollender@eawag.ch and benedikt.lauper@eawag.ch

Analysing abiotic transformation of micropollutants across different sediment-water test systems

Starting Date: Between July and September 2019

To date, it remains impossible to fully prevent bioactive and therefore potentially harmful substances, such as pharmaceuticals and pesticides, from entering the aquatic environment. Some pathways through which those pollutants reach surface waters are hardly controllable, e.g. agricultural runoff. The manufacturing and use of chemicals, however, could be controlled more easily. From the regulatory side, there are testing guidelines in place that dictate to assess the transformation potential of chemicals that may enter surface waters prior to their registration on the market. However, there is scientific consensus that transformation kinetics observed in those regulatory studies do not directly project into compound behaviour in the environment. A thorough understanding of relevant system-specific differences that govern the transferability from laboratory to field systems is still missing. This Master Thesis is embedded in the context of a project that targets the connection between a substance’s persistence measured in different regulatory water-sediment test systems and its fate in the environment.  

We follow the dissipation of 44 micropollutants, mostly pharmaceuticals and pesticides, in various laboratory test systems. We then use the observed concentration-time series to estimate transformation rate constants. The ultimate goal is to generate substance-specific and therefore system-independent parameters that can be used to predict transformation kinetics in rivers based on experimental data from lab studies.  

For surface water bodies, the fate and transport of individual compounds is controlled by complex interactions between abiotic and biotic processes such as hydrolysis, direct and indirect phototransformation, microbial biotransformation and sorption. While we are currently investigating biotransformation in running experiments, we are missing information on the abiotic transformation pathways of our target compounds. To close this knowledge gap, we are seeking a student who will perform sorption and phototransformation experiments in the lab.  

Briefly, sorption experiments will be carried out with two different sediments in various solids-to-water ratios at two micropollutant concentration levels representative of lab and field conditions. Samples will then be analysed for solid-sorbed and dissolved compound concentrations using liquid chromatography - mass spectrometry (LC-MS/MS). A sun-simulator will be used to determine direct and indirect phototransformation in samples containing the investigated micropollutant mixture and different contents of dissolved organic matter. In addition to this experimental work, a thorough literature study on previous research on sorption and phototransformation behaviour will be necessary to bring the results in context with existing data.  

We encourage motivated master students with a background in environmental science, analytical chemistry, or related fields to apply. During your time at Eawag, you will perform independent lab work and acquire skills in sample preparation, LC-MS/MS analysis and potentially simple kinetic modelling.  

Keywords: micropollutants, sorption, phototransformation, LC-MS/MS

Advisors: Carolin Seller, Dr. Elisabeth Janssen, Prof. Dr. Kathrin Fenner

Contact:  carolin.seller@eawag.ch or kathrin.fenner@eawag.ch

 

 

Assessing inactivation rates of extracellular enyzmes in surface waters

Starting Date: as soon as possible

Various organisms including bacteria and algae produce extracellular enzymes. These extracellular enzymes are also referred to as “master recyclers” as they turn over vast amounts of organic matter in surface waters, driving carbon and nutrient cycling. The enzymes are released by heterotrophic microorganisms to the environment via active secretion or cell lysis. Once released, extracellular enzymes are exposed to numerous environmental factors that can lead to their inactivation. The activity of an enzyme depends critically on the integrity of its macromolecular structure. Inactivation of extracellular enzymes and therefore changes in their structure need to be better understood to predict their impacts on biogeochemical cycles. Besides studying transformation processed driven by microbes and light, the enzymes can also be inactivated by changing environmental conditions and chemicals that act as inhibitors.

In this Thesis project, the student will learn about mechanisms that inactivate critically important extracellular enzymes in our surface waters. Briefly, the student will work with model enzymes and enzymes from natural biofilms. The student will use colorimetric activity assays to follow the functioning of the enzymes and then test the stability of these enzymes under different solution condition (pH, temperature, etc.) and in the presence of inhibitors. Here, the student will test whether a group of cyclic peptides can inhibit these enzymes including natural toxins produced by cyanobacteria and peptide-based pharmaceuticals.

In this Thesis project, the student will learn about 

  • enzymatic activity assays (HPLC, colorimetric plate reader assays, kinetic rates)
  • dose response batch tests (IC50 values, mixture effects)
  • research environment at the Environmental Chemistry Department at Eawag
  • project planning, experimental routine and data interpretation, writing and presentation skills

For a successful thesis, you should 

  • have strong interest in environmental science and chemistry
  • ideally have first experience (practical courses, internships) regarding laboratory work
  • be proficient in speaking and writing in English
  • be enthusiastic and motivated

Keywords: enyzmes, inactivation rates, inhibition by toxins and pharmaceuticals

Advisors: Christine Egli, Dr. Elisabeth Janssen

contact:
elisabeth.janssen@eawag.chchristine.egli@eawag.ch

Identification of ozonation transformation products during wastewater treatment

Starting date: as soon as possible

Short description:

Ozonation is increasingly applied for the abatement of micropollutants, such as pharmaceuticals and personal care products, during drinking water and wastewater treatment. However, the micropollutants are not fully mineralized but ozonation transformation products (OTPs) are formed. Persistent OTPs might enter the aquatic environment with the effluent of wastewater treatment plants. However, it can be hypothesized that OTPs are better biodegradable during biological post-treatment as their parent micropolluants. Unfortunately, up to now only a few single substance studies are available that test this hypothesis. 

Project:

Within the TRANSFO3RM project, laboratory-based ozonation batch experiments with mixtures of micropollutants were performed and measured with liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to identify OTP signals. These OTPs were screened for in samples of both a full-scale wastewater and a pilot drinking water treatment plant with biological post-treatment after ozonation. Within this project, the following topics can be tackled for the master thesis, I) differentiation between persistent and biodegradable OTP peaks , II) behavior of biodegradable OTPs over the sandfilter III) structure elucidation of prioritized OTPs, or IV) identification and structure elucidation of persistent OTPs entering the aquatic environment after wastewater treatment. The exact topic of the thesis can be discussed together with the master student depending on her / his interests. Since all experiments are already performed, this master thesis does not involve lab work, but insight can be gained. 

keywords:   LC-HRMS, wastewater treatment with ozone, suspect screening, structure elucidation, (lab work already finished)

contact:
rebekka.gulde@eawag.chchrista.mcardell@eawag.ch

Pollution fingerprint of waste water – development of a novel combinatory approach linking LC-ICP-MS and LC-Orbitrap

keywords:   LC-HRMS, LC-ICPMS, non-target screening, industrial and   municipal wastewater

contact:
heinz.singer@eawag.chjulie.tolu@eawag.chlenny.winkel@eawag.ch

Application of effect-directed analysis for the identification of genotoxic byproducts potentially formed during ozonation of wastewater

Starting date: January or later (spring 2020)

Organic micropollutants (MPs) such as pharmaceuticals, hormones, or pesticides can cause undesired effects when they are discharged into the aquatic environment. Wastewater discharges are one of the major sources of introduction of MPs into watercourses. While conventional wastewater treatment plants (WWTPs) are efficient in removing organic material and nutrients, their removal of MPs is insufficient. In Switzerland, a new Water Protection Act, in force since 2016, requires major WWTPs to upgrade their treatment technologies so that treated wastewater contains on average no more than 20% of MPs in influent wastewater. Ozonation is one of the treatments that has been and continues to be used in upgrading WWTPs to fulfill the requirement of abatement of MPs. However, this chemical process leads to the formation of ozonation transformation products (OTPs) and ozonation byproducts (OBPs), among which some are potentially toxic. Accordingly, a better understanding of the chemistry of reactions taking place in ozonated wastewater as well as the identification of precursors and factors which may lead to or favor the formation of toxic OTPs and/or OBPs is required. This is especially relevant for WWTPs receiving considerable industrial inputs, where the profile of precursors and hence of the formed OTPs and OBPs, could be different from those encountered in WWTPs without industrial inputs, engendering stronger toxicological concerns.

In the proposed master thesis, the student will investigate the formation of genotoxic compounds during ozonation of wastewater. In brief, the student will carry out bioanalytical evaluation of laboratory- or pilot-plant-ozonated wastewater samples in order to identify genotoxic compounds and ultimately identify their precursors. The bioanalytical strategy is based on an integrative approach that combines genotoxicity assays (particularly Ames test), genotoxic sample fractionation, and chemical analysis using high-resolution mass spectrometry (Orbitrap technology).

In this project, the student will be able to develop multidisciplinary skills related to: 

  • Preparing and enriching samples (e.g. solid-phase extraction)
  • Conducting bioassays (e.g. working in sterile conditions, preparing bacteria for Ames test)
  • Performing mass spectrometry data analysis
  • Research project planning and data interpretation

Competent candidates are those with strong interests towards multidisciplinary research in particular related to toxicology and analytical chemistry.

keywords: wastewater treatment, ozonation, genotoxicity, suspect and non-target screening, effect-directed analysis

Contact:
tarek.manasfi@eawag.chchrista.mcardell@eawag.ch

Designing and comparing experimental set-ups to study biotransformation in river biofilms

Keywords: Field work, native stream biofilm, artificial substrates, development of experimental setup, pesticides, pharmaceuticals, microorganisms

Description: Stream biofilms consisting of bacteria, algae and other microorganisms play an essential role in aquatic ecosystems. They have been shown to possess the ability to reduce the concentrations of chemical pollutants via e.g. biotransformation, usually demonstrated in controlled laboratory experiments. Naturally, stream biofilms grow on rocks and pebbles, but also on any other submerged surfaces. In order to evaluate processes, such as, e.g., biotransformation, in a consistent way, it is crucial to develop experimental setups that allow for reproducible research. Therefore, stream biofilms that are used for laboratory experiments are often grown on artificial substrates like, e.g., glass plates. In order to increase the biomass/volume ratio, those biofilms are then usually suspended in water, instead of using them in their attached form. Regardless, the consequences of such modifications from the natural appearance are not well investigated. Also, it is not well understood if results gained with such modified systems still can be translated back to behavior in the natural environment.

This project addresses those knowledge gaps by i) developing test systems that are more representative of naturally grown biofilms than the ones used so far, ii) doing biotransformation experiments with the newly developed systems and iii) comparing the results to a standardized testing system.

Project: One goal of this project is the development of an experimental setup that allows using stream biofilms in their native form, i.e. on a solid surface (glass slide, stones/pebbles) in contrast to suspending the biofilms. This experimental system will then be used to assess biotransformation of a mixture of environmentally relevant polar organic micropollutants. To do so, test reactors will be spiked with a set of micropollutants and the decrease of these pollutants will be monitored over time by means of high-performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS). Finally the outcomes of the different experimental setups will be evaluated (i.e. glass vs. stone vs. suspension). The MSc student will learn how to properly plan and set up an experiment, will gain knowledge in sophisticated analytical methods, and learns how to handle and evaluate large chemical datasets.

Advisors: Werner Desiante, Prof. Dr. Kathrin Fenner

Contact: werner.desiante@eawag.ch or kathrin.fenner@eawag.ch