Description du sujet de thèse

Bacteria are widely used in environmental technologies. In wastewater treatment, for example, the most widespread process for treating soluble organic pollution is the "activated sludge" process. In this process, the wastewater to be treated is brought into contact with a selected aerobic microbial ecosystem. Coupled with conventional two-stage nitrification-denitrification treatment of nitrogenous matter, this process offers perfectly acceptable depollution yields. However, the energy required to aerate these biological basins is excessive, accounting for around half the total energy consumption of a wastewater treatment plant, to which must be added the organic carbon substrate required for the denitrification stage. Increasing concern about environmental protection, disappearance of fossil fuels and global warming are encouraging the development of new, more efficient and less energy-devouring technologies. Utilization of anaerobic processes thereby is among the possible improvements and offer considerable advantages. These processes, by definition do not require aeration, which considerably reduces the treatment's energy bill. Furthermore they convert part of the organic matter into biogas that can be used directly as an energy carrier (H2 or CH4). The anaerobic digestion process for the remediation of organic molecules can be combined with a partial nitritation process followed by anaerobic ammonium oxidation (anammox), which reduces aeration costs by 60% and organic carbon addition costs by 100% compared to conventional nitrogen treatment. Indeed, the second step relies on autotrophic bacteria only which use CO2 as their carbon source instead of organic carbon while the preceding partial nitritation takes place in the limited presence of oxygen. Overall this process consumes little energy, results in fixation of part of the CO2 produced during the organic carbon treatment and produces very little sludge due to the very slow growth of anammox bacteria.
Anammox-type bacteria (for anaerobic ammonium oxidation), initially discovered in a water treatment plant in the Netherlands (1995), are quite unique, and have significant potential for effluent treatment processes. In the natural environment they contribute to the conversion of up to half of fixed nitrogen into N2. Despite their great ecological and economic interest our knowledge of their metabolism is scarce (Kartal et al 2013). In particular, the link between nitrogen metabolism, ATP formation and CO2 fixation for growth remains unexplored. During this thesis the combination of metabolic and molecular approaches aims at a better understanding of anammox metabolism. In particular, a membrane enzyme at the crossroads of nitrogen metabolism, CO2 fixation and energy conversion, the Rieske/cytb complex, will be purified and studied by biochemical, electrochemical and spectroscopic approaches (Bergdoll et al 2016). In anammox bacteria this wide spread enzyme indeed features additional subunits that may allow electron transfer reactions with NADPH and nitrogen compounds.
A further originality of this project is the use of microcalorimetry coupled to high-pressure chromatography to study the metabolism of the living cell. Calorimetry enables determination of the enthalpy related to metabolism and growth. It allows an overall view of the metabolic flux associated with the conversion of nitrogen and/or carbon molecules. The enthalpy measured can then be compared with theoretical value calculated from metabolic network models based on biochemical and biophysical data to determine reaction pathways.
Once a specific calorimetric signature of anammox bacteria established, the variability of their metabolism can be addressed and the impact of nutrients other than ammonium, nitrite and CO2 studied. A change in the expression profile of the Rieske/cytb complexes in response to these different growth conditions will provide further information on the involvement of this key enzyme in the metabolism.
The results of this interdisciplinary work should shed light on our understanding of the peculiar anammox metabolism at the level of global fluxes, the impact of different carbon substrates, and at the molecular level define the role of the Rieske/cytb complexes as a potential metabolic node. In a wider perspective, control and development of waste water treatment processes which are currently steered empirically, will benefit from the knowledge gained during this thesis.
References:

Bergdoll L, ten Brink F, Nitschke W, Picot D and Baymann F (2016) From low- to high-potential bioenergetic chains: thermodynamic constraints on Q-cycle function. Biochim.Biophys.Acta Bioenergetics 1857, 1569-1579.
Kartal B, De Almeida NM, Maalcke WJ, Op den Camp HJM, Keltjens JT (2013) How to make a living from anaerobic ammonium oxidation. FEMS Microbiol Rev 37, 428-461.
Tafoukt D, Soric A, Sigoillot JC, Ferrasse JH (2017) Determination of kinetics and heat of hydrolysis for non-homogenous substrate by isothermal calorimetry, Bioproc and Biosys Eng, 40(4), 643-650.

Wang Y, Wang G. Moitessier N, Mittermaier AK (2020) Enzyme Kinetics by Isothermal Titration Calorimetry: Allostery, Inhibition, and Dynamics. Front. Mol. Biosci. , Sec. Biological Modeling and Simulation, Volume 7.
Lackner S, Gilbert EM, Vlaeminck SE, Joss A, Horn H, van Loosdrecht MCM (2014), Full-scale partial nitritation/anammox experiencese - An application survey, Water research 5 5, 2 9 2 -3 0 3.

Contexte de travail

To carry out this project, the PhD student can rely on the biomass production of the anammox bacterium Candidatus Kuenenia stuttgartiensis in a planktonic culture in a 95% enriched chemostat located at the IMM fermentation platform in Marseille. The project will take place in the M2P2 and the BIP laboratory.
The M2P2 Water and Waste Treatment team under the direction of Audrey Soric works in process engineering for the design of wastewater treatment processes, including bioprocesses. The team has developed strong skills in the study of gas-liquid transfer mechanisms and kinetics linked to the degradation of organic loads. In the frame of this project the development of calorimetry, a technic Jean-Henry Ferrasse is an expert in, is planned to give access to kinetic characteristics in addition to conventional techniques.
The BIP is an interdisciplinary laboratory bringing together physicists, chemists and biologists to decipher the metabolism, including energy metabolism, of various bacterial systems. Two teams are involved in the project, one specializing in deciphering metabolic pathways and purification of proteins complexes under the direction of Marie-Thérèse Giudici-Orticoni, the other one in evolution and thermodynamics of the bioenergetic system. In the frame of this thesis purification and characterization of the Rieske/cytb complex, an enzyme family for which Frauke Baymann is a specialist, will be carried out at the BIP.
The candidate will benefit from the actions set up as part of the PhD program PLINIUS and will be enrolled in two doctoral schools: ED 353 (Engineer science) and ED 62 (life sciences).

Contraintes et risques

The project will take place on two sites, i.e. on the Arbois campus where the M2P2 laboratory is located, and in the BIP laboratory on the Joseph Aiguier campus. The two sites are connected by public transport, and the thesis will be carried out in eight-month periods on both sites.
Salary and environment of the thesis will be financed by a PRIME project funded by the CNRS.